YTHDF2 upregulation and relocation dictate CD8 T cell polyfunctionality in tumor immunity

31 Epigenetic traits impact the antitumor function of CD8 T cells, yet whether and how RNA methylation 32 programs engage in T cell immunity is poorly understood. Here we show that the N6-methyladenosine 33 (m6A) RNA reader YTHDF2 is highly expressed in early effector or effector -like CD8 T cells and is 34 partially distributed in the nucleus. YTHDF2 loss in T cells exacerbates tumor progression and confers 35 unresponsiveness to PD -1 blockade in mice and humans. In addition to initiating RNA decay for 36 mitochondrial fitness, YTHDF2 can orchestrate chromatin regulation to promote T cell polyfunctionality. 37 YTHDF2-mediatd preservation of gene transcription arises from the interaction of YTHDF2 with IKZF1/3. 38 Accordingly, immunotherapy-induced efficacy could be largely restored in YTHDF2 -deficient T cells 39 through combinational use of lenalidomide. Moreover, m 6A recognition is fundamental for YTHDF2 40 translocation to the nucleus and autoregulation at the RNA level. Thus, YTHDF2 coordinates 41 epitranscriptional and transcriptional networks to potentiate T cell immunity. 42 43

YTHDF2; Teff and Teff-like cells; Immunotherapy; Lenalidomide 44 45 Highlights: 46 • YTHDF2 expression and distribution underpin the threshold for bona fide CD8 T cell effector 47 response 48 • Canonical YTHDF2 -mRNA decay pathway alleviates mitochondrial stress and CD8 T cell 49 exhaustion 50 • Nuclear YTHDF2 sequesters IKZF1/3-mediated transcriptional repression to safeguard CD8 T 51 cell polyfunctionality 52 • The tumoricidal activity of YTHDF2-deficient CD8 T cells could be repaired through the synergy 53 of anti-PD-1 and lenalidomide 54 55 56 57 58 59 60 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint

61 Among more than 170 types of RNA modifications, N6-methyladenosine (m 6A) represents the most 62 prevalent and abundant modification in eukaryotic mRNA. By enlisting the “writer” (methyltransferase), 63 “eraser” (demethylase) and “reader” proteins, dynamic m 6A modification regulates nearly every step of 64 mRNA metabolism and interferes with various biological processes 1. Emerging evidence shows that 65 m6A RNA modifiers within tumor cells or surrounding myeloid cells largely affect tumor immunity and 66 immunotherapy efficacy2,3. For instance, the tumor-intrinsic m6A demethylase FTO can either subvert 67 the host immune reaction by facilitating the expres sion of the immune checkpoint gene LILRB41 or 68 restrict T cell activation by altering the metabolic microenvironment 4. The methyltransferase complex 69 component METTL14 disrupts the interferon-γ signalling within microsatellite instability-low tumors and 70 limits the response to immune checkpoint blockade (ICB) therapy 5; however, METTL14 governs m6A 71 RNA stabilization in a subpopulation of tumor-associated macrophages to prevent T cell dysfu nction6. 72 The m 6A reader YTH domain family 1 (YTHDF1) in classic dendritic cells impedes the cross -73 presentation of tumor antigens and the cross-priming of CD8 T cells 7. Two recent reports showed that 74 myeloid cell YTHDF2 is also associated with tumor immunosuppression8,9. These findings indicate that 75 the m 6A machinery posttranscriptionally controls cancer -immune set points, which may be useful 76 targets for overcoming immunotherapy resistance. 77 Tumor-reactive CD8 T cells are key to both natural and therapy-induced antitumor immunity. However, 78 chronic exposure to tumor antigens or environmental stimuli may render CD8 T cells dysfunctional and 79 limit the outcomes of cancer therapy 10,11. Remarkably, T cell activatio n and differentiation are often 80 concomitant with epigenetic processes, many of which account for the molecular rewiring of T cell 81 dysfunction imposed by the tumor microenvironment (TME)12. Therefore, manipulating T cell epigenetic 82 programs may foster cancer therapeutic efficacy owing to the acquisition of long -term T cell 83 persistence13,14. Although epigenetic changes accompanying differentiation have been extensively 84 studied15, how T cell effector polyfunctionality is epigenetically safeguarded in the early phase remains 85 elusive. In addition, recent insights have revealed that a progenitor exhausted T (T pex) cell population 86 can partially differentiate into effector-like transitory exhausted T ( Tex) cells, which serve as a cardinal 87 force of T cell immunity when responding to anti-PD-1 therapy16,17, highlighting the need for knowledge 88 assimilation to better understand and harness this function al process. Given both distinct and shared 89 epigenetic circuits between different T cell subsets 18, we embark on identifying a novel regulator that 90 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint governs early epigenetic events exclusively for tumoricidal effector and effector -like T cells. m 6A-91 mediated RNA methylation and destabilization have been recognized as an ingenious mechanism for 92 T cell homeostasis 19 and survival 20, but the implication of m 6A RNA modifiers in antitumor T cells 93 remains an enigma. Therefore, it is imperative to illustrate the m 6A machinery underlying T cell 94 activation and therapy-induced rejuvenation. 95 RNA m 6A is cotranscriptionally installed by the methyltransferase complex in the nucleus 21. Recent 96 studies have suggested that m 6A modification has an interplay with and an impact on the chromatin 97 state. In mouse embryonic stem cells, METTL14 can recognize transcription elongation mark histone 98 H3 trimethylation at Lys36 (H3K36me3), which guides m 6A deposition on actively transcribed nascent 99 RNAs22. Conversely, m 6A-modified nuclea r RNAs can direct chromatin organization and gene 100 expression. The nuclear m6A reader YTHDC1 plays a sophisticated role in such a context; it can either 101 recruit the histone demethylase KDM3B to erase the repressive histone mark H3K9me2 , or dictate 102 nuclear R NA decay to restrict the chromatin activity and downstream transcriptio n 23-25. These 103 discoveries raise questions about whether the m 6A machinery adapts the binding interface for core 104 transcription factors and whether T cell activation necessitates the crosstalk between m6A modification 105 and chromatin organization. 106 YTHDF2, a highly effective m6A reader, specifically recognizes and degrades m6A-containing mRNAs 107 in the cytoplasm, where it primarily resides 26. Otherwise, under heat shock stress, nucle us-localized 108 YTHDF2 protects the 5’ untranslated region (5’UTR) of stress -induced transcripts from FTO-mediated 109 demethylation, resulting in cap-independent translation initiation27. In the present study, we reveal that 110 YTHDF2 is uniquely express ed and distributed during early T cell activation and therapy -induced 111 rejuvenation. Despite its short -term upregulation and nuclear localization, YTHDF2 ensures the 112 longevity and tumoricidal activity of CD8 T effector (T eff) and T eff-like cells. In quiescent T cells, 113 cytoplasmic YTHDF2 potentially destabilizes its cognate coding mRNA via m6A recognition, self -114 maintaining a low expression level under nonpathological conditions. When encountering robust 115 antigen stimuli or ICB therapy, a portion of YTHDF2 switches to a non-autoregulating state followed by 116 nuclear translocation , allowing cognate mRNA translation in early polyfunctional T cells . The 117 accumulation of YTHDF2 in the cytoplasm results in the degradation of the redundant mitochondrial 118 component-encoding mRNAs , withstanding mitochondrial stress and T cell exhaustion . In a more 119 tumor-rejecting manner, the nuclear import of YTHDF2 directs chromatin organization and effector 120 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint polyfunctionality by limiting IKZF1 and IKZF3 from the transcriptional inhibition of T cell receptor (TCR) 121 signalling. Conversely, YTHDF2 deficiency in T cells thwarts both endogenous and ICB-induced tumor 122 immunity in mice and is correlated with a poor therapeutic response in cancer patients. Nevertheless, 123 owing to the dominant transcriptional repression by IKZF1/3 in YTHDF2-null T cells, lenalidomide, a 124 clinically available IKZF1/3 inhibitor, could achieve a compensatory immune response in synergy with 125 ICB. Collectively, these data provide proof-of-concept evidence that YTHDF2 integrates RNA and DNA 126 epigenetics to potentiate T cell antitumor immunity. 127 128

129 YTHDF2 is selectively upregulated and redistributed in early Teff and Teff-like cells 130 To begin, we interrogated several transcriptomic datasets and assessed the expression of m 6A 131 machinery components in T cells that undergo diverse signals. When stimulated with anti -CD3/CD28 132 antibodies, both CD4 and CD8 T cells showed a predominant increase in Ythdf2 expression during 133 early activation (Supplementary Fig. 1 a, b). In an in vitro system mimicking different states of human 134 CD8 T cells 28, a high Ythdf2 mRNA level was observed during 3 –48 h of anti -CD3/CD28 stimulation, 135 but its expression decreased at later time points of activation as well as toward s exhaustion-like and 136 memory-like stages (Fig. 1a). Similarly, among the in vitro-generated CD4 T cell subsets , a group of 137 inducible tolerant T cells exhibited the lowest YTHDF2 level (Supplementary Fig. 1c). In immunotherapy 138 settings, PD-1 blockade induced the upregulation of YTHDF2 in CD8 T cells from responding tumors; 139 however, this upregulation occurred only during early tumor regression but not in the late regression or 140 progression stage (Fig. 1b). As shown in these datasets, the expression of the gene encoding another 141 important m6A reader, YTHDF1, could also be upregulated upon T cell activation and rejuvenation, but 142 its expression level was relatively low in tumor-infiltrating CD8 T cells (Supplementary Fig. 1d). A recent 143 report indicated that the loss of YTHDF1 in CD8 T cells does not affect antitumor immunity7. Therefore, 144 the potential function of YTHDF2 in T cell-mediated tumor immunity was the focus of this study. 145 We obtained both human peripheral and mouse splenic CD8 T cells for in vitro activation and validated 146 the inducible upregulation of YTHDF2 expression at the protein level ( Supplementary Fig. 1e, f ). By 147 performing liquid chromatography -tandem mass spectrometry (LC–MS/MS) analysis, we observed a 148 significant loss of m6A modification in the transcriptome after CD8 T cell activation (Supplementary Fig. 149 1g). We then conducted antibody -based m 6A sequencing (m 6A-seq) and noted that m 6A-150 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint hypomethylated peaks were mostly distributed in 3’ untranslated regions (3’UTRs) (Supplementary Fig. 151 1h). In addition to the possibility of mRNA 3′UTR shortening pending T cell activation 29, this observed 152 hypomethylation might also be explained by YTHDF2-induced destabilization of m 6A-containing 153 mRNAs26. 154 As shown by flow cytometry analysis, in vitro-generated early-phase effector CD8 T cells manifested 155 a much higher YTHDF2 level than did exhausted CD8 T cells (Fig. 1c). In C57BL/6 mice subcutaneously 156 challenged with ovalbumin (OVA) -expressing B16F10 cells, tumor-infiltrating T cells expressed more 157 YTHDF2 than did splenic T cells, among which CD44 +KLRG1+ terminal effector CD8 T cells were 158 predominant (Fig. 1d). Consistent with the transcriptomic data, PD -1+TIM3+TCF1- terminal exhausted 159 CD8 T cells exhibited a much lower YTHDF2 level than did PD-1+TCF1+SLAMF6+TIM3- Tpex and 160 CD44+PD-1-TCF1+CD127+ memory T cell (T mem) subsets ( Fig. 1e ). In addition, early anti-PD-1 161 treatment led to a 3-fold increase in YTHDF2 expression in Tpex and their progeny PD-1+TCF1-KLRG1+ 162 transitory Tex cells (also known as terminal T eff-like cells30, which are believed to expan d upon ICB for 163 tumor killing16,17,31 (Fig. 1f ). These observations imply that YTHDF2 may widely impact tumor-164 experienced CD8 T cells, particularly the early effector and effector-like subsets. 165 Whereas it is well-accepted that YTHDF2 primarily resides in the cytosol, where mRNA decay occurs, 166 previous work has shown that heat shock stress c an lead to the relocation of YTHDF2 to the nucleus 167 through an unknown mechanism 27. In the present study, we as sessed whether T cel l activation or 168 reinvigoration could alter the subcellular localization of YTHDF2. Surprisingly, wild-type and OT-1 CD8 169 T cells accumulat ed nuclear YTHDF2 when stimulated with anti -CD3/CD28 antibodies and OVA 170 peptides, respectively, for 12 –48 h (Fig. 1g, h and Supplementary Fig. 1i, j ). In the case of the Jurkat 171 human T lymphoma cell line , we detect ed an inherent nuclear fraction of YTHDF2, which modestly 172 increased after phytohaemagglutinin (PHA) treatment (Supplementary Fig. 1k, l). Akin to the temporary 173 YTHDF2 relocation observed in vitro , tumor-infiltrating CD8 T cells showed nuclear expression of 174 YTHDF2 in regressing but not progressed lesions (Fig. 1i). We then implanted OT-1 (expressing a TCR 175 specific to MHC -I-restricted OVA residues) transgenic mice with B16F10 or B16F10 -OVA cells but 176 discovered YTHDF2-redistributed CD8 T cells only within tumors formed by the latter ( Supplementary 177 Fig. 1m), showing that such a phenotype depends on antigen -specific T cell reaction s. As expected, 178 early anti-PD-1 treatment triggered the overexpression and nuclear relocation of YTHDF2 within a small 179 portion of CD8 + tumor-infiltrating lymphocytes ( TILs) (Supplementary Fig. 1n ). Specifically, PD-180 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 1+SLAMF6+TIM3- CD8 Tpex sorted from B16 -OVA tumors exhibited YTHDF2 overexpression and 181 nuclear relocation when cultured in the presence of anti-PD-1 (Fig. 1j ). Mirroring the selective 182 overexpression pattern, these data highlight subcellular YTHDF2 distribution as an acute T cell 183 phenotype underlying natural or therapy-induced tumor eradication, which prompted us to explore the 184 multiple functions of YTHDF2 in T cell immunity. 185 186 YTHDF2 is essential for the antitumor effects of CD8 T cells 187 We crossed Ythdf2Flox/Flox (hereafter Ythdf2F/F) mice 32 with dLckCre transgenic mice (expressing Cre 188 recombinase under the distal Lck promoter)33 to conditionally knockout Ythdf2 in T cel ls (Ythdf2CKO) 189 (Supplementary Fig. 2a, b). First, we compared the thymuses, spleens and peripheral blood from 6 -190 week old Ythdf2F/F, dLckCre and Ythdf2CKO mice and found no obvious difference s in their T cell 191 compositions (Supplementary Fig. 2c). Gene knockout efficiency was demonstrated by comparing the 192 intratumoral CD8 T cells from Ythdf2F/F and Ythdf2CKO mice (Supplementary Fig. 2d). When inoculated 193 with hepatocellular carcinoma Hepa1 -6 cells, melanoma B16F10 cells, or colorectal carcinoma MC38 194 cells, Ythdf2CKO mice exhibited much faster tumor growth than did Ythdf2F/F or dLckCre mice (Fig. 2a–c 195 and Supplementary Fig. 2 e). Correspondingly, Ythdf2 deficiency led to lower numbers of tumor-196 infiltrating CD8 T cells but did not affect CD4 T cells or regulatory T cells ( Fig. 2d and Supplementary 197 Fig. 3a, c). Dextramer staining further indicated that the frequency of the tumor-specific CD8 T cell 198 subpopulation was substantially decreased in the absence of YTHDF2 ( Supplementary Fig. 3a). 199 Reduced CD8 T cell numbers and percentages were also seen in the tumor-draining lymph nodes 200 (dLNs) of Ythdf2 CKO mice (Supplementary Fig. 3b). Moreover, the percentage of terminal effector CD8 201 T cells was lower in Ythdf2CKO mice at day 12 after MC38 tumor inoculation, consistent with increased 202 apoptosis and impaired cytokine production and proliferative capacity of a broader CD8 T cell population 203 (Fig. 2e, f). In contrast, PD -1+TIM3+CD101+ terminally exhausted CD8 T cells were more frequently 204 found in the Ythdf2CKO group at a later time point (Fig. 2g and Supplementary Fig. 3d). In addition to the 205 crucial role of CD8 T cell immunity, we were also curious about whether CD4 T cells were affected in 206 Ythdf2CKO mice. H owever, a ntibody-mediated neutralization confirmed that YTHDF2 loss mainly 207 jeopardizes CD8 (but not CD4) T cell-mediated antitumor immunity (Supplementary Fig. 3e), possibly 208 because YTHDF2 expression by regulatory T cells is conductive to tumor growth34. 209 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint To determine whether YTHDF2 engages in regulating tumor-specific CD8 T cells, we further bred 210 Ythdf2CKO (or Ythdf2F/F) mice with OT-1 transgenic mice to generate a Ythdf2CKO;OT-1 (or Ythdf2F/F;OT-211 1) line. Strikingly, OVA-expressing B16F10 cells were resisted in Ythdf2F/F;OT-1 mice but rapidly grown 212 up in the Ythdf2CKO;OT-1 counterparts (Fig. 2h). To align the initial immune state, an equivalent number 213 of in vitro-activated CD8 T cells of Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1 origin were transferred into mice 214 inoculated with B16F10-OVA cells. As anticipated, YTHDF2-deficient T cells exhibited inferior antitumor 215 efficacy in this setting (Fig. 2i). 216 Further, to substantiate the importance of YTHDF2 in ICB -induced T cell immunity, we subjected the 217 above mice to grow MC38 or Hepa1 -6 cells, both of which are thought to respond vigorously to anti -218 PD-1 monotherapy. Nonetheless, unlike Ythdf2F/F mice, the Ythdf2CKO littermates produced a 219 compromised response to PD-1 blockade in the MC38 model and gained no aid of killing effect toward 220 the Hepa1-6 tumors (Fig. 2j, k). In keeping with this, anti -PD-1 therapy led to a much lower frequency 221 of tumor-specific or CX3CR1+Tim3+CD101- Teff-like CD8 T cells as well as less cytokine production by 222 these cells in Ythdf2CKO mice (Fig. 2l and Supplementary Fig. 3f), indicating an indispensable role for 223 YTHDF2 in implementing ICB-elicited T cell functionality. 224 Together, these observations have indicated that YTHDF2 expression is fundamental for antitumor 225 effector and effector-like CD8 T cells, which constitute natural and ICB-induced immunity, respectively. 226 227 YTHDF2 prevents T cell mitochondrial dysfunction through m6A-dependent RNA decay 228 To understand how YTHDF2 de pletion results in T cell dysfunction and to minimize the disturbance 229 posed by the TME, we assessed in vitro-activated Ythdf2F/F and Ythdf2CKO CD8 T cells under different 230 conditions. Consistent with our in vivo results, Ythdf2-deficient CD8 T cells yielded a decline in survival 231 and cytokine production as well as a susceptibility to exhaustion, but did not differ in memory T cell 232 differentiation (Supplementary Fig. 4). 233 In terms of a selective expression pattern, by performing RNA sequencing, we asked whether the 234 abnormalities caused by YTHDF2 loss were rooted in early effector CD8 T cells while doing RNA 235 sequencing. Among the 611 genes upregulated upon YTHDF2 ablation, gene ontology (GO) analysis 236 revealed dominant enrichment for gene sets related to mitochondrial organization and mRNA 237 translation ( Supplementary Table 1 and Fig. 3a, b ). Ythdf2CKO CD8 T cells exhibited perturbed 238 mitochondrial membrane potential and accumulated mitochondrial mass and reactive oxygen species 239 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint (ROS) both in vitro and in vivo (Fig. 3c–f). We further probed the metabolic phenotype of OVA-activated 240 Ythdf2CKO;OT-1 CD8 T cells. Ythdf2CKO;OT-1 CD8 T cells exhibited decreased extracellular acidification 241 rate (ECAR) and oxygen consumption rate (OCR) (Supplementary Fig. 5a, b) during a mitochon drial 242 stress test. Morphologically, activated Ythdf2CKO;OT-1 CD8 T cells had swollen and fewer mitochondria 243 with disorganized crist ae, while Ythdf2F/F;OT-1 CD8 T cells had compact mitochondria with tightly 244 packed cristae (Supplementary Fig. 5c). These observations suggest that YTHDF2 loss -associated 245 redundant mRNA translation and mitochondrial mass resulted in T cell stress, which can explain the 246 susceptibility to exhaustion during chronic TCR stimulation 35 (Supplementary Fig. 4d). Given the 247 established causal relationship between mitochondrial malfunction and T cell exhaustion 24,36, YTHDF2 248 might be critical for ensuring mitochondrial fitness and T cell persistence . SinceYthdf2CKO CD8 T cells 249 did not preferentially express genes related to programmed cell death, we reasoned that the perturbed 250 cell survival might also be a result of mitochondrial stress ( Supplementary Table 1 and Fig. 3b). We 251 then employed N -acetylcysteine (NAC) to s cavenge mitochondrial ROS in in vitro stimulated CD8 T 252 cells. Nonetheless, no effect on T cell proliferation or cytokine production was observed, but NAC was 253 sufficient to prevent excessive exhaustion and cell death caused by YTHDF2 loss (Supplementary Fig. 254 5d, e, g and Fig. 3g, h). 255 To elucidate the underlying molecular mechanism, we performed RNA -immunoprecipitation 256 sequencing (RIP -seq) to map the target transcripts bound by YTHDF2 in CD8 T cells. Integrative 257 analyses of RIP-seq, m6A-seq and RNA-seq data indicated that 47.9% of the differentially expressed 258 genes caused by YTHDF2 deficiency were eligible for YTHDF2 recognition and m 6A modification (Fig. 259 3a and Supplementary Table 2). As potential RNA decay targets, the aforementioned mitochondr ia-260 related genes (including mitochondrial ribosomal protein-encoding genes) were frequently found among 261 the YTHDF2- and m6A-bound transcripts (Fig. 3i). Noticeably, YTHDF2-RIP-seq and m6A-seq identified 262 overlapping peaks on the downstream coding regions of Coa3, Mrpl16, Mrps12 and Tefm mRNAs 263 (Supplementary Fig. 6a ). Consistent with the increased mitochondrial stress , the half -lives of these 264 transcripts were significantly prolonged in the absence of YTHDF2 (Supplementary Fig. 6b). 265 Similarly, the knockdown of YTHDF2 in Jurkat cells also led to excessive mitochondri a-related gene 266 expression and ROS accumulation ( Supplementary Fig. 6c–f and Supplementary Table 3). To 267 determine the dependency of this regulatory process on the m6A machinery, we exogenously 268 reconstituted YTHDF2 in Jurkat-shYTHDF2 cells. The overexpression of wild-type YTHDF2, but not its 269 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint inactive mutants that fail in recognition of m 6A37, preserved mitochondrial fitness ( Supplementary Fig. 270 6g–k). However, dampening METTL3, which constructs the m 6A methylome in T cells 19, rekindled 271 mitochondrial malfunction despite the presence of abundant YTHDF2 expression (Supplementary Fig. 272 6l–n). These results demonstrate that the m6A machinery i s essential for YTHDF2-regulated 273 mitochondrial fitness in T cells. 274 275 YTHDF2 sequesters IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T cells 276 Despite identifying the YTHDF2-mediated regulation of CD8 T cell persistence, the mechanism by 277 which YTHDF2 promotes CD8 T cell polyfunctionality remains to be addressed. Interestingly, YTHDF2 278 depletion was thought to mainly stabilize its target genes, but RNA profiling revealed a comparable 279 number of downregulated genes, which integrally reflected an inactive chromatin state in Ythdf2CKO 280 CD8 T cells (Supplementary Table 1 and Fig. 4a). Provided that recent studies have uncovered a m6A-281 responsible crosstalk between RNA modification and chromatin regulation 22,23,25, we tested whether 282 gene transcription was affected by YTHDF2 in early effe ctor CD8 T cells. Consistent with the 283 distinguishing gene signature, compared with Ythdf2F/F (or Ythdf2F/F;OT-1) CD8 T cells when shortly 284 activated in vitro, Ythdf2CKO (or Ythdf2CKO;OT-1) CD8 T cells manifested a remarkable decrease in the 285 abundance of nascent transcripts (Supplementary Fig. 7a, b), hinting that YTHDF2 plays an important 286 role in chromatin remodelling. No genes encoding general epigenetic regulators or transcription factors 287 were found among those putative decay targets ( Supplementary Table 2), excluding the possibility of 288 indirect chromatin regulation rooted in the canonical function of YTHDF2. Interestingly, in the presence 289 of an RNA polymerase II-selective inhibitor α-Amanitin38, nascent transcripts were significantly reduced 290 in Ythdf2F/F but not Ythdf2CKO CD8 T cells (Supplementary Fig. 7c ), suggesting that YTHDF2 mainly 291 promotes RNA polymerase II-dependent transcription. 292 Nuclear YTHDF2 was known to promote translation initiation of stress-inducible transcripts27. However, 293 ribosome profiling indicated no difference in translation efficiency between activated Ythdf2F/F and 294 Ythdf2CKO CD8 T cells, even for YTHDF2 -targeted and m 6A-marked transcripts ( Supplementary Fig. 295 7d). To investigate the mechanism underlying YTHD F2-directed transcriptional adap tation, we 296 performed immunoprecipitation followed by mass spectrometry (IP –MS) and identified proteins that 297 were bound to YTHDF2 in the scenario of early T cell activation. Mirroring its potential nuclear 298 functionality, YTHDF2-binding partners included the lymphoid transcription factor Ikaros (IKZF1) and 299 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Aiolos (IKZF3) 39 (Supplementary Table 4). Fitting their roles as transcription repressors, IK ZF1 and 300 IKZF3 were expressed at lower levels in activated CD8 T cells than in naï ve CD8 T cells (Supplementary 301 Fig. 7e). The results of a proximity ligation assay (PLA) showed that YTHDF2 interacted with IKZF1 and 302 IKZF3 in short -term primed mouse or human CD8 T cells and untreated Jurkat cells ( Fig. 4b and 303 Supplementary Fig. 7f). Subsequent coimmunoprecipitation (CO-IP) assays further confirmed these 304

(Fig. 4c). Of note, upon early CD8 T cell activation, a majority of IKZF1 or IKZF3 protein could 305 be bound to YTHDF2. To probe whether RNA species facilitates the interaction between YTHDF2 and 306 IKZF1/3, endogenous YTHDF2 immunoprecipitants from acutely stimulated CD8 T cells were incubated 307 with RNase or DNase. As shown by Supplementary Fig. 7g, neither RNase nor DNase repressed 308 YTHDF2 binding with IKZF1/3. 309 To assess whether YTHDF2 affects chromatin openness, we performed an assay for transposase -310 accessible chromatin with high-throughput sequencing (ATAC-seq). Unexpectedly, YTHDF2 deficiency 311 resulted in enhanced chromatin accessibility in both activated CD8 T cells and Jurkat cells (Fig. 4d, e). 312 Notably, among the ‘ATAC gain’ regions conditioned by YTHDF2 depletion, an analysis of transcription 313 factor (TF) motifs revealed that the ACAGGAAG element, which is capable of binding IKZF1 or IKZF3, 314 was the top hit (Supplementary Fig. 7h, i), which is capable of binding IKZF1 or IKZF3. Using existing 315 chromatin immunoprecipitation sequencing (ChIP -seq) datasets 39,40, we determined that the specific 316 loci for these two transcription factors selectively displayed greater chromatin accessibility in YTHDF2-317 deficient T cells than in YTHDF2 -competent T cells ( Fig. 4f and Supplementary Fig. 7j). Epigenomic 318 mapping through cleavage under targets and release using nuclease (CUT&RUN), however, 319 underscored that the transcription start sites (TSS s) of IKZF1/3-regulated genes in YTHDF2 -deficient 320 T cells were marked by much lower amounts of histone H3 lysine 4 methylation (H3K4Me ) (Fig. 4g), 321 which is an active form of chromatin modification. These unusual chromatin changes were profoundly 322 observed for genes responsible for TCR signalling, such as Stat5a9 and Rasgrp141, which were 323 confirmed to undergo transcriptional silencing in early activated Ythdf2CKO T cells ( Fig. 4h–j). Genes 324 encoding epigenetic modulators were also widely affected, many of which were simultaneously found 325 to have ‘ATAC gain’ regions predicted for IKZF1/3 binding (Fig. 4j). 326 Since the protein levels of IKZF1/3 were comparable between Ythdf2F/F and Ythdf2CKO CD8 T cells 327 (Supplementary Fig. 8a), we asked whether YTHDF2 deficiency had an impact on IKZF-DNA binding 328 activity. As expected , CUT&RUN assay detected stronger IKZF1/3 binding signals at the promoter 329 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint regions of genes expressed under a YTHDF2 -deficient condition (Supplementary Fig. 8b ). As 330 suggested by a recent report 42, nucleosome occupancy at IKZF motifs could inactivate effector gene 331 transcription without reducing T cell chromatin op enness. The molecular basis might be IKZF1/3-332 mediated recruitment of histone deacetylase (HDAC) complexes 43,44. Here i n an early activation 333 scenario, Ythdf2CKO but not Ythdf2F/F CD8 T cells enabled strong IKZF-HDAC1 interaction 334 (Supplementary Fig. 8c), raising the possibility of nucleosome occupancy and chromatin inactivation 335 upon YTHDF2 loss. Together, these data highlight that YTHDF2 loss may result in IKZF1/3-associated 336 transcriptional repression. 337 Lenalidomide rescues the polyfunctionality of YTHDF2-deficient CD8 T cells 338 We next sought to unravel the dependency of YTHDF2 loss-associated T cell malfunction on IKZF1 339 and IKZF3. As shown in Supplementary Fig. 8d, the knockdown of IKZF1 and IKZF3 restored nascent 340 transcription in Jurkat -shYTHDF2 cells but did not change that in control cells. The myeloma drug 341 lenalidomide has been shown to cause the proteasomal degradation of IKZF1 and IKZF345,46. We asked 342 whether this clinically available drug could re store the effector function of YTHDF2 -deficient T cells by 343 targeting IKZF1/3. When lenalidomide was added to short -term activated CD8 T cells or untreate d 344 Jurkat cells, it did not obviously enforce T cell function under a YTHDF2-competent condition; in contrast, 345 it restored nascent RNA synthesis as well as the proliferation of Ythdf2CKO CD8 T cells (Supplementary 346 Fig. 8e–h and Fig. 5a–d), manifesting a context-dependent mode of action. Although there was no effect 347 on preventing T cell exhaustion , lenalidomide profoundly provoked cytokine production by Ythdf2CKO 348 CD8 T cells (Supplementary Fig. 8i and Fig. 5e). Supporting a retrieved functional state, Stat5a and 349 Rasgrp1 expression in Ythdf2CKO CD8 T cells returned to normal in the presence of lenalidomide 350 (Supplementary Fig. 8j). 351 Taking into consideration of ICB-induced YTHDF2 relocation in effector-like CD8 T cells, we evaluated 352 the dependency of these cells on IKZF1/3 signalling and the therapeutic effect of lenalidomide in vivo. 353 Although lenalidomide monotherapy was less efficient than anti -PD-1 therapy when administered to 354 MC38-bearing Ythdf2F/F mice, it had a slightly better effect on Ythdf2CKO mice; promisingly, lenalidomide 355 combined with PD-1 blockade largely rescued tumor-eradicating immunity in Ythdf2CKO mice (Fig. 5f, 356 g). In line with this, flow cytometry analysis showed that combination therapy increased cytokine 357 production by PD-1+ Ythdf2CKO CD8+ TILs to a level equivalent to that in the Ythdf2F/F group receiving 358 anti-PD-1 or combination therapy (Fig. 5h). While it is true lenalidomide is more than an IKZF degrader, 359 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint these data have supported the likelihood that the unresponsiveness of YTHDF2-deficient T cells was 360 caused by IKZF1/3. Together, we conclude that YTHDF2 may achieve polyfunctionality in effector or 361 effector-like CD8 T cells by preventing IKZF1/3-associated transcriptional repression. 362 363 YTHDF2 relies on m6A-bound transcripts for nuclear translocation 364 Regarding YTHDF2 nuclear relocation in early (re)activated T cells ( Fig. 1e–h), we inferred that newly 365 synthesized RNA substrates at this stage might be necessary for YTHDF2 trafficking or acting in the 366 nucleus. In support of this notion, the addition of the transcription inhibitor actinomycin D (ActD) largely 367 blocked the interaction between YTHDF2 and IKZF3 in the T cell nucleus (Fig. 6a). Further, the finding 368 that YTHDF2 was recruited to the m6A sites within mRNAs transcribed from its DNA targets raised the 369 possibility of cotranscriptional regulation ( Supplementary Fig. 9a and Supplementary Table 5 ). To 370 determine whether the m 6A machinery is required for nuclear YTHDF2 distribution and function, we 371 depleted METTL3 in Jurkat cells and mouse T cells. Compared with Mettl3F/F mice, Mettl3CKO littermates 372 exhibited accelerated tumor growth, accompanied by decreased proliferation and cytokine production 373 in tumor-infiltrating CD8 T cells ( Supplementary Fig. 9b–d). Of importance, YTHDF2 failed to localize 374 to the nucleus of METTL3 -deficient CD8 T cells following in vitro priming (Fig. 6b). In line with this, 375 METTL3 loss interrupted the binding of YTHDF2 to IKZF3 in both Jurkat cells and activated mouse T 376 cells ( Fig. 6c, d ). Mutation of a catalytic residue ( W386A or W432A) in the hydrophobic pocket of 377 YTHDF2, which specifically recognize s m6A, had a similar effect as METTL3 depletion (Fig. 6e and 378 Supplementary Fig. 9e). In parallel, the disruption of METTL3 in YTHDF2 -overexpressing cells 379 abrogated its ability to facilitate nascent RNA synthesis and proliferation ( Fig. 6f, g). We then explored 380 whether augmented m6A mRNA modification could conversely reinforce YTHDF2 nuclear function by 381 repressing the m 6A demethylase FTO in activated T cells. However, treatment with an FTO inhibitor 382 (FB23-2)47 did not promote nascent RNA synthesis regardless of the YTHDF2 concentration 383 (Supplementary Fig. 9f, g), suggesting that increasing m 6A deposition per se is not sufficient to boost 384 the nuclear function of YTHDF2. These observations suggest that m 6A recognition might be a 385 prerequisite for YTHDF2 trafficking to the nucleus. 386 387 YTHDF2 expression is posttranscriptionally autoregulated via the m6A machinery 388 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Although Ythdf2 mRNA expression was increased at early timepoints of CD8 T cell priming 389 (Supplementary Fig. 9h), its chromatin accessibility at TSS regions remained unchanged after 5 h of in 390 vitro priming (Fig. 6h). The addition of ActD to CD8 T cell s did not weaken the fold change in Ythdf2 391 mRNA expression, thus excluding a direct transcriptional regulation (Fig. 6i). By incorporating YTHDF2-392 RIP-seq with m 6A-seq data from both primed CD8 T cells and untreated Jurkat cells, we noticed that 393 YTHDF2 directly bound to its own mRNA and identified overlapping peaks on its m 6A-occupied exons 394 (Supplementary Fig. 9i). Therefore, one interpretation of these findings is that YTHDF2 recognizes and 395 destabilizes its cognate mRNA in the cytoplasm; once a portion of YTHDF2 is dissociated and enters 396 the nucleus in response to T cell activation, its mRNA translation can be partially unleashed. To test 397 this hypothesis, we generated a mouse line ( Ythdf2-249) carrying a n N-terminal truncated form of 398 YTHDF2, which is unable to translocate to mRNA decay sites but retains the domain for the recognition 399 of methylated RNA 26. A dditionally, this gene engineering strategy d id not affect the internal mRNA 400 regions that are able to undergo m6A modification. Compared to the wild -type control, naï ve Ythdf2-249 401 CD8 T cells expressed a much higher level of Ythdf2 mRNA, which did not further increase following 6 402 h of in vitro priming ( Fig. 6j ). A prolonged Ythdf2 mRNA lifetime in Ythdf2-249 CD8 T cells further 403 confirmed the preexistence of autoregulated RNA decay (Fig. 6k). Ythdf2-249 CD8 T cells also exhibited 404 increased protein expression in both the cytosol and the nucleus (Fig. 6l). Similarly, at the Ythdf2 gene 405 locus of Ythdf2CKO CD8 T cells, RNA-seq captured a higher level of pseudogene expression than that 406 of Ythdf2 mRNA in Ythdf2F/F CD8 T cells (Supplementary Fig. 9k). Taken together, these data have 407 shown an unprecedented mode of YTHDF2 autoregulation in CD8 T cells. 408 409 YTHDF2 expression and distribution in human tumor-infiltrating T cells 410 To explore the clinical relevance of our findings, we reanalyzed single-cell RNA-seq data from human 411 colorectal carcinoma (CRC)48 (GSE146771), pancreatic ductal adenocarcinoma (PDA)49 (GSE155698), 412 or anti -PD-1-treated hepatocellular carcinoma (HCC) 50 (GSE206325). We divided the single-cell 413 transcriptomes of CD8+ TILs into two groups using a customi zed polyfunctional ity signature score 414 (based on Ifng, Gzma, Gzmb, and Prf1 gene expression). As shown in Fig. 7a, Ythdf2 mRNA expression 415 was much greater in CD8 T cells assigned with high er scores. Notably, post -ICB datasets of human 416 melanoma (GSE12057551) or HCC (GSE206325) showed that Ythdf2 level was much greater in CD8+ 417 TILs of responders than that of nonresponders (Fig. 7b), suggesting that YTHDF2 is involved in ICB -418 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint induced antitumor immunity. Moreover, among the 7 different CD8 T cell subsets from HCC patients 419 who responded to anti-PD-1 therapy, cytotoxic CD8 T cells exhibited the greatest Ythdf2 expression 420 level (Fig. 7c), which is consistent with the results of our mouse experiments. However, those 421 nonresponder-derived cytotoxic CD8 T cells exhibited a Ythdf2 level comparable to that of the terminally 422 exhausted population (Fig. 7c). 423 We further investigated YTHDF2 protein expression using tissue samples from HCC or CRC patients. 424 For those who had not received preoperative treatment, YTHDF2 protein was moderately expressed in 425 cancer cells 32 but undetectable in CD8 + TILs. Results from our clinical trials demonstrate d that 426 chemoimmunotherapy significantly improved overall survival (OS) and progression-free survival (PFS) 427 of patients with advanced cancer 52,53. YTHDF2-expressing CD8 T cells were substantially more 428 common in patients who received neoadjuvant chemoimmunotherapy ( Fig. 7d, h ), supporting its 429 inducible upregulation concomitant with therapy-induced CD8 T cell infiltration and activation. Although 430 a greater number of CD8 + TILs could not distinguish responders from nonresponders to neoadjuvant 431 therapy, YTHDF2 positivity and nuclear accumulation in CD8 T cells were more frequently found in 432 patients who achieved a complete or partial response ( Fig. 7d–k). These observations suggest that 433 YTHDF2-expressing CD8 T cells confer therapy -induced antitumor immunity, potentially serving as a 434 clinical indicator of cancer prognosis. 435 436

437 T cell -mediated tumor-eradicating immunity forms the basis of successful cancer treatments 10,20. 438 Intense efforts have now been invested in elucidating and breaking the T cell -intrinsic barriers to 439 rejuvenation. Differing from e ffector and memory T cells, exhausted T cells display distinct functional 440 properties, which are attributed greatly to epigenetic and transcriptional mechanisms implicated in T 441 cell differentiation 12,54. TOX transcriptionally induces exhaustion -associated gene expression and 442 meanwhile recruits chromatin modifiers to repress gene expression involved in T eff differentiation55. 443 Similarly, NR4A1 restrains effector gene transcription by shielding AP -1 from its binding chr omatin 444 regions and promotes acetylation of histone 3 at lysine 27 (H3K27ac) for activating genes related to T 445 cell dysfunction56. Depletion of either TOX or NR4A1 provisions CD8 T cells with an effector phenotype 446 and boosts antitumor immunity. Besides, CD8 T cells acquire DNMT3A -dependnet de novo DNA 447 methylation events upon effector -to-exhaustion transition even when subjected to PD -1 blockade 448 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint therapy. Co-targeting of this epigenetic program during ICB yields a more effective antitumor response57. 449 As characterized in chro nic infection and cancer, proliferation -competent T pex cells, which co -express 450 the transcription factor TCF -1 and exhaustion markers, represent the major therapeutic targets of 451 immune interventions 58, such that epigenetic imprints in this T cell compartment irrevocably affect 452 immunotherapy efficacy. For instance, the SWI/SNF chromatin remodelling complex PBAF 59 and the 453 NFAT family transcription factor NFAT5 60 can specifically drive T pex transition to terminal T ex cells, 454 therefore limiting the outcome of T cell -based immunotherapy. In the present work, we embark on 455 investigating RNA epigenetics in terms of both endogenous and ICB -induced T cell immunity , thus 456 uncovering an indispensable role for the m 6A reader YTHDF2. Aligning with robust antitumor immune 457 responses, YTHDF2 expression is selectively upregulated and redistributed within both terminal Teff and 458 Teff-like cells. In accordance, the loss of YTHDF2 in T cells dampens both endogen ous and therapy -459 induced tumor immunity. Unlike previously reported epigenetic events, YTHDF2 depletion does not 460 necessarily affect T cell differentiation, but functionally erodes T cell proliferation and cytokine 461 production. Longitudinal analyses of CD8 + TILs suggest that YTHDF2 deficiency can impair effector 462 functionality and durability, thus yielding hyporesponsiveness to anti -PD-1 therapy. Coupled with 463 phenotypic observation and multi-dimensional sequencing, our data further demonstrate that YTHDF2 464 expression impacts both effector and exhaustion phases through dual mechanisms. 465 YTHDF2 has been found to exert context-dependent functions in cancer. While verifying the oncogenic 466 roles of m6A methylation in some cancer types, several studies postulated the a ccompanying position 467 of YTHDF2 as an executer for the RNA decay of tumor suppressors61,62. In fact, YTHDF2 could also 468 destabilize oncogene-coding mRNAs in a m6A-dependent manner63,64. As demonstrated in our previous 469 work, YTHDF2 inhibits mouse and human HCC by processing the decay of Il11 and Serpine2 mRNAs, 470 which are responsible for inflammation -related cancer progression and metastasis 32. Otherwise, 471 YTHDF2 overexpressed in leukemic stem cells can decrease the ha lf-life of apoptosis gene Tnfrsf2, 472 thereby descending to a cancer-promoting position65. With immunobiology appearing as one of the most 473 promising frontiers, the m6A machinery in finetuning tumor immunity has been explored2. YTHDF2 has 474 been found to dominate immunosuppressive myeloid cell function in both natural and therapy -475 experienced cancer contexts 8,9. On the other hand, YTHDF2 promotes NK cell immunity partially by 476 inhibiting the mRNA stability of Tardbp, a negative regulator of cell division and proliferation66. Uniquely, 477 here we reveal that YTHDF2 dictates both posttranscriptional and transcriptional programs to reinforce 478 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint the antitumor function of CD8 T cells. In a naï ve state, low-level YTHDF2 limits the stability of its cognate 479 encoding mRNA, which harbors bona fide m 6A sites. Acute a ctivation or rejuvenation signals incite 480 YTHDF2 relocation to the nucleus, albeit temporarily, paving an individual way for its abundant 481 expression in the early phase. In the circumstance of natural or ICB-induced tumor-eradicating immunity, 482 nuclear YTHDF2 combines and curbs the transcriptional repressor IKZF1 /3 to safeguard gene 483 transcription and T cell function. Meanwhile, cytoplasmic YTHDF2 -mediated mRNA decay can help 484 improve the mitochondrial fitness and persistence of tumor-reactive T cells, opposing their progressive 485 trajectory toward exhaustion. Further, we’ve shown the necessity of m6A recognition for YTHDF2 486 repositioning into the nucleus, which highlights the important crosstalk between RNA modification and 487 chromatin regulation within antitumor CD8 T cells. 488 RNA-binding proteins have been proven to pe rvasively participate in transcriptional control 67. 489 Consistent with this concept, YTHDC1 was found to regulate gene transcription by recruiting a histone 490 modifier or by processing regulatory RNA species in close proximity to active chromatin regions 24,25. 491 Our data show that YTHDF2 depletion renders T cell chromatin more accessible to IKZF1 and IKZF3 492 and trapped in an inactive state, which influences the downstream effector genes such as Stat5a and 493 Rasgrp1. Instead of regulating chromatin modifier transcripts in the cytoplasm, nuclear -localized 494 YTHDF2 directly interacts with repressive transcription factors. Despite this, its relocation depends on 495 m6A-labelled nascent RNAs, reminiscent of the previous observation that m6A deposition was essential 496 for partitioning the stress-conditioned mRNA -YTHDF2 complexes into phase -separated subcellul ar 497 compartments68. Similar to the perturbed YTHDF2 trafficking in METTL14 -deficient mouse embryonic 498 stem cells68, nuclear YTHDF2 is hardly detectable when METTL3 is depleted in either activated CD8 T 499 cells or untreated Jurkat cells . Importantly , the genes encoding YTHDF2 -bound transcripts largely 500 overlap with the IKZF1/3 targets, suggesting that m 6A may cotranscriptionally enlist YTHDF2 in 501 chromatin remodelling. As such, m 6A participates in every step of this non -canonical YTHDF2 signal 502 undertaken in T cells committed to effector or effector-like function. 503 We pre viously reported that YTHDF2 expression could be transcriptionally silenced in the hypoxic 504 TME32. Here we show its low expression level in human intratumoral T cells might also be explained by 505 the insufficiency of immune response . Only in cancer patients with a better therapeutic response can 506 YTHDF2-expressing CD8 T cells be profoundly detected, whic h are supposed to address therapy -507 induced immunity through a positive feedback loop . Otherwise, the paucity of YTHDF2 unmasks an 508 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint overlooked T cell epigenetic mechanism, posing threat of immunotherapy resistance. Rationally, the 509 immunomodulatory drug lenali domide, which targets IKZF1 /3 for degradation, facilitates ICB-induced 510 rejuvenation of YTHDF2-deficient T cells. Thus, our understanding of the YTHDF2-centered regulatory 511 circuit in antitumor T cells may inspire novel paths for the development of immunotherapies. 512 513

514 Animals 515 Wild-type (WT) C57BL/6 mice were purchased from Charles River (Beijing, China) for Medical 516 Research. Ythdf2flox/flox (Ythdf2F/F) mice in C57BL/6 background were described previously 32. 517 Mettl3flox/flox (Mettl3F/F) mice were kindly provided by Prof. Z. Yin (Jin an University). dLckCre and OT-1 518 TCR transgenic mice were purchased from the Jackson Laboratory. Ythdf2F/F or Mettl3F/F mice were 519 then crossed with dLckCre transgenic mice to obtain Ythdf2CKO or Mettl3CKO mice with Ythdf2 or Mettl3 520 conditionally knocked out in T cells. For animal experiments referring to Ythdf2CKO or Mettl3CKO mice, 521 littermate controls with normal YTHDF2 (Ythdf2F/F) or METTL3 ( Mettl3F/F) expression were used. 522 Ythdf2CKO mice were also crossed with OT -1 TCR transgenic mice to obtain Ythdf2CKO;OT-1 mice. 523 Ythdf2-249 transgenic mice were generated by depleting a 249-amino acid fragment from the N-terminus 524 of Ythdf2 gene coding region. Correctly targeted mice were determined by PCR and gene sequence. 525 Primers used for genotyping of Ythdf2-249: Forward–5’-TGTGAATGATGTGGAAGGAA-3’ and Reverse–526 5’-CAACAGCAGAGCCTACAA-3’. All mice were maintained under specific pathogen -free conditions. 527 Mice with 8–12 weeks of age were used for all animal experiments. Animals were randomly allocate d 528 to experimental groups. 529 Cell cultures 530 Peripheral naï ve CD8 T cells were isolated from the mouse spleen by negative selection magnetic 531 beads (STEM CELL). CD8 T cells were cultured in complete RPMI medium (RPMI 1640, 10% FBS, 20 532 mM HEPES, 1 mM sodium pyruvate, 0.05 mM 2 -mercaptoethanol, 2 mM glu tamine, 100 μg/ml 533 streptomycin and 100 units/ml penicillin) and stimulated with plate -bound anti -CD3/CD28 in the 534 presence of 10 ng/ml IL -2 (Peprotech) as indicated. To detect T cell proliferation, naï ve CD8 T cells 535 were stained with 0.5 μM CellTracker Violet fluorescent dye (Thermo Fisher) in serum-free medium for 536 20 min at 37 ° C, and then washed three times in PBS. Stained cells were activated by plate-bound anti-537 CD3/CD28 (biolegend) for 24–120 h and detected in the BV421 channel by FACS. To detect T cell 538 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint activation-induced apoptosis, naï ve CD8 T cells were activated by plate -bound anti -CD3/CD28 for 539 different number of hours, then analyzed with an annexin V/propidium iodide kit (BD). 540 For in vitro T cell exhaustion assay 69, CD8 T cells were seeded at a concentration of 1 million/ml on 541 plates coated with of anti -CD3 (5 μg/ml) and anti -CD28 (2 μg/ml). After 48 h of activation , chronic 542 stimulation was performed using plates coated with anti -CD3 (5 μg/ml). Cells were passaged onto a 543 fresh anti-CD3-coated plate every two days, maintained at 1 million/ml (in the continued presence of 10 544 ng/mL IL-2), and analyzed via flow cytometry on Day 8. 545 To induce CD8 T cells with a memory phenotype in vitro, CD8 T cells were activated in a ‘‘transient’’ 546 stimulation condition in which beads were removed after the initial 3-day incubation as reported28. CD8 547 T cells were seeded at a concentration of 1 million/ml in presence of mouse T-activator CD3/CD28 548 Dynabeads (Thermo Fisher Scientific) and IL-2 (10 ng/ml) at 1:1 beads-to-cells ratio. After 2 days, cells 549 were split 1:2. Beads were removed on day 3 and cells were maintained in culture for 6 days (with fresh 550 media added every 2 days) in the presence of 10 ng/ml IL-2. Cells were analyzed via flow cytometry on 551 Day 9. 552 To obtain mouse tumor-infiltrating CD8 T pex, B16-OVA tumor-derived single cell suspension s were 553 stained with Zombie NIR and then sorted for live CD45+CD8+PD1+Tim3-SLAMF6+ cells by the BD FACS 554 Aria II Cell Sorter. Sorted Tpex were cultured in U-bottom plates and stimulation assays were performed 555 using T-activator CD3/CD28 Dynabeads and IL -2 (10 ng/ml) at 1: 2 beads-to-cells ratio with 10 μg/ml 556 anti-PD-1 (RMP1-14, BioXCell) or cIg for 48 h. 557 Human peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers by 558 gradient centrifugation with Lymphoprep (STEM CELL) . CD8 T cells were then purified by EasySep 559 Human CD8 T Cell Isolation Kit (STEM CELL) . For in vitro T cell activation experiments, cells were 560 plated at 1 million/ml in the presence of Human T -Activator CD3/CD28 Dynabeads (Thermo Fisher 561 Scientific) at 1:1 beads-to-cells ratio supplemented with 30U/ml human IL-2 (Peprotech). 562 The MC38 (mouse colon adenocarcinoma) cell line was originally from Prof. Y.-X. Fu laboratory 563 (University of Texas Southwestern Medical Center) . The B16F10 (mouse melanoma) cell line (ATCC, 564 CRL-6475) was purchased from the American Type Culture Collection and the B16F10 -OVA cell line 565 was generated by EGFP -OVA (SIINFEKEL) lentivirus transduction. Mouse he pa1-6 (hepatoma cells) 566 and human Jurkat (Clone E6-1, T lymphoblast) cells were purchased from the Cell bank of the Chinese 567 Academy of Sciences. 568 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint MC38, Hepa1 -6, B16F10, B16F10-OVA, Jurkat cells w ere grown in Dulbecco’s modified Eagle’s 569 medium (DMEM) (Invitrogen) or RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine 570 serum (FBS) (Gibco), 10 mM HEPES (Gibco) and 1% Penicillin/Streptomycin (Gibco). All cell lines were 571 maintained at 37°C, 5% CO2 and routinely tested negative for Mycoplasma. 572 Short hairpin RNAs (shRNAs) targeting human Mettl3, Ikzf1 and Ikzf3 were used to generate gene 573 knockdown in Jurkat cells. Target sequences are listed in Supplementary Table 6. Relevant shRNA-574 expressing lentiviruses were produced by Obio technology or Gene Chem. YTHDF2-overexpressing or 575 -mutant (the m 6A recognition sites W432 and W486 were mutated into A) lentiviruses were designed 576 and synthesized as previously described32. Briefly, Jurkat cells were seeded 1 × 10 6/ml in a 12 well -577 plate. HitransG P transfection reagent (GENE) and the corresponding lentiviruses were added. After 578 centrifugation at 1,200 rpm, 37° C for 60 min, cells were incubated at 37° C, 5% CO 2 overnight then 579 culture medium was changed. To obtain stably transfected clones, these cell s were treated with 580 puromycin (3 μg/ml) for 1 week and maintained in 1 μg/ml. The knockdown or overexpression efficiency 581 was confirmed by quantitative PCR and western blot analysis before the cells were used for subsequent 582 experiments. 583 In some settings, Jurkat cells were treated with 100 μΜ lenalidomide (Selleck) or vehicle for 24 h. 584 Primed CD8 T cells were treated with 10 μΜ lenalidomide or vehicle for 24, 48 or 72 h as indicated. For 585 FTO inhibition, Jurkat cells or primed CD8 T cells were treated with10 μΜ FB23-2 (Selleck) or vehicle 586 for 72 h. To neutralize ROS, primed CD8 T cells were treated with 10 mΜ NAC(Sigma) or vehicle for 587 48 or 72 h as indicated. To selectively inhibit Pol II, primed CD8 T cells were treated with 2 μg/mL α-588 amanitin (MCE) or vehicle for 12 h as indicated. 589 Tumor growth and treatments 590 MC38 (1 × 106), Hepa1-6 (1 × 106), B16F10 (5 × 105) or B16-OVA (1 × 106) tumor cells were injected 591 subcutaneously (s.c.) into the right flank of mice. Tumor growth was monitored every 2 or 3 days. Tumor 592 volumes were measured by length (a) and width (b) and calculated as tumor volume = ab2/2. 593 For anti-PD-1 treatment, MC38 or Hepa1 -6 tumors were allowed to grow for five or six days then 594 intraperitoneally (i.p.) injected with 250 μg /dose anti-PD-1 (RMP1-14, BioXCell) or control IgG (cIg). 595 Anti-PD-1 or cIg was given on days 6, 9,12, 15 for MC38 tumors while on days 5, 8,11, 14 for hepa1-6 596 tumors. For adoptive cell transfer therapy, B16F10-OVA (1 × 106) tumor cells were s.c. injected into the 597 right flank of C57BL/6 WT mice (female, 8 weeks). On day 6, tumor-bearing mice were randomly divided 598 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint into three groups (n = 5) and intravenously injected with either PBS or 1 × 106 OVA-primed (72 h) OT-599 1 CD8 T cells from Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1. Tumor growth was monitored every 2 or 3 days 600 from day 6. For in vivo lenalidomide treatment, tumor-bearing mice were i.p. injected once daily with 10 601 mg/kg lenalidomide70 (Selleck) dissolved in DMSO and diluted in 100 μl PBS or with DMSO in 100 μl 602 PBS. 603 For antibody-mediated T cell depletion, 200 µ g anti-CD4 (BioXCell, BE0003-1, clone GK1.5) or anti -604 CD8 (BioXCell, BE0061, clone 2.43) was given by i.p. 3 days before MC38 tumor inoculation and on 605 day 1, 2, 4, 8, 12, 16 and 19, relative to tumor injection (day 0). 606 Patient samples 607 Human HCC tissue specimens were collected from 45 patients receiving surgery with informed consent 608 at Sun Yat-sen University Cancer Center (SYSUCC) from 2019 to 2021, who had been administrated 609 with neo -adjuvant chemo -(immuno-) therapy (regional chemotherapy using a FOLFOX (oxaliplatin, 610 leucovorin, and fluorouracil) regimen, supplemented with or without anti -PD-1 therapy). Human CRC 611 tissue specimens were collected from 30 patients receiving surgery at SYSUCC from 2016 to 2019, 612 who had been administrated with neo -adjuvant chemotherapy (systemic chemotherapy using a 613 FOLFIRI (irinotecan, leucovorin, and fluorouracil) regimen). All patients were followed up on a regular 614 basis. The study was approved by the Medical Ethics Committee of SYSUCC. Written informed consent 615 was obtained from the patients who provided samples. All patients had a histological diagnosis of HCC 616 or CRC. 617 Flow cytometry 618 Tumors, tumor draining lymph nodes, livers, peripheral blood and spleens were harvested from mice 619 as indicated in figure legends. Tumors were sliced into small pieces and put into a gentleMACS C Tube 620 (Miltenyi) containing 100 ml Enzyme D, 50 ml Enzyme R, 12.5 ml E nzyme A (Miltenyi) and 2.35 ml 621 RPMI 1640. The C tube was then processed on a gentleMACS Octo Dissociator with Heaters (Miltenyi) 622 for 30 min. The resulting cell suspension was passed through a 70 -mm cells strainer (Miltenyi), then 623 washed with PBS buffer con taining 0.04% BSA. Single cell suspensions of spleens were depleted of 624 erythrocytes. Cells were re-suspended in staining buffer (PBS with 2% FBS and 1 mM EDTA). To block 625 mouse Fc receptors, cells were incubated with anti-CD16/CD32 antibody (BD) for 10 min. Subsequently, 626 specific antibodies for cell surface epitope staining were added and staining was continued for 30 min 627 at 4° C in the dark. For mitochondrial staining, cells were incubated with 25 nM MitoTracker Orange 628 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint (ThermoFisher), 50nM MitoTracker Green (ThermoFisher) and 5 uM MitoSOX red (ThermoFisher) in 629 RPMI with 2% FBS for 30 min at 37 ° C after staining surface markers. For intracellular staining, cells 630 were stimulated ex vivo with Cell Stimulation Cocktail plus protein transport inhibitors (eBioscience) for 631 4 h before surface staining 71. Following incub ation, cells were washed twice with buffer before 632 proceeding to intracellular staining. Cells were then fixed and permeabilized using the Foxp3/ 633 transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's protocol and 634 stained with intracellular antibodies or respective isotype antibodies. Cells were analyzed by the Cytek 635 Aurora (Cytek) or CytoFLEX (Beckman) machine. Analysis of flow cytometry data was performed using 636 Flowjo 10.7.1 (Treestar). Dead cells stained by live dead blue ( eBioscience) or Zombie Aqua 637 (BioLegend) were excluded from analysis. Gating was confirmed with fluorescence -minus-one (FMO) 638 controls for low-density antigens. 639 RIP-seq 640 RIP-seq was conducted in accordance with a previously reported protocol with minor modifications26. 641 Mouse activated CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) or Jurkat-YTHDF2 OE cells were collected 642 then the pellet was treated with cell lysis buffer. The 10% lysis sample was saved as input, 80% was 643 used in immunoprecipitation reactions with anti-YTHDF2 (Abcam) or anti-Flag (CST) antibody, and 10% 644 was incubated with rabbit IgG (CST) as a negative control. The RIP step was performed by using Epi 645 RNA immunoprecipitation kit (Epibiotek) following the manufacturer’s protocols. RNA was then 646 extracted using TRIzol reagent (Invitrogen) . Input and immunoprecipitated RNA of each sample were 647 used to generate the library using a TruSeq stranded mRNA sample preparation kit (Illumina). Libraries 648 quality were determined on Qseq100 Bio -Fragment Analyzer (Bioptic). The strand -specific libraries 649 were sequenced on Illumina Novaseq 6000 system with paired-end 2 x 150 bp read length. 650 m6A-seq 651 Total RNA in CD8 T cells or Jurkat cells was extracted by using TRIzol Reagent. DNase I (Invitrogen) 652 treatment was adopted to remove DNA contamination. Additional phenol -chloroform isolation and 653 ethanol precipitation treatments were performed to remove enzyme contamination. Following meRIP -654 Seq was carried out as previously described 72. Briefly, 20 μg purified RNA was fragmented into ~200 655 nucleotide-long fragments by incubating in magnesium RNA fragmentation buffer for 6 min at 70° C. 656 The fragmentation was stopped by adding EDTA. Then, RNA clean and concentrator-5 kit (Zymo) was 657 used to purify fragmented total RNA. Next, m 6A immunoprecipitation was performed by using Epi m 6A 658 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint immunoprecipitation kit (Epibiotek). Fragmented total RNA (Input) and immunoprecipitated RNA (IP) 659 were subjected to library construction by using Epi mini longRNA -seq kit (Epib iotek) according to the 660 manufacturer’s protocols. Briefly, reverse transcription was performed using random primers and the 661 ribosome cDNA was removed after cDNA synthesis using probes specific to mammalian rRNA. The 662 directionality of the template-switching reaction not only preserves the 5’ end sequence information of 663 RNA but the strand orientation of the original RNA. Libraries for immunoprecipitated RNA were PCR 664 amplified for 18 cycles. The quality of libraries was determined on Qseq100 Bio -Fragment Analyzer 665 (Bioptic). The strand-specific libraries were sequenced on Illumina Novaseq 6000 system with paired -666 end 2 x 150 bp read length. 667 LC–MS/MS quantification of m6A 668 Total RNA of naï ve or activated CD8 T cells (anti-CD3/CD28, 5 μg /ml, 24 h) was extracted by using 669 TRIzol Reagent. Quantification of m 6A in mRNAs was carried out as previously described 32.100 ng of 670 mRNA was digested by nuclease P1 (NEB) in 25 μl of buffer containing 25 mM NaCl, and 2.5 mM ZnCl2 671 at 42 ° C for 2 h, followed by the addition of NH4HCO3 and alkaline phosphatase and incubation at 37 ° C 672 for 2 h. The sample was then fi ltered (0.22 μm, Millipore) and injected into the LC -MS/MS. The 673 nucleosides were separated by reverse -phase ultraperformance liquid chromatography on a C18 674 column using an Agilent 6410 QQQ triple -quadrupole LC mass spectrometer in positive electrospray 675 ionization mode. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions 676 of 282 to 150 (m 6A) and 268 to 136 (A). Quantification was carried out by comparison with a standard 677 curve obtained from pure nucleoside standards run with the same batch of samples. The m 6A/A ratio 678 was calculated based on the calibrated concentrations. 679 Bulk RNA-seq 680 RNA-seq was performed as previously described32. Briefly, RNA was isolated from CD8 T cells or Jurkat 681 cells using TRIzol for subsequent RNA library construction. The libraries were sequenced on Illumina 682 nova 6000 in a 150-bp pair-end run (PE150). 683 ATAC-seq 684 ATAC libraries were generated as described with minor modifications 73. In brief, mouse CD8 T cells or 685 Jurkat cells were harvested and c ounted. Nuclei from 50,000 cells were isolated using a lysis solution 686 composed of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630. Immediately after 687 cell lysis, nuclei were pelleted in low-bind 1.5-ml tubes and resuspended in transposition mix (10 µ l 5 x 688 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint TD buffer, 5 µ l Tn5 transposase, 35 µ l nuclease-free water). The transposition reaction was performed 689 at 37° C for 45 min. DNA fragments were purified from enzyme solution using Zymo DNA Clean and 690 Concentrator TM -5 kit (Zymo). Libraries were barcoded (Nextera Index Kit, Illumina) and amplified with 691 NEBNext High Fidelity PCR Mix (New England Biolabs). Size selection of the PCR product were 692 performed by using DNA clean beads (Epibiotek). The quality of libraries was determined on Qseq100 693 Bio-Fragment Analyzer (Bioptic) and sequenced on Illumina Novaseq 6000 system with paired-end 2 x 694 150 bp read length. 695 Ribo-seq 696 Ribosome profiling libraries were prepared as described with minor changes74. Briefly, CD8 T cells were 697 exposed to cycloheximide (CHX, 100 μg/ml) for 15 min, washed twice with 5 ml cold PBS with CHX 698 (100 μg/ml), pelleted, and lysed in Lysis Buffer (20 mM Tris -HCl, pH 7.8, 100 mM KCl, 10 mM MgCl 2, 699 1% Triton X-100, 2 mM DTT, 100 μg/ml cycloheximide, 1:100 protease inhibitor, 40 U/ml SUPERasin). 700 The following Ribo-Seq experiment were performed by using Epi TM Ribosome Profiling Kit (Epibiotek) 701 according to the manufacturer’s protocols. Released RNA fragments were purified using Zymo RNA 702 Clean&ConcentratorTM-5 kit (Zymo) an d ribosomal RNA was deleted by using RiboRNA Depletion Kit 703 (Epibiotek). Ribosome protected fragments (RPF) were recovered by using Zymo RNA 704 Clean&ConcentratorTM-5 kit (Zymo). RNA fragments were prepared into libraries using a QIAseq miRNA 705 Library kit (Qiag en). Size selection of the library products were performed by using Native -PAGE 706 electrophoresis to capture fragments range from 178 -180bp. Libraries quality were determined on 707 Qseq100 Bio -Fragment Analyzer (Bioptic) and sequenced on Illumina Novaseq 6000 s ystem with 708 single-end 1 x 75 bp read length. 709 Immunoprecipitation (IP) 710 Cells were washed with 2 ml of phosphate -buffered saline twice and then lysed with IP lysis buffer 711 (Beyotime). After incubation on ice for 15 min and centrifugation at 12,000 rpm at 4° C for 20 min, the 712 supernatant was saved and the protein concentration was determined with the BCA assay 713 (ThermoFisher). Proteins were either directly analyzed by immunoblotting as input or used for 714 immunoprecipitation analysis. Briefly, the proteins were fir st incubated with the corresponding 715 antibodies (anti-YTHDF2 (Abcam, ab246514), anti-Ikaros (CST, 14859) or anti-Aiolos (CST, 15103)) 716 overnight and then mixed with Protein G beads (MCE) and incubated for 2 more hours. The beads were 717 collected with a magnetic stand (ThermoFisher) and washed five times with Wash Buffer. After the final 718 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint wash, the beads were resuspended and heated in loading buffer and the supernatant was 719 electrophoresed through SDS-PAGE. 720 Mass spectrometry (MS) 721 Protein lysates from naï ve and ac tivated CD8 T cells were immunoprecipitated with anti -YTHDF2 722 (Abcam, ab246514) antibody, separated by SDS-PAGE, and finally visualized with Coomassie brilliant 723 blue staining. Protein in-gel digestion and nano-HPLC MS/MS were carried out as described75. 724 CUT&RUN 725 CUT&RUN was carried out as previously described 76. Briefly,105 primed Ythdf2F/F and Ythdf2CKO CD8 726 T cells (anti -CD3/CD28, 5 μg/ml, 24 h) were washed and bound to concanavalin A -coated magnetic 727 beads, then permeabilized with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine 728 and protease in hibitor cocktails from Sigma -Aldrich) containing 0.05% digitonin (Dig Wash), and 729 incubated with anti-H3K4Me (Active Motif, 39635), anti-Ikaros (CST, 14859) or anti-Aiolos (CST, 15103) 730 overnight at 4° C. Cell-bead slurry was washed twice with 1 ml Dig Wash, incubated with Protein A -731 MNase (pA-MN) for 1h at 4C, then washed twice more with Dig Wash to remove unbound PA/G-MNase 732 protein. Slurry was then placed on a pre -cooled metal block and incubated with cold Dig Wash 733 containing 2 mM CaCl 2 to activate pA-MN digestion. After 30 min incubation, one volume of 2 x Stop 734 Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 50 μg/ml glycogen, 50 μg/ml RNase 735 A, 4 pg/ml heterologous spike -in DNA) was added to stop the reaction, and fragments were released 736 by incubating the tubes on a heat block at 37C for 30 min. Samples were centrifuged 5 minutes at 737 16000xg at 4° C, and supernatant was recovered and DNA extracted via phenol-chloroform extraction 738 and ethanol precipitation. Extract DNA was processed for library generation using the QIAseq Ultralow 739 Input Library Kit (QIAGEN) following the manufacturer’s protocol. Libraries quality were determined on 740 Qseq100 Bio -Fragment Analyzer (Bioptic) and sequenced on Illumina Novaseq 6000 system with 741 paired-end 2× 150 bp read length. 742 Proximity Ligation Assays (PLA) Fluorescence Assay 743 The DuoLink In Situ Red Starter Kit Mouse/Rabbit (Sigma -Aldrich) was used to detect interacting 744 proteins. The assay was performed according to the manufacturer’s instructions. Glass bottom cell 745 culture dishes were treated with poly-lysine (Sigma-Aldrich) at 37° C for 4 hours. Then cells were seeded 746 to the culture dishes and settled for 15 min. Cells were fixed with 4% paraformaldehyde solution for 20 747 min. Then the dishes were permeabilized with 0.05% Trit on X-100 and blocked with Duolink Blocking 748 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Solution in a pre-heated humidified chamber for 60 min at 37° C. The primary antibodies (anti-YTHDF2 749 (Abcam, ab246514), anti-DYKDDDDK Tag antibody (Cell Signaling Technology , 14793), anti-Ikaros 750 (Proteintech, 66966), anti-Aiolos ( Lesding Biology , AMM16470VCF), and anti -HDAC1 (Proteintech, 751 66085)) were added to the dishes and incubated overnight at 4° C. Then the dishes were washed with 752 Wash Buffer A and subsequently incubated with the PLA probes for 60 min, the Ligation-Ligase solution 753 for 30 min, and the Amplification-Polymerase solution for 100 min in a pre -heated humidified chamber 754 at 37° C. Before imaging, the dishes were mounted with a cover slip using Duolink In Situ Mounting 755 Medium with DAPI. Fluorescence images were acquired using a ZEISS LSM880 with fast airyscan 756 confocal microscope. 757 Nuclear and cytoplasmic protein extraction 758 Mouse CD8 T cells or Jurkat cells were harvested and washed twice in PBS. Cytoplasmic and nuclear 759 fractions were separated using the Minute Cytoplasmic and Nuclear Fractionation kit (Invent) according 760 to the manufacturer’s instructions. Briefly, cells were lysed in the cytoplasmic l ysis buffer on ice for 5 761 min and then centrifuged at 12000 rpm for 5 min at 4 °C. The supernatant containing the cytoplasmic 762 proteins were harvested. The pellet containing the cell nucleus were wash with ice-cold PBS for 5 times. 763 Then the pellet was lysed with the nuclear lysis buffer on ice for 40 min and with violent vortex for 15s 764 every 10 min. The nuclear lysate was centrifuged at 12,000 rpm for 5 min and the supernatant 765 containing the nuclear proteins were harvested. The concentrations of both fractions were normalized 766 with BCA assay and subjected to western blot analysis. 767 Western blot analysis 768 Cells were lysed on ice for 15 min using lysis buffer (Beyotime) supplemented with a protease inhibitor 769 cocktail (ThermoFisher). The cell lysate was centrifuged at 12,000 rpm at 4° C for 20 min. The protein 770 concentrations were normalized with a BCA assay kit (ThermoFisher). Equivalent proteins were loaded 771 into 10% SDS-PAGE Gel and transferred to PVDF membranes (Life Technologies). Membranes were 772 blocked for 1 h in TBST buffer with 5% skim milk and then incubated with primary antibodies in the 773 blocking buffer at 4° C overnight. After being washed three times in TBST, membranes were incubated 774 with secondary antibodies for 1 h at room temperature. The quantitative densi tometry of immunoblots 775 was analyzed by using Image J software. Relevant antibodies are listed in Supplementary Table 6. 776 Nascent RNA labeling assay 777 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint CD8 T cells or Jurkat were cultured on pre -coated glass over slides. A nascent RNA synthesis assay 778 was conduc ted using Click -It RNA Imaging Kits (Invitrogen) following the manufacturer’s protocols. 779 Images were captured with LSM 880 (Zeiss) confocal and intensity of signal was quantified using Zen 780 2.6 software (Zeiss). 781 Quantitative PCR (qPCR) 782 RNAs were extracted f rom primary CD8 T cells and Jurkat cells by using RNA -Quick Purification Kit 783 (ESscience), according to the manufacturer’ protocol, and were reverse transcri bed using the 784 PrimeScript RT Master Mix (Takara). The GoTaq qPCR Master Mix (Promega) was used to pe rform 785 quantitative real-time PCR on the LightCycler 480 System (Roche). The primer sequences are listed in 786 Supplementary Table 6. 787 Measurement of RNA lifetime 788 Cells were treated with Act D (500 μg/ml, MCE) for 1, 2, or 4 h. Untreated cells were used as 0 h. Cells 789 were collected at the indicated timepoints. The total RNA was purified by EasySep™ Total Nucleic Acid 790 Extraction Kit (STEM CELL) with an additional DNase -I digestion step (Invitrogen). The quality of the 791 total RNA w as assessed using a Bioanalyzer 2100 instrument and the RNA 6000 Nano Assay Kit 792 (Agilent). RNA quantities were determined using qPCR. 793 Immunofluorescent Staining 794 YTHDF2 and CD8 in mouse samples or human specimen were detected by Tyramide SuperBoost kits 795 (Alexa Fluor 488 -labeled tyramide, Cy3 -labeled tyramide) (ThermoFisher), according to the 796 manufacturers’ protocol. Briefly, paraffin -embedded tissue specimen was first dewaxed at 70° C for 20 797 min. After antigen retrieval and blocking, tissue sections were incuba ted with corresponding primary 798 antibodies overnight at 4° C and then incubated with poly -HRP-conjugated secondary antibody and 799 Alexa Fluor tyramide reagent. Finally, HRP reaction was stopped and the tissue sections were 800 multiplexed for second and third sign al detection. Nucleus was counterstained with DAPI. Whole slide 801 overview images at 40x magnification were obtained using Pannoramic MIDI (3DHISTECH). 802 Seahorse metabolic assay 803 Seahorse assay was performed to measure OCR and ECAR of primed Ythdf2F/F;OT-1 or Ythdf2CKO;OT-804 1 CD8 T cells . CD8 T cells were washed in assay media (XF RPMI medium pH 7.4 (Agilent)) and 805 seeded in a 96-well Seahorse Cells Culture Plate (Agilent) in a non-CO2 incubator at 37 ° C for 40 min. 806 OCR and ECAR were measured by a Seahorse XFe96 Extracellular Flux Analyzer (Agilent) following 807 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint the manufacturer’s instructions. During a mito-stress assay, cells were treated with oligomycin (1.5μM, 808 Sigma-Aldrich), carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1.5 μM, Sigma-Aldrich), 809 rotenone (0.5 μM, Sigma-Aldrich) and antimycin A (0.5 μM, Sigma-Aldrich). During a glycolysis assay, 810 cells were treated with glucose (10 mM, Sigma -Aldrich), oligomycin (1 μM, Sigma -Aldrich) and 2 -DG 811 (50 mM, Sigma -Aldrich). Each condition was performed with 3 –6 replicates in a single experiment. 812 OXPHOS and glycolysis were calculated according to the previous report 77. 813 Transmission electron microscopy 814 Cell pellets were fixed in 2.5% glutaraldehyde for 4h at 22 °C. Following pre -fixation, samples were 815 washed in PBS and post fixed in 1% osmium tetroxide for 1h at 22°C. After several washes in PBS and 816 dehydration in acetone, samples were embedded in Epon. Ultrathin sections of 100 nm were prepared 817 on a Leica EM UC7 Ultramicrotome (Leica Microsystems) and stained with uranyl acetate and lead 818 citrate. Images of mitochondria morphology were captured using a Tecnai G2 Spirit transmission 819 electron microscope (FEI Company). 820 Processing of single-cell RNA sequencing data 821 Human single-cell RNA sequencing datasets (GSE206325 50, GSE146771 48, GSE155698 49, 822 GSE12057551) were pretreated by R (v4.2.2). The metadata was loaded and pre -processed using the 823 R package Matrix (v1.4 -1). For the analysis of tumor-infiltrating CD8 T cells in the violin plot, we 824 excluded cells that met the criteria as reported 78. Single-cell data processing was carried out using the 825 R package Seurat (version 4.3.0). The defining gene sets for the polyfunctionality score of CD8 T cells 826 include Ifng, Gzma, Gzmb, and Prf1. The scRNA-seq gene set functional score was generated using 827 the R package Seurat (version 4.3.0) function 'AddModuleScore'79-82. We then categorized intratumoral 828 CD8 T cells based on the median of the polyfunctionality score a nd compared the Ythdf2 expression 829 level between the high and low polyfunctionality groups. The resulting graphs were plotted using the R 830 package ggplot2 (version 3.3.6). In the GSE206325 dataset, the cells were subjected to quality control, 831 standardization, clustering, and dime nsionality reduction, resulting in 34 cell clusters. Using hallmark 832 genes, these clusters were further categorized into seven subpopulations: Terminal 1, Progenitor 2, 833 Proliferating 3, Effector 4, Effector 5, Memory 6, and Cytotoxic 7. We examined the diff erences in 834 YTHDF2 expression levels among the seven subpopulations or based on the computed 835 polyfunctionality score. 836 Statistical analysis 837 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Histological analyses of both mouse and human tissue were performed in a blinded fashion. Immunoblot 838 and immunofluorescence images are representative of experiments that have been repeated at least 839 three times with similar results. Data from cell culture -based flow cytometry and qPCR assays were 840 generated from two or three independent experiments. Data are presented as me an ± standard error 841 of the mean (SEM). The statistical significance of differences was evaluated by two -tailed unpaired 842 Student’s t test, two -sided Wilcoxon tests or one(two) -way ANOVA. P values of less than 0.05 were 843 considered statistically significant. All statistical analyses were carried out using R (v4.2.2) or Graphpad 844 Prism 8 (GraphPad Software). 845 Study approval 846 All patient samples were obtained from Sun Yat -sen University Cancer Center (SYSUCC). T he 847 collection of tissue specimens was approved by the internal review and ethics boards of SYSUCC. All 848 animal care and handling procedures were performed in accordance with the NIH’s Guide for the Care 849 and Use of Laboratory Animals (National Academies Press, 2011) and were approved by the ethics 850 committees of Sun Yat-sen University and University of Macau. 851 Data availability 852 Data for bulk RNA -seq, RIP -seq, meRIP -seq, ATAC -seq, Ribo -seq and CUT& RUN are available 853 through the BioProject portal (BioProject ID: PRJNA748842). All other data are available from the 854 corresponding author on reasonable request. 855 856

857 1 Su, R. et al. Targeting FTO Suppresses Cancer Stem Cell Maintenance and Immune Evasion. 858 Cancer Cell 38, 79-96 e11, doi:10.1016/j.ccell.2020.04.017 (2020). 859 2 Shulman, Z. & Stern-Ginossar, N. The RNA modification N-6-methyladenosine as a novel 860 regulator of the immune system. Nature Immunology 21, 501-512, doi:10.1038/s41590-020-861 0650-4 (2020). 862 3 Han, D. & Xu, M. M. RNA Modification in the Immune System. Annu Rev Immunol 41, 73-98, 863 doi:10.1146/annurev-immunol-101921-045401 (2023). 864 4 Liu, Y. et al. Tumors exploit FTO-mediated regulation of glycolytic metabolism to evade 865 immune surveillance. Cell Metab 33, 1221-1233 e1211, doi:10.1016/j.cmet.2021.04.001 866 (2021). 867 5 Wang, L. et al. m(6) A RNA methyltransferases METTL3/14 regulate immune responses to 868 anti-PD-1 therapy. EMBO J 39, e104514, doi:10.15252/embj.2020104514 (2020). 869 6 Dong, L. et al. The loss of RNA N(6)-adenosine methyltransferase Mettl14 in tumor-870 associated macrophages promotes CD8(+) T cell dysfunction and tumor growth. Cancer Cell, 871 doi:10.1016/j.ccell.2021.04.016 (2021). 872 7 Han, D. et al. Anti-tumour immunity controlled through mRNA m(6)A methylation and 873 YTHDF1 in dendritic cells. Nature 566, 270-274, doi:10.1038/s41586-019-0916-x (2019). 874 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 8 Ma, S. et al. YTHDF2 orchestrates tumor-associated macrophage reprogramming and 875 controls antitumor immunity through CD8(+) T cells. Nat Immunol 24, 255-266, 876 doi:10.1038/s41590-022-01398-6 (2023). 877 9 Wang, L. et al. YTHDF2 inhibition potentiates radiotherapy antitumor efficacy. Cancer Cell 878 41, 1294-1308 e1298, doi:10.1016/j.ccell.2023.04.019 (2023). 879 10 Thommen, D. S. & Schumacher, T. N. T Cell Dysfunction in Cancer. Cancer Cell 33, 547-562, 880 doi:10.1016/j.ccell.2018.03.012 (2018). 881 11 Wherry, E. J. & Kurachi, M. Molecular and cellular insights into T cell exhaustion. Nat Rev 882 Immunol 15, 486-499, doi:10.1038/nri3862 (2015). 883 12 Henning, A. N., Roychoudhuri, R. & Restifo, N. P. Epigenetic control of CD8(+) T cell 884 differentiation. Nat Rev Immunol 18, 340-356, doi:10.1038/nri.2017.146 (2018). 885 13 Chan, J. D. et al. Cellular networks controlling T cell persistence in adoptive cell therapy. 886 Nature Reviews Immunology, doi:10.1038/s41577-021-00539-6 (2021). 887 14 Topper, M. J., Vaz, M., Marrone, K. A., Brahmer, J. R. & Baylin, S. B. The emerging role of 888 epigenetic therapeutics in immuno-oncology. Nat Rev Clin Oncol 17, 75-90, 889 doi:10.1038/s41571-019-0266-5 (2020). 890 15 Belk, J. A., Daniel, B. & Satpathy, A. T. Epigenetic regulation of T cell exhaustion. Nat 891 Immunol 23, 848-860, doi:10.1038/s41590-022-01224-z (2022). 892 16 Miller, B. C. et al. Subsets of exhausted CD8(+) T cells differentially mediate tumor control 893 and respond to checkpoint blockade. Nat Immunol 20, 326-336, doi:10.1038/s41590-019-894 0312-6 (2019). 895 17 Siddiqui, I. et al. Intratumoral Tcf1(+)PD-1(+)CD8(+) T Cells with Stem-like Properties 896 Promote Tumor Control in Response to Vaccination and Checkpoint Blockade 897 Immunotherapy. Immunity 50, 195-211 e110, doi:10.1016/j.immuni.2018.12.021 (2019). 898 18 Giles, J. R. et al. Shared and distinct biological circuits in effector, memory and exhausted 899 CD8(+) T cells revealed by temporal single-cell transcriptomics and epigenetics. Nat Immunol 900 23, 1600-1613, doi:10.1038/s41590-022-01338-4 (2022). 901 19 Li, H. B. et al. m(6)A mRNA methylation controls T cell homeostasis by targeting the IL-902 7/STAT5/SOCS pathways. Nature 548, 338-342, doi:10.1038/nature23450 (2017). 903 20 Ito-Kureha, T. et al. The function of Wtap in N(6)-adenosine methylation of mRNAs controls 904 T cell receptor signaling and survival of T cells. Nat Immunol 23, 1208-1221, 905 doi:10.1038/s41590-022-01268-1 (2022). 906 21 Slobodin, B. et al. Transcription Impacts the Efficiency of mRNA Translation via Co-907 transcriptional N6-adenosine Methylation. Cell 169, 326-337, doi:10.1016/j.cell.2017.03.031 908 (2017). 909 22 Huang, H. et al. Histone H3 trimethylation at lysine 36 guides m(6)A RNA modification co-910 transcriptionally. Nature 567, 414-419, doi:10.1038/s41586-019-1016-7 (2019). 911 23 Li, Y. et al. N(6)-Methyladenosine co-transcriptionally directs the demethylation of histone 912 H3K9me2. Nat Genet 52, 870-877, doi:10.1038/s41588-020-0677-3 (2020). 913 24 Yu, Y. R. et al. Disturbed mitochondrial dynamics in CD8(+) TILs reinforce T cell exhaustion. 914 Nat Immunol 21, 1540-1551, doi:10.1038/s41590-020-0793-3 (2020). 915 25 Liu, J. et al. N (6)-methyladenosine of chromosome-associated regulatory RNA regulates 916 chromatin state and transcription. Science 367, 580-586, doi:10.1126/science.aay6018 917 (2020). 918 26 Wang, X. et al. N6-methyladenosine-dependent regulation of messenger RNA stability. 919 Nature 505, 117-120, doi:10.1038/nature12730 (2014). 920 27 Zhou, J. et al. Dynamic m(6)A mRNA methylation directs translational control of heat shock 921 response. Nature 526, 591-594, doi:10.1038/nature15377 (2015). 922 28 Battistello, E. et al. Stepwise activities of mSWI/SNF family chromatin remodeling complexes 923 direct T cell activation and exhaustion. Mol Cell 83, 1216-1236 e1212, 924 doi:10.1016/j.molcel.2023.02.026 (2023). 925 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 29 Sandberg, R., Neilson, J. R., Sarma, A., Sharp, P. A. & Burge, C. B. Proliferating cells express 926 mRNAs with shortened 3' untranslated regions and fewer microRNA target sites. Science 927 320, 1643-1647, doi:10.1126/science.1155390 (2008). 928 30 Globig, A. M. et al. The beta(1)-adrenergic receptor links sympathetic nerves to T cell 929 exhaustion. Nature, doi:10.1038/s41586-023-06568-6 (2023). 930 31 Hudson, W. H. et al. Proliferating Transitory T Cells with an Effector-like Transcriptional 931 Signature Emerge from PD-1(+) Stem-like CD8(+) T Cells during Chronic Infection. Immunity 932 51, 1043-1058 e1044, doi:10.1016/j.immuni.2019.11.002 (2019). 933 32 Hou, J. et al. YTHDF2 reduction fuels inflammation and vascular abnormalization in 934 hepatocellular carcinoma. Mol Cancer 18, 163, doi:10.1186/s12943-019-1082-3 (2019). 935 33 Zhang, D. J. et al. Selective expression of the Cre recombinase in late-stage thymocytes using 936 the distal promoter of the Lck gene. J Immunol 174, 6725-6731, 937 doi:10.4049/jimmunol.174.11.6725 (2005). 938 34 Zhang, L. et al. YTHDF2/m(6) A/NF-kappaB axis controls anti-tumor immunity by regulating 939 intratumoral Tregs. EMBO J 42, e113126, doi:10.15252/embj.2022113126 (2023). 940 35 Scharping, N. E. et al. Mitochondrial stress induced by continuous stimulation under hypoxia 941 rapidly drives T cell exhaustion. Nat Immunol 22, 205-215, doi:10.1038/s41590-020-00834-9 942 (2021). 943 36 Bengsch, B. et al. Bioenergetic Insufficiencies Due to Metabolic Alterations Regulated by the 944 Inhibitory Receptor PD-1 Are an Early Driver of CD8(+) T Cell Exhaustion. Immunity 45, 358-945 373, doi:10.1016/j.immuni.2016.07.008 (2016). 946 37 Zhu, T. et al. Crystal structure of the YTH domain of YTHDF2 reveals mechanism for 947 recognition of N6-methyladenosine. Cell Res 24, 1493-1496, doi:10.1038/cr.2014.152 (2014). 948 38 Bensaude, O. Inhibiting eukaryotic transcription: Which compound to choose? How to 949 evaluate its activity? Transcription 2, 103-108, doi:10.4161/trns.2.3.16172 (2011). 950 39 Zhang, J. et al. Harnessing of the nucleosome-remodeling-deacetylase complex controls 951 lymphocyte development and prevents leukemogenesis. Nat Immunol 13, 86-94, 952 doi:10.1038/ni.2150 (2011). 953 40 Schwickert, T. A. et al. Stage-specific control of early B cell development by the transcription 954 factor Ikaros. Nat Immunol 15, 283-293, doi:10.1038/ni.2828 (2014). 955 41 Salzer, E. et al. RASGRP1 deficiency causes immunodeficiency with impaired cytoskeletal 956 dynamics. Nat Immunol 17, 1352-1360, doi:10.1038/ni.3575 (2016). 957 42 Tay, T. et al. Degradation of IKAROS prevents epigenetic progression of T cell exhaustion in a 958 novel antigen-specific assay. bioRxiv, 2024.2002.2022.581548, 959 doi:10.1101/2024.02.22.581548 (2024). 960 43 Koipally, J., Renold, A., Kim, J. & Georgopoulos, K. Repression by Ikaros and Aiolos is 961 mediated through histone deacetylase complexes. EMBO J 18, 3090-3100, 962 doi:10.1093/emboj/18.11.3090 (1999). 963 44 Song, C. et al. Epigenetic regulation of gene expression by Ikaros, HDAC1 and Casein Kinase II 964 in leukemia. Leukemia 30, 1436-1440, doi:10.1038/leu.2015.331 (2016). 965 45 Kronke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple 966 myeloma cells. Science 343, 301-305, doi:10.1126/science.1244851 (2014). 967 46 Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction 968 of Ikaros proteins. Science 343, 305-309, doi:10.1126/science.1244917 (2014). 969 47 Huang, Y. et al. Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid 970 Leukemia. Cancer Cell 35, 677-+, doi:10.1016/j.ccell.2019.03.006 (2019). 971 48 Zhang, L. et al. Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in 972 Colon Cancer. Cell 181, 442-459 e429, doi:10.1016/j.cell.2020.03.048 (2020). 973 49 Steele, N. G. et al. Multimodal Mapping of the Tumor and Peripheral Blood Immune 974 Landscape in Human Pancreatic Cancer. Nat Cancer 1, 1097-1112, doi:10.1038/s43018-020-975 00121-4 (2020). 976 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 50 Magen, A. et al. Intratumoral dendritic cell-CD4(+) T helper cell niches enable CD8(+) T cell 977 differentiation following PD-1 blockade in hepatocellular carcinoma. Nat Med 29, 1389-978 1399, doi:10.1038/s41591-023-02345-0 (2023). 979 51 Sade-Feldman, M. et al. Defining T Cell States Associated with Response to Checkpoint 980 Immunotherapy in Melanoma. Cell 175, 998-1013 e1020, doi:10.1016/j.cell.2018.10.038 981 (2018). 982 52 Luo, H. et al. Effect of Camrelizumab vs Placebo Added to Chemotherapy on Survival and 983 Progression-Free Survival in Patients With Advanced or Metastatic Esophageal Squamous 984 Cell Carcinoma: The ESCORT-1st Randomized Clinical Trial. JAMA 326, 916-925, 985 doi:10.1001/jama.2021.12836 (2021). 986 53 Mai, H. Q. et al. Toripalimab or placebo plus chemotherapy as first-line treatment in 987 advanced nasopharyngeal carcinoma: a multicenter randomized phase 3 trial. Nat Med 27, 988 1536-1543, doi:10.1038/s41591-021-01444-0 (2021). 989 54 Franco, F., Jaccard, A., Romero, P., Yu, Y. R. & Ho, P. C. Metabolic and epigenetic regulation 990 of T-cell exhaustion. Nature Metabolism 2, 1001-1012, doi:10.1038/s42255-020-00280-9 991 (2020). 992 55 Khan, O. et al. TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. 993 Nature 571, 211-218, doi:10.1038/s41586-019-1325-x (2019). 994 56 Chen, J. et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature 995 567, 530-+, doi:10.1038/s41586-019-0985-x (2019). 996 57 Ghoneim, H. E. et al. De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell 997 Rejuvenation. Cell 170, doi:10.1016/j.cell.2017.06.007 (2017). 998 58 Zehn, D., Thimme, R., Lugli, E., de Almeida, G. P. & Oxenius, A. 'Stem-like' precursors are the 999 fount to sustain persistent CD8(+) T cell responses. Nat Immunol 23, 836-847, 1000 doi:10.1038/s41590-022-01219-w (2022). 1001 59 Baxter, A. E. et al. The SWI/SNF chromatin remodeling complexes BAF and PBAF 1002 differentially regulate epigenetic transitions in exhausted CD8(+) T cells. Immunity 56, 1320-1003 1340 e1310, doi:10.1016/j.immuni.2023.05.008 (2023). 1004 60 Tille, L. et al. Activation of the transcription factor NFAT5 in the tumor microenvironment 1005 enforces CD8(+) T cell exhaustion. Nat Immunol 24, 1645-1653, doi:10.1038/s41590-023-1006 01614-x (2023). 1007 61 Chen, M. N. et al. RNA N6-methyladenosine methyltransferase-like 3 promotes liver cancer 1008 progression through YTHDF2-dependent posttranscriptional silencing of SOCS2. Hepatology 1009 67, 2254-2270, doi:10.1002/hep.29683 (2018). 1010 62 Guo, X. Y. et al. RNA demethylase ALKBH5 prevents pancreatic cancer progression by 1011 posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner. Molecular 1012 Cancer 19, doi:ARTN 91 10.1186/s12943-020-01158-w (2020). 1013 63 Lin, X. Y. et al. RNA m(6)A methylation regulates the epithelial mesenchymal transition of 1014 cancer cells and translation of Snail. Nature Communications 10, doi:ARTN 2065 1015 10.1038/s41467-019-09865-9 (2019). 1016 64 Su, R. et al. R-2HG Exhibits Anti-tumor Activity by Targeting FTO/m(6)A/MYC/CEBPA 1017 Signaling. Cell 172, 90-+, doi:10.1016/j.cell.2017.11.031 (2018). 1018 65 Paris, J. et al. Targeting the RNA m(6)A Reader YTHDF2 Selectively Compromises Cancer 1019 Stem Cells in Acute Myeloid Leukemia. Cell Stem Cell 25, 137-+, 1020 doi:10.1016/j.stem.2019.03.021 (2019). 1021 66 Ma, S. et al. The RNA m6A reader YTHDF2 controls NK cell antitumor and antiviral immunity. 1022 J Exp Med 218, doi:10.1084/jem.20210279 (2021). 1023 67 Xiao, R. et al. Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based 1024 Regulation of Transcription. Cell 178, 107-+, doi:10.1016/j.cell.2019.06.001 (2019). 1025 68 Ries, R. J. et al. m(6)A enhances the phase separation potential of mRNA. Nature 571, 424-+, 1026 doi:10.1038/s41586-019-1374-1 (2019). 1027 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 69 Belk, J. A. et al. Genome-wide CRISPR screens of T cell exhaustion identify chromatin 1028 remodeling factors that limit T cell persistence. Cancer Cell 40, 768-786 e767, 1029 doi:10.1016/j.ccell.2022.06.001 (2022). 1030 70 Kronke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) 1031 MDS. Nature 523, 183-188, doi:10.1038/nature14610 (2015). 1032 71 Li, X. Y. et al. Targeting CD39 in Cancer Reveals an Extracellular ATP- and Inflammasome-1033 Driven Tumor Immunity. Cancer Discov 9, 1754-1773, doi:10.1158/2159-8290.CD-19-0541 1034 (2019). 1035 72 Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M., Amariglio, N. & Rechavi, G. 1036 Transcriptome-wide mapping of N-6-methyladenosine by m(6)A-seq based on 1037 immunocapturing and massively parallel sequencing. Nature Protocols 8, 176-189, 1038 doi:10.1038/nprot.2012.148 (2013). 1039 73 Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of 1040 native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding 1041 proteins and nucleosome position. Nat Methods 10, 1213-1218, doi:10.1038/nmeth.2688 1042 (2013). 1043 74 Loayza-Puch, F. et al. p53 induces transcriptional and translational programs to suppress cell 1044 proliferation and growth. Genome Biology 14, doi:10.1186/gb-2013-14-4-r32 (2013). 1045 75 Chen, Y., Kwon, S. W., Kim, S. C. & Zhao, Y. Integrated approach for manual evaluation of 1046 peptides identified by searching protein sequence databases with tandem mass spectra. J 1047 Proteome Res 4, 998-1005, doi:10.1021/pr049754t (2005). 1048 76 Skene, P. J., Henikoff, J. G. & Henikoff, S. Targeted in situ genome-wide profiling with high 1049 efficiency for low cell numbers. Nat Protoc 13, 1006-1019, doi:10.1038/nprot.2018.015 1050 (2018). 1051 77 Guo, Y. G. et al. Metabolic reprogramming of terminally exhausted CD8(+) T cells by IL-10 1052 enhances anti-tumor immunity. Nature Immunology 22, 746-+, doi:10.1038/s41590-021-1053 00940-2 (2021). 1054 78 Yan, C. et al. Exhaustion-associated cholesterol deficiency dampens the cytotoxic arm of 1055 antitumor immunity. Cancer Cell 41, 1276-1293 e1211, doi:10.1016/j.ccell.2023.04.016 1056 (2023). 1057 79 Stuart, T. et al. Comprehensive Integration of Single-Cell Data. Cell 177, 1888-1902 e1821, 1058 doi:10.1016/j.cell.2019.05.031 (2019). 1059 80 Satija, R., Farrell, J. A., Gennert, D., Schier, A. F. & Regev, A. Spatial reconstruction of single-1060 cell gene expression data. Nat Biotechnol 33, 495-502, doi:10.1038/nbt.3192 (2015). 1061 81 Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell 1062 transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 1063 36, 411-420, doi:10.1038/nbt.4096 (2018). 1064 82 Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573-3587 e3529, 1065 doi:10.1016/j.cell.2021.04.048 (2021). 1066 1067

1068 We thank Prof. Z. Yin (Jinan University) for providing the Mettl3F/F mice. We thank Dr. R. Su (Beckman 1069 Research Institute of City of Hope), Dr. Z. Chen (Harvard University) and Dr. H. Huang (Sun Yat -sen 1070 University) and Dr. K. Wu (University of Macau) for helpful discussion. This work was supported by 1071 Guangdong Provincial Science Fund for Distinguished Young Scholars (2021B1515020007, to J.H.), 1072 General Program of National Natural Science Foundation of China (8271881 & 81871970, to J.H.), 1073 Macau Science and T echnology Development Fund (FDCT) (0071/2023/RIA2, to J.H. ), CAMS 1074 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Innovation Fund for Medical Sciences (CIFMS) (2019-I2M-5-036, to R.-H. X.), Science and Technology 1075 Program of Guangdong (2019B020227002 , to R. -H. X. ), Hundred Talent s Program of Sun Yat -sen 1076 University (2019079, to J.H.) and Ministry of Education Frontiers Science Centre for Precision Oncology, 1077 University of Macau (SP2023-00001-FSCPO, to J.H.). 1078 1079 Author contributions 1080 H.Z performed the animal and immunological experiments, interpreted the data and prepare the 1081 manuscript. X.L., Z.W., Zhicong Zhao, X.P. , J.H.L., and W.W. performed the cellular and molecular 1082 experiments. W.Y., X.Z., and Q.S. performed the animal experiments. C.C., Q.Z. and Y.C. analysed the 1083 multiple sequencing data. M.Q., Zhaolei Zeng and M.C. collected the clinical samples and patient 1084 information. C.D., J.C., B.S., J.L. and H.-Q.J. provided key suggestions. R.-H.X. and J.H. co-supervised 1085 the study. J.H. conceived the project, designed the study, interpreted the data and wrote the manuscript. 1086 The order of the co–first authors was determined according to the time spent on this project. 1087 1088 Competing interests 1089 J.C. is a scientific founder of Genovel Biotech Corp. and holds equities with the company, and is also a 1090 Scientific Advisor for Race Oncology. Other authors declare no conflict of interests. 1091 1092 Figure Legends 1093 Fig. 1 | YTHDF2 is selectively upregulated and redistributed in early Teff and Teff-like cells. 1094 a–b Transcriptomic data were mined from existing datasets. mRNA expression of m 6A modifiers in 1095 CD3/CD28 bead -based stimulation of human CD8 T cells at various time points indicating 1096 naive/memory, activated, and exhausted populations (GSE212357) (a). mRNA expression of m 6A 1097 modifiers in total tumor-infiltrating CD8 T cells from HKP1 lung cancer-bearing mice upon anti-PD-1 or 1098 cIg treatment (GSE114300) (b). c Quantification of YTHDF2 MFI between in vitro-generated effector 1099 CD8 T cells (n = 5) and exhausted CD8 T cells (n = 5). d–e Quantification of YTHDF2 MFI in various 1100 cell populations from late time point (Day 13) B16F10 -OVA tumor (n = 5) and spleen (left panels) or 1101 from early time point (D6) B16F10 -OVA tumor (n = 5) by flow cytometry. f Quantification of YTHDF2 1102 MFI in Tpex cells and transitory Tex cells from B16F10-OVA tumor treated with anti-PD-1 (250μg/mouse) 1103 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint (n = 5) or control IgG (cIg) (n = 5). g Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of 1104 mouse (WT or OT-1) and human CD8 T cells stimulated with anti-CD3/CD28 (5 μg/ml) or OVA (10 nM). 1105 h Representative confocal immunofluorescence images of YTHDF2 (red) and DAPI (blue) in naï ve (n 1106 = 5) or activated (anti -CD3/CD28, 5 μg/ml, 12 –96h) CD8 T cells (n = 5) . Scale bar, 10 μm. i 1107 Representative immunofluorescent staining of CD8 (red) and YTHDF2 (green) in regressing (n = 5) or 1108 progressed (n = 5) B16-OVA tumors. A dashed box represents the 4 × enlarged area shown in the 1109 bottom panels with separate channels. White arrows point to cells positive for YTHDF2 and CD8. Scale 1110 bar, 10 μm. Middle panel, frequencies of YTHDF2 -positive CD8 T cells. Right panel, quantification of 1111 the nuclear to cytoplasmic ratios of YTHDF2 intensity in YTHDF2-positive CD8 T cells. j Representative 1112 confocal immunofluorescence images of YTHDF2 (red) and DAPI (blue) in PD -1+SLAMF6+TIM3- CD8 1113 Tpex cells from B16-OVA tumors (Day 16) after in vitro anti-PD-1 (10 μg/ml, 48 h) or cIg stimulation (n = 1114 6). Scale bar, 10 μm. Error bars, mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. One-way analysis 1115 of variance (ANOVA) (d, e) or two-tailed unpaired Student’s t-test (c, f, i, j). 1116 Fig. 2 | YTHDF2 is essential for the antitumor effects of CD8 T cells. 1117 a Male Ythdf2F/F (n = 6) or Ythdf2CKO (n = 6) mice were injected subcutaneously with 106 Hepa1-6 cells. 1118 Tumor growth was monitored ever 2 or 3 days. b–c FemaleYthdf2F/F (n = 6) and Ythdf2CKO (n = 5–6) 1119 mice were injected subcutaneously with 5 × 105 B16F10 (b) or 106 MC38 (c) cells. Tumor growth was 1120 monitored ever 2 or 3 days. d–f Tumor-infiltrating lymphocytes (TILs) were isolated from Ythdf2F/F (n = 1121 6) and Ythdf2CKO (n = 6) mice 12 days after MC38 tumor inoculation. Numbers of immune subsets (CD8 1122 T, CD4 T and T reg cells) within TILs (d) and frequencies of CD8 T cell subpopulations positive for 1123 CD44+KLRG1+ (e) active caspase-3 (Casp-3), granzyme B (Gzm B), IFN -γ or Ki-67 (f) were assessed 1124 by flow cytometry. g Frequencies of PD-1+TIM3+CD101+CD8 T cell subpopulations from MC38 tumor -1125 bearing Ythdf2F/F (n = 6) and Ythdf2CKO (n = 6) mice (Day 18). h FemaleYthdf2F/F;OT-1 (n = 8) or 1126 Ythdf2CKO;OT-1 (n = 6) mice were injected subcutaneously with 10 6 B16F10-OVA cells and monitored 1127 for tumor growth. i Adoptive transfer therapy using PBS control or OVA-primed (72 h) Ythdf2F/F;OT-1 or 1128 Ythdf2CKO;OT-1 CD8 T cells against B16F10 -OVA melanoma (n = 5 per group). j–k Female Ythdf2F/F 1129 (n = 12) or Ythdf2CKO (n = 10) mice were injected subcutaneously with 106 MC38 cells (j). Male Ythdf2F/F 1130 (n = 16) or Ythdf2CKO (n = 12) mice were injected subcutaneously with 10 6 Hepa1-6 cells (k). Tumor-1131 bearing mice were treated with anti-PD-1 (250 μg/mouse) or cIg. l TILs were isolated from Ythdf2F/F (n 1132 = 6) and Ythdf2CKO (n = 6) mice 12 days after MC38 tumor inoculation with a nti-PD-1 treatment. 1133 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Frequencies of CD8 T cell subpopulations positive for PD-1+Dextramer+, CX3CR1+Tim3+CD101-PD-1+ 1134 were assessed by flow cytometry. Error bars, mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P 1135 < 0.0001. Two-way ANOVA (a–c, h–k) or two-tailed unpaired Student’s t-test (d-g, l). 1136 Fig. 3 | YTHDF2 prevents mitochondrial stress and T cell exhaustion. 1137 a Volcano plots of genes with differential expression in activated Ythdf2F/F or Ythdf2CKO CD8 T cells 1138 (anti-CD3/CD28, 5 μg/ml, 24 h) (n = 2 per group), putative YTHDF2 targets that were enriched in both 1139 RIP-seq and m6A-seq are marked with yellow circles. b GO enrichment analysis of upregulated genes 1140 in Ythdf2CKO compared with Ythdf2F/F CD8 T cells after priming (anti -CD3/CD28, 5 μg /ml, 24 h). c 1141 Mitochondrial membrane potential and mitochondrial mass was measured by MitoTracker Orange (MO) 1142 and MitoTracker Green (MG) staining in activated Ythdf2F/F and Ythdf2CKO CD8 T cells (n = 5 per group). 1143 Mitochondrial fitness was evaluated according to the MO/MG ratio. d Mitochondrial ROS was measured 1144 by MitoSOX staining in activated CD8 T cells from Ythdf2F/F or Ythdf2CKO mice (n = 5 per group) . e 1145 MitoSOX staining in MC38 tumor-infiltrating CD8 T cells from Ythdf2F/F and Ythdf2CKO mice (n = 5 per 1146 group). f Quantification of the MG MFI in MC38 tumor-infiltrating CD8 T cells from Ythdf2F/F or Ythdf2CKO 1147 mice (n = 5 per group) . g–h Quantification of Tim3 + PD-1+ (g), Zombie NIR + (h) f requencies among 1148 primed Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 48 h) in the presence of 10 mΜ 1149 NAC or veh (n = 5 per group) . i Heatmap showing the relative expression of representative genes 1150 (mitochondrion-related and up-regulated in Ythdf2CKO) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells 1151 from RNA-seq data. Putative YTHDF2 targets are depicted by filled circles . Error bars, mean ± s.e.m. 1152 *P < 0.05; **P < 0.01. Two-way ANOVA (g, h) or two-tailed unpaired Student’s t-test (c–f). 1153 Fig. 4 | YTHDF2 segregates IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T 1154 cells. 1155 a GO enrichment analysis of downregulated genes in Ythdf2CKO compared with Ythdf2F/F CD8 T cells 1156 after priming (anti -CD3/CD28, 5 μg/ml, 24 h). b Proximity ligation assay (PLA) analysis of YTHDF2 1157 associated with IKZF1 or IKZF3 in primed mouse (WT or OT-1) and human CD8 T cells stimulated with 1158 anti-CD3/CD28 (5 μg/ml, 24 h) or OVA (10 nM, 24 h). Scale bar, 10 μm. c Coimmunoprecipitation 1159 assays of YTHDF2 associated with IKZF1 or IKZF3 in primed WT CD8 T cells (anti-CD3/CD28, 5 μg/ml, 1160 24 h). d Volcano plot of genes with differential chromatin accessibility between activated Ythdf2F/F and 1161 Ythdf2CKO CD8 T cells (anti -CD3/CD28, 5 μg/ml, 24 h). e Volcano plot of genes with differential 1162 chromatin accessibility bet ween Jurkat -shCtrl (Vec) and Jurkat -shYTHDF2 (KD) cells. f–g ChIP-seq 1163 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint datasets for IKZF1 (GSM1296538) and IKZF3 (GSM803106) in mouse T cells were obtained using 1164 Cistrome Data Browser. ATAC -seq (f) or H3K4me CUT&RUN (g) profiles of activated Ythdf2F/F and 1165 Ythdf2CKO CD8 T cells were represented on IKZF1/3 -bound loci or IKZF1/3 -associated promoters. h 1166 Stat5a (left) and Rasgrp1 (right) mRNA levels detected by RT -qPCR in activated Ythdf2F/F and 1167 Ythdf2CKO CD8 T cells (anti -CD3/CD28, 5 μg/ml, 24 h) (n = 3 per group). i ATAC-seq and H3K4me 1168 CUT&RUN tracks on the gene loci of Stat5a (top) and Rasgrp1 (bottom) in activated Ythdf2F/F and 1169 Ythdf2CKO CD8 T cells. j Heatmap showing the relative expression of representative genes (down -1170 regulated in Ythdf2CKO) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells from RNA-seq data. Enhanced 1171 chromatin accessibility or putative IKZF1/3 binding is depicted by filled circle. Error bars, mean ± s.e.m. 1172 ***P < 0.001. Two-tailed unpaired Student’s t-test (h). 1173 Fig. 5 | Lenalidomide retrieves the antitumor function of YTHDF2-deficient CD8 T cells. 1174 a Click-it RNA imaging and analysis of nascent RNA synthesis (green) in Ythdf2F/F and Ythdf2CKO CD8 1175 T cells primed ( anti-CD3/CD28, 5 μg/ml, 24 h ) in the presence of 10 μΜ lenalidomide (len) or vehicle 1176 (veh) (n = 5 per group). Scale bar, 10 μm. b Quantification of Ki-67 MFI among Ythdf2F/F and Ythdf2CKO 1177 CD8 T cells primed in the presence of 10 μΜ len or veh (n =4 per group). c Click-it RNA imaging and 1178 analysis of nascent RNA synthesis (green) in Ythdf2F/F;OT-1 and Ythdf2CKO;OT-1 CD8 T cells primed 1179 (OVA, 10 nM, 24 h) in the presence of 10 μΜ len or veh (n = 5 per group) . Scale bar, 10 μm. d 1180 Quantification of Ki -67 MFI among Ythdf2F/F;OT-1 and Ythdf2CKO;OT-1 CD8 T cells primed (OVA, 10 1181 nM, 72 h) (n = 5 per group) in the presence of 10 μΜ len or veh. e Quantification of Gzm B, IFN-γ MFI 1182 among Ythdf2F/F and Ythdf2CKO CD8 T cells primed (anti -CD3/CD28, 5 μg/ml, 48 h) in the presence of 1183 10 μΜ len or veh (n = 4 per group). f MC38-bearing Ythdf2F/F (n = 21) or Ythdf2CKO (n = 18) mice were 1184 treated with anti-PD-1 (250 μg/mouse) and/or len (10 mg/kg) and monitored for tumor growth. g Hepa1-1185 6 tumor-bearing Ythdf2F/F (n = 12) or Ythdf2CKO (n = 12) mice were treated with anti-PD-1 (250 μg/mouse) 1186 and/or len (10 mg/kg) and monitored for tumor growth. h Quantification of Gzm B+ CD8 T or IFN-γ+ CD8 1187 T frequencies within TILs from Hepa1-6-bearing Ythdf2F/F (n = 6) or Ythdf2CKO (n = 6) mice treated with 1188 anti-PD-1 (250 μg/mouse) and/or len (10 mg/kg) (D13). Error bars, mean ± s.e.m. *P < 0.05; **P < 0.01; 1189 ***P < 0.001. Two-way ANOVA (a–h). 1190 Fig. 6 | The m6A machinery regulates both YTHDF2 relocation and expression. 1191 a PLA analysis of YTHDF2 associated with IKZF3 in Jurkat cells treated with or without ActD (500ug/ml, 1192 4h). Scale bar, 10 μm. b Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of Mettl3F/F or 1193 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Mettl3CKO CD8 T cells stimulated with anti -CD3/CD28 (5 μg/ml, 24 h). c PLA analysis of YTHDF2 1194 associated with IKZF3 in Jurkat-shCtrl and Jurkat-shMETTL3 cells. Scale bar, 5 μm. d PLA analysis of 1195 YTHDF2 associated with IKZF3 in primed Mettl3F/F or Mettl3CKO CD8 T cells (5 μg/ml, 24 h). Scale bar, 1196 10 μm. e PLA analysis of Flag associated with IKZF3 in Jurkat cells introduced with Flag-tagged WT or 1197 mutant YTHDF2. Scale bar, 10 μm. f Quantification of Ki -67 MFI among METTL3 -knockdown and 1198 control Jurkat cells with or without YTHDF2 overexpression (OE) (n = 5 per group) . g Click-it RNA 1199 imaging and analysis of nascent RNA synthesis in METTL3-knockdown and control Jurkat cells with or 1200 without YTHDF2 overexpression (OE) (n = 5 per group). Scale bar, 10 μm. h ATAC-seq tracks of Ythdf2 1201 loci on naï ve or activated human T cells (anti-CD3/CD28, 5 h) (GSE116696). i Ythdf2 mRNA levels 1202 detected by qPCR in CD8 T cells stimulated with or without anti -CD3/CD28 (5 μg/ml) and ActD (500 1203 ug/ml) for 24 h (n = 5 per group). j Ythdf2 mRNA levels detected by qPCR in naï ve (0 h) or activated (6 1204 h) WT (n = 5) and Ythdf2-249 (n = 5) CD8 T cells. K Naï ve Ythdf2-249 and WT CD8 T cells were treated 1205 with ActD (500 μg/ml) and RNAs were collected at different time points after ActD treatment. Ythdf2 1206 mRNA levels were measured using qPCR and represented as mRNA remaining after transcription 1207 inhibition (TI) (n = 3 per group) . l Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of 1208 naï ve (0 h) or activated (24 h) WT compared with Ythdf2-249 CD8 T cells. Error bars, mean ± s.e.m. NS, 1209 no significance; * P < 0.05; ** P < 0.01; *** P < 0.001. One -way (f, i, g) or two -way ANOVA (j) or non-1210 linear regression (k). 1211 Fig. 7 | YTHDF2 expression and distribution are associated with T cell function in human cancers. 1212 a Violin plot comparing Ythdf2 gene expression levels of CD8 T cells assigned with high or low 1213 polyfunctionality signature scores that were derived from single-cell RNA -seq datasets . L eft, CRC. 1214 Middle, PDAC. Right, HCC. b Violin plot comparing pre - or post -treatment Ythdf2 gene expression 1215 levels of CD8 T cells between responders and nonresponders. Left, melanoma. Right, HCC. c Violin 1216 plot comparingYthdf2 gene expression levels among CD8 T cell clusters generated from neoadj uvant 1217 anti-PD-1-treated HCC. d–i Tissue sections from patients with hepatocellular carcinoma (HCC) (n = 45) 1218 or colorectal carcinoma (CRC) (n = 30) were stained for CD8 (red), YTHDF2 (green) and DAPI (blue). 1219 d–h Representative immunofluorescent images of sections from HCC (d) and CRC (h) patients showing 1220 different responses to neo -adjuvant chemo -(immuno-) therapy. A dashed box represents the 4 × 1221 enlarged area shown in the bottom panels with separate channels. Whit e arrows point to cells positive 1222 for YTHDF2 and CD8. Quantification of CD8 T cells (e, i), frequencies of YTHDF2-positive CD8 T cells 1223 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint (f, j) and quantification of YTHDF2 intensity of CD8 T cells (g, k). CR, complete response; PR, partial 1224 response; SD, stable disease; PD, progressed disease. Scale bar, 10 μm. Error bars, mean ± s.e.m. *P 1225 < 0.05; **P < 0.01. Two-tailed unpaired Student’s t-test (e–g, i–k). 1226 1227 Supplementary Figure Legends 1228 Supplementary Fig. 1 | YTHDF2 is characteristically expressed upon T cell activation and 1229 reinvigoration. 1230 a–d Transcriptomic data were mined from existing datasets. mRNA expression of m 6A modifiers in 1231 mouse naï ve or activated WT CD8 (a) and CD4 (b) T cells. mRNA expression of m 6A modifiers in T 1232 tolerant (Ttol), T helper (T H1, TH2 and TH17), natural regulatory T (nT reg) and naï ve T cells (c). mRNA 1233 expression of m6A modifiers in OVA-specific CD8 TILs from anti-PD-1- or cIg-treated mouse tumors (d). 1234 e–f Protein expression of m 6A modifiers in CD8 T cells primed for the indicated amoun t of time. One 1235 representative of three independent experiments is shown. Human CD8 T cells from peripheral blood 1236 were stimulated with 5 μg/ml anti -CD3/CD28 (e). Wild -type (WT) (top) or OT -1 mouse CD8 T cells 1237 (bottom) were stimulated with 5 μg/ml anti -CD3/CD28 or 10 nM OVA (f). g LC-MS/MS-based m 6A 1238 quantification in mRNAs from naï ve and activated CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) (n = 3 1239 per group). h Metagene distribution of the m6A peaks of naï ve and active CD8 T cells (anti-CD3/CD28, 1240 5 μg/ml, 24 h) along the whole transcriptome. The enriched consensus motifs were detected within m6A 1241 peaks. i Representative confocal Z-stack images of YTHDF2 (red), and DAPI (blue) in naï ve or activated 1242 (anti-CD3/CD28, 5 μg/ml, 0–48 h) CD8 T cells. j Quantification of YTHDF2 intensity of CD8 T cells (left) 1243 and frequencies of nuclear YTHDF2 + cells from naï ve or activated (anti-CD3/CD28, 5 μg/ml, 0 –96 h) 1244 (right) CD8 T cells (n = 5 per group). k Immunoblotting analysis of YTHDF2 in the cytosol and nucleus 1245 of Jurkat cells stimulated with or without PHA (150 ng/ml, 24 h). respectively. Relative YTHDF2 levels 1246 were calculated using densitometry values for β -actin or Lamin B as calibrators. l Representative 1247 confocal Z-stack images of YTHDF2 (red) and DAPI (blue) in unstimulated Jurkat cells. Scale bar, 10 1248 μm. m Representative multiplexable immunofluorescent staining of CD8 (red) and YTHDF2 (green) 1249 within B16F10 or B16F10-OVA tumors grown in OT-1 mice (n = 5 per group). A dashed box represents 1250 the 4× enlarged area shown in the bottom panels with separate channels. White arrows point to cells 1251 positive for YTHDF2 and CD8. Scale bar, 10 μm. Middle panel, frequencies of YTHDF2 -positive CD8 1252 T cells. Right panel, quantification of the nuclear to cytoplasmic ratios of YTHDF2 intensity in YTHDF2-1253 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint positive CD8 T cells. n Representative multiplexable immunofluorescent staining of CD8 (red) and 1254 YTHDF2 (green) within B16F10-OVA tumors treated with cIg or anti -PD-1(n = 5 per group) . A dashed 1255 box represents the 4× enlarged area shown in the bottom panels with separate channels. White arrows 1256 point to cells positive for YTHDF2 and CD8. Scale bar, 10 μm. Middle panel, frequencies of YTHDF2 -1257 positive CD8 T cells. Right panel, quantification of the nuclear to cytoplasmic ratios of YTHDF2 intensity 1258 in YTHDF2-positive CD8 T cells. Error bars, mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-1259 tailed unpaired Student’s t-test (j, m, n). 1260 Supplementary Fig. 2 | Phenotyping for the YTHDF2 conditional knockout miceYTHDF2 1261 deprivation hinders antitumor T cell immune response. 1262 a Immunoblotting analysis of YTHDF2 expression in heart, lung, liver, stomach, pancreas, intestine and 1263 thymus tissues from Ythdf2F/F and Ythdf2CKO mice. b Protein expression of YTHDF2 in naï ve and 1264 activated CD8 T cells (anti -CD3/CD28, 5 μg/ml, 24 h) derived from Ythdf2F/F and Ythdf2CKO mouse 1265 spleens. c Flow cytometry analysis of immune cells within the thymuses, spleens, peripheral blood from 1266 Ythdf2F/F, dLckCre and Ythdf2CKO mice (n = 6 per group). d Quantification of YTHDF2 expression in B16-1267 OVA tumor-infiltrating CD8 T cells from Ythdf2F/F and Ythdf2CKO mice (n = 5 per group). e Female dLckCre 1268 (n = 5) and Ythdf2CKO (n = 5) mice were injected subcutaneously with 10 6 MC38 cells. Tumor growth 1269 was monitored ever 2 or 3 days. Error bars, mean ± s.e.m. *P < 0.05; *** P < 0.001; ****P < 0.0001. 1270 One-way (c) or two-way ANOVA (e) or two-tailed unpaired Student’s t-test (d). 1271 Supplementary Fig. 3 | YTHDF2 deprivation hinders antitumor T cell immune response. 1272 a TILs were isolated from Ythdf2F/F (n = 5) and Ythdf2CKO (n = 5) mice 12 days after MC38 tumor 1273 inoculation. Frequencies of immune subsets (CD8 T, CD4 T and Treg cells) within TILs and frequencies 1274 of PD -1+KILTFDRL-Dextramer+ CD8 T cell subpopulations were assessed by flow cytometry. b 1275 Numbers and frequencies of immune subsets within tumor -draining lymph nodes (dLN) from MC38 1276 tumor-bearing Ythdf2F/F (n = 6) and Ythdf2CKO (n = 6) mice. c TILs were isolated from Ythdf2F/F (n = 6) 1277 and Ythdf2CKO (n = 6) mice 10 days after Hepa1 -6 tumor inoculation. Frequencies of immune subsets 1278 (CD8 T, CD4 T and T reg cells) within TILs and frequencies of PD -1+KILTFDRL-Dextramer+ CD8 T cell 1279 subpopulations were assessed by flow cytometry. d Frequencies of PD -1+TIM3+CD101+ CD8 T cell 1280 subpopulations from Hepa1 -6 tumor-bearing Ythdf2F/F (n = 6) and Ythdf2CKO (n = 6) mice (Day 14). e 1281 MC38-bearing Ythdf2F/F (n = 5) and Ythdf2CKO (n = 5) mice were treated with anti-CD8 (200 μg/mouse) 1282 (left) or anti -CD4 antibody (200 μg/mouse) (right) and monitored for tumor growth. f Frequencies of 1283 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint CX3CR1+Tim3+CD101- CD8 T cell subpopulations positive for Gzm B, IFN-γ or Ki-67 from MC38 tumor-1284 bearing Ythdf2F/F (n = 6) and Ythdf2CKO (n =6) mice with anti-PD-1 treatment (D12). Error bars, mean ± 1285 s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Two-way ANOVA (e) or two-tailed unpaired 1286 Student’s t-test (a–d, f). 1287 Supplementary Fig. 4 | YTHDF2 maintains CD8 T cell expansion and activation in vitro. 1288 a–c Naï ve CD8 T cells isolated from Ythdf2F/F (n = 5) or Ythdf2CKO (n = 5) mice were stimulated with 1289 anti-CD3/CD28 (2.5 or 5 μg/ml as indicated) for the indicated time. Apoptosis of Ythdf2F/F or Ythdf2CKO 1290 CD8 T cells was measured by Annexin V and propidium iodide (PI) staining (a). Proliferation of Ythdf2F/F 1291 or Ythdf2CKO CD8 T cells was assessed by Celltracker V (CTV) dilution (b). Quantification of frequencies 1292 of IFN-γ+, GZM B+, and Ki-67+ subpopulations within activated Ythdf2F/F or Ythdf2CKO CD8 T cells (c). d 1293 Naï ve CD8 T cells isolated from Ythdf2F/F (n = 5) or Ythdf2CKO (n = 5) mice to induce T cell exhaustion 1294 by chronic stimulation (plates coated with anti-CD3, 5 mg/mL, 8 days). Quantification of frequencies of 1295 IFN-γ+, GZM B+, and Ki-67+ subpopulations within Ythdf2F/F or Ythdf2CKO CD8 T cells from in vitro T cell 1296 exhaustion assay. e Quantification of frequencies of CD62L +, CCR7+, and CD122 + subpopulations at 1297 Day 9 of transient stimulation (CD3/CD28 beads,1:1 beads-to-cells ratio, 72h) in Ythdf2F/F or Ythdf2CKO 1298 CD8 T cells from in vitro memory-like T cell induction assay (n = 6 per group). Error bars, mean ± s.e.m. 1299 *P < 0.05; **P < 0.01. Two-tailed unpaired Student’s t-test (a–e). 1300 Supplementary Fig. 5 | YTHDF2 deficiency impairs the mitochondria function of CD8 T cells. 1301 a–b Extracellular acidification rate (ECAR) levels (a) and oxygen consumption rate ( OCAR) levels of 1302 primed Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1 CD8 T cells (OVA, 10 nM, 72 h) were measured in real-time 1303 with Seahorse assay (n = 3 per group). c Transmission electron microscopic (TEM) analysis of 1304 mitochondrial morphology in activated Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1 CD8 T cells (OVA, 10 nM, 72 1305 h). Red arrows in the left panels show the position of mitochondria in respective higher magnification in 1306 the right panels. Scale bar, 200 nm. d–e Quantification of Ki-67 MFI (d), IFN-γ+, GZM B+ (e) frequencies 1307 among Ythdf2F/F and Ythdf2CKO CD8 T cells primed in the T cell exhaustion assay (plates coated with 1308 anti-CD3, 5 mg/mL, 8 days) with 10 mΜ NAC or veh (72 h) (n = 4 per group). Error bars, mean ± s.e.m. 1309 *P < 0.05. Two-tailed unpaired Student’s t-test (a–c). Two-way ANOVA (d, e). 1310 Supplementary Fig. 6 | The m 6A machinery is necessary for YTHDF2 -regulated mitochondrial 1311 fitness. 1312 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint a m6A-seq and RIP-seq tracks of Coa3, Mrpl16, Mrps12 and Tefm mRNA loci on primedYthdf2F/F and 1313 Ythdf2CKO CD8 T cells. b Activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) 1314 were treated with ActD (500 μg/ml) and RNAs were collected at different time points. Coa3, Mrpl16, 1315 Mrps12 and Tefm mRNA levels were measured using qPCR and represented as mRNA remaining after 1316 ActD treatment (n = 3 per group) . c Immunoblotting analysis of YTHDF2 expression in Jurkat -shCtrl 1317 and Jurkat -shYTHDF2 cells. d GO enrichment analysis of upregulated genes in Jurkat-shCtrl and 1318 Jurkat-shYTHDF2 cells. e–f Quantification of MitoSOX (e) and MG (f) MFI in Jurkat-shCtrl and Jurkat-1319 shYTHDF2 cells (n = 4 per group). g Immunoblotting analysis of YTHDF2 and Flag expression in Jurkat 1320 cells with or without YTHDF2 overexpression (OE) (n = 4 per group). h Immunoblotting analysis of Flag 1321 expression in Jurkat cells transduced with wild-type (OE) or mutant YTHDF2 (W432A and W486A) or 1322 empty vector lentiviruses. i–j Quantification of MitoSOX (i) and MG (j) MFI in Jurkat cells with or without 1323 YTHDF2 OE (n = 4 per group). k Quantification of the MO/MG ratio in Jurkat cells transduced with wild-1324 type or mutant YTHDF2 or empty vector lentiviruses (n = 3 per group). l Immunoblotting analysis of 1325 METTL3, YTHDF2 and Flag expression in YTHDF2 -OE Jurkat cells with or without knockdown of 1326 METTL3. m MitoSOX staining among METTL3 -knockdown and contr ol Jurkat cells with or without 1327 YTHDF2 overexpression (n = 3 per group). n Quantification of the MO/MG ratio among METTL3 -1328 knockdown and control Jurkat cells with or without YTHDF2 overexpression (n = 3 per group). Error 1329 bars, mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed unpaired Student’s t-test (f, i, j) or 1330 one-way ANOVA (k, m, n) or non-linear regression (b). 1331 Supplementary Fig. 7 | Acute T cell activation enlists nuclear functionality of YTHDF2. 1332 a–b Click-it RNA imaging and analysis of nascent RNA (green) synthesis in activated Ythdf2F/F or 1333 Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) (a) and activated Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1334 1 CD8 T cells (OVA, 10 nM, 24 h) (b). Scale bar, 10 μm. c Click-it RNA imaging and analysis of nascent 1335 RNA (green) synthesis in activated Ythdf2F/F or Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) 1336 in the presence of α -amanitin (2μg/mL, 12 h) or vehicle (n = 5 per group) . Scale bar, 10 μm. d 1337 Cumulative distribution of the fold change in translational efficiency of YTHDF2 -targeted and m 6A-1338 marked transcripts between activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 1339 h). e Ikzf1 and Ikzf3 mRNA levels detected by qPCR in naï ve or activated (anti-CD3/CD28, 5 μg/ml, 24 1340 h) CD8 T cells (n = 3 per group). f PLA analysis of YTHDF2 associated with IKZF1 or IKZF3 in 1341 unstimulated Jurkat cells. Scale bar, 10 μm. g Whole-cell lysates of activated WT CD8 T cells (anti -1342 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint CD3/CD28, 5 μg/ml, 24 h) were subjected to immunoprecipitation using anti -YTHDF2 antibody. The 1343 immunoprecipitants were incubated with RNase A (1 ug/ul) or DNase I (0.4 U/ul) DNase followed by 1344 immunoblot analysis. h–i Motif discovery analysis of the genomic sequences under A TAC-seq peaks 1345 conditioned by YTHDF2 depletion using HOMER. Red rectangle shows the IKZF1/3-binding motif within 1346 ATAC-sequenced Ythdf2F/F and Ythdf2CKO CD8 T cells (h) or Jurkat-shCtrl and Jurkat-shYTHDF2 cells 1347 (i). j ChIP-seq datasets for IKZF1 (GSM935442) in human T cells were obtained using Cistrome Data 1348 Browser. ATAC -seq profiles of Jurkat -shCtrl and Jurkat -shYthdf2 cells were represented on IKZF1 -1349 bound loci. Error bars, mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed unpaired Student’s 1350 t-test (a, b, e) or two-way ANOVA (c). 1351 Supplementary Fig. 8 | IKZF1/3 -associated transcriptional repression in YTHDF2 -deficient T 1352 cells 1353 a Quantitative flow cytometry analysis of IKZF1 (top) or IKZF3 (bottom) expression in activated Ythdf2F/F 1354 and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) (n = 3). b IKZF1 (top) or IKZF3 (bottom) 1355 CUT&RUN profiles of activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) 1356 were represented at the gene promoter regions. c PLA analysis of HDAC1 associated with IKZF1 or 1357 IKZF3 in activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h). Scale bar, 10 1358 μm. d Click-it RNA imaging and analysis of nascent RNA synthesis (red) in Jurkat -shCtrl, Jurkat -1359 shIKZF1/3 cells, Jurkat -shYTHDF2 cells and Jurkat -shYTHDF2/IKZF1/3 cells (n = 5 per group). e 1360 Immunoblotting analyses of IKZF1 and IKZF3 in Jurkat cells treated with len (10 or 100 μΜ) or veh for 1361 6 or 24 h. f Click-it RNA imaging and analysis of n ascent RNA synthesis (red) in Jurkat -shCtrl and 1362 Jurkat-shYTHDF2 cells in the presence of 100 μΜ len or veh for 24 h (n = 5 per group). g–h 1363 Representative confocal immunofluorescence images of IKZF1 (g) or IKZF3 (h) (green) and DAPI (blue) 1364 in activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) in the presence of 10 1365 μΜ len or veh (n = 5 per group). Scale bar, 10 μm. i Quantification of Tim3+ PD-1+ frequencies among 1366 primedYthdf2F/F and Ythdf2CKO CD8 T cells (anti -CD3/CD28, 5 μg/ml, 48 h) in the presence of 10 μΜ 1367 len or veh (n = 4 per group). j Stat5a (left) and Rasgrp1 (right) mRNA levels detected by qPCR in 1368 activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) in the presence of 10 μΜ 1369 len or veh (n = 3 per group). Error bars, mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed 1370 unpaired Student’s t-test (a). One (d) or two-way (f–j) ANOVA. 1371 Supplementary Fig. 9 | YTHDF2 distribution and expression are governed by the m6A machinery. 1372 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint a Venn diagram showing the overlap of putative YTHDF2 -target genes and IKZF1/3-target genes with 1373 both enhanced chromatin accessibility and reduced mRNA expression found in Ythdf2CKO CD8 T cells. 1374 b Immunoblotting analysis of METTL3 and GAPDH expression in ac tivated Mettl3F/F or Mettl3CKO CD8 1375 T cells (anti -CD3/CD28, 5 μg/ml, 24 h). c Female Mettl3F/F (n = 6) and Mettl3CKO (n = 6) mice were 1376 injected subcutaneously with 10 6 MC38 cells. Tumor growth was monitored ever 2 or 3 days. d TILs 1377 were isolated from Mettl3F/F (n = 5) and Mettl3CKO (n = 5) mice 12 days after MC38 tumor inoculation. 1378 Frequencies of CD8 T cell subpopulations positive for Gzm B, IFN -γ, or Ki-67 were assessed by flow 1379 cytometry. e Click-it RNA imaging and analysis of nascent RNA (red) synthesis in Jurkat cells introduced 1380 with WT or mutant YTHDF2. Scale bar, 10 μm (n = 5 per group). f Click-it RNA imaging and analysis of 1381 nascent RNA synthesis (red) in Jurkat -shCtrl and Jurkat -shYTHDF2 cells in the presence of 10 μΜ 1382 FB23-2 or veh for 72 h (n = 5 per group). Scale bar, 10 μm. g Click-it RNA imaging and analysis of 1383 nascent RNA synthesis (green) in Ythdf2F/F and Ythdf2CKO CD8 T cells primed in the presence of 10 μΜ 1384 FB23-2 or veh for 72 h (n = 5 per group). Scale bar, 10 μm. h Ythdf2 mRNA levels detected by qPCR 1385 in CD8 T cells stimulated with anti -CD3/CD28 (5 μg/ml) for the indicated amount of time (n = 3 per 1386 group). i m6A-seq and RIP-seq tracks of Ythdf2 mRNA loci on mouse CD8 T cells (left) and Jurkat cells 1387 (right). j RNA-seq tracks of Ythdf2 mRNA loci on primedYthdf2F/F and Ythdf2CKO CD8 T cells. Error bars, 1388 mean ± s.e.m. NS, no significance; *P < 0.05; ****P < 0.0001. Two-tailed unpaired Student’s t-test (d). 1389 One-way (e, h) or two-way ANOVA (c, f, g). 1390 Supplementary Fig. 10 | Schematic diagra m of YTHDF2 functioning in antitumor CD8 T cells. 1391 Contrary to the autoregulated Ythdf2 mRNA decay in a quiescent state, YTHDF2 protein is partially 1392 relocated and swiftly accumulated upon early CD8 T cell activation or reinvigoration. While cytoplasmic 1393 YTHDF2 degrades redundant mitochondrial component-encoding mRNAs to sustain T cell persistence, 1394 its nuclear translocation is likely to safeguard T cell effectiveness and ICB responsiveness by minimizing 1395 IKZF1/3-mediated transcriptional repression . Converse ly, YTHDF2 defect-associated ICB resistance 1396 could be overcome by targeting IKZF1/3. 1397 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Fig. 1 YTHDF2 is selectively upregulated and redistributed in early Teff and Teff-like cells Tumor-infiltrating CD8 T GSE114300 b YTHDF2 Lamin B GAPDH 0 24 0 24 hAnti-CD3/28 Cytosol Nucleus WT CD8 T 70 70 35 0 24 0 24 h g h GSE212357In vitro-generated CD8 T cell subsets a c d YTHDF2 D2 Teff D8 Tex Isotype YTHDF2 CD44+KLRG1+ CD44+KLRG1- CD44-KLRG1- Gating on CD8 T cells YTHDF2 Spleen CD8 Tumor CD4 Tumor CD8 Gating on CD3+ T cells PD-1+TCF1-KLRG1+ CD8 T cIg PD-1 0 1 2 3 4MFI of YTHDF2 (104) ✱✱✱ PD-1 cIg PD-1+TCF1+TIM3- CD8 T PD-1 cIg cIg PD-1 0 2 4 6 8 10 MFI of YTHDF2 (104) ✱✱ Gating on CD8 T cells YTHDF2 B16-OVA tumor D6 B16-OVA tumor D13 Tpex Tmem Tex Wtap Rbm15 Alkbh5 Fto Ythdf1 Ythdf2 Ythdf3 Igf2bp1 Igf2bp2 Igf2bp3 Ythdc1 Ythdc2 Mettl16 Mettl14 Mettl3 2 1 0 -1 -2 0h 3h 24h 48h 72h D6 D9 Exhaustion-like D9 Memory-like Wtap Rbm15 Alkbh5 Fto Ythdf1 Ythdf2 Ythdf3 Igf2bp1 Igf2bp2 Igf2bp3 Ythdc1 Ythdc2 Mettl16 Mettl14 Mettl3 cIg, progressing, early PD-1, regressing, early PD-1, progressing, early PD-1, progressing, late PD-1, partially regressing, late PD-1, progressing, late 2 1 0 -1 -2 YTHDF2YTHDF2 Spleen CD8 Tumor CD4 Tumor CD8 0.0 0.5 1.0 1.5 2.0 2.5 MFI of YTHDF2 (104) ✱✱✱ ✱✱ Tex Tmem Tpex 0 1 2 3MFI of YTHDF2 (104) ✱✱✱ ✱✱ CD44 - KLRG1 - CD44 + KLRG1 - CD44 + KLRG1 + 0 5 10 15 20 MFI of YTHDF2 (104) ✱✱✱ ✱✱ ✱✱ Tex Teff 0 5 10 15 MFI of YTHDF2 (104) ✱✱✱ e f B16-OVA tumor D13 j 48 240 12 DAPIYTHDF2 96h72 i Progressed Regressed 0.0 0.1 0.2 0.3 0.4 N/C ratio ✱ Progressed Regressed 0 10 20 30 YTHDF2+/ CD8 T (%) ✱ CD8 DAPIYTHDF2 Progressed YTHDF2 Lamin B GAPDH 0 24 0 24 hAnti-CD3/28 Cytosol Nucleus Human CD8 T 70 70 35 DAPIYTHDF2 cIgGanti-PD-1 cIgG anti-PD-1 0 10 20 30 40 Fluorescence intensity of YTHDF2 ✱✱✱ cIgG anti-PD-1 0 5 10 15 20 25 Nuclear YTHDF2+ (%) ✱✱✱ Regressing YTHDF2 Lamin B GAPDH OVA Cytosol Nucleus OT-1 CD8 T 70 70 35 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Fig. 2 YTHDF2 is essential for the antitumor effects of CD8 T cells h 0 10 20 30 0 500 1000 1500 2000 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F;OT-1 Ythdf2CKO;OT-1 *** ** **** B16F10-OVA 0 5 10 15 20 25 0 500 1000 1500 2000 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F Ythdf2CKO **** **** **** MC38B16F10 0 5 10 15 20 25 0 500 1000 1500 2000 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F Ythdf2CKO **** **** **** ****b c d e CD45 CD45 Ki-67 22.6 12.3 12.8 7.58 IFN-γ 21.1 12.7 Gzm B 46.2 32.6 Casp-3 Ythdf2F/F Ythdf2CKO Ythdf2F/F Ythdf2CKO MC38 TIL-CD8 T cells 46.2 0 5 10 15 20 25 0 500 1000 1500 2000 Days after tumor inoculation Tumor size (mm3) PBS Ythdf2F/F;OT-1 Ythdf2CKO;OT-1 ******** **** B16F10-OVAi 0 5 10 15 20 25 0 500 1000 1500 2000 2500 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F+cIg Ythdf2F/F+anti-PD-1 Ythdf2CKO+cIg Ythdf2CKO+anti-PD-1 **** ******* j 0 5 10 15 20 0 500 1000 1500 2000 2500 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F+cIg Ythdf2F/F+anti-PD-1 Ythdf2CKO+cIg Ythdf2CKO+anti-PD-1 **** **** MC38 Hepa1-6k Ythdf2F/F Ythdf2CKO CD8 T CD4 T Treg 0.0 0.5 1.0 1.5 2.0 2.5 Cell count / mg tumor mass （ 103） ✱ g a l Ythdf2F/F Ythdf2CKO Dextramer CD8 18.3 6.63 MC38 TIL-PD-1+ CD8 T cells TIM-3 CX3CR1 69.5 32.6 0 5 10 15 20 0 500 1000 1500 2000 2500 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F Ythdf2CKO **** **** **** Hepa1-6 f Casp-3 IFN- Gzm B Ki-67 0 10 20 30 40 50 Frequency within CD8 T cells (%) Ythdf2F/F Ythdf2CKO ✱✱✱ ✱ ✱✱ ✱✱ 30.1 18.2 CD44 KLRG1 Ythdf2 F/F Ythdf2 CKO 0 10 20 30 40 Frequency within CD8 T (%) ✱✱ Ythdf2F/F Ythdf2CKO MC38 TIL-CD8 T cells CD44+KLRG1+ CD101 TIM-3 4.79 10.1 Ythdf2 F/F Ythdf2 CKO 0 2 4 6 8 10 Frequency within CD8 T (%) ✱✱ Ythdf2F/F Ythdf2CKO PD-1+TIM-3+CD101+ MC38 TIL-PD-1+ CD8 T cells Dextramer CX3CR1 + TIM-3 + 0 20 40 60 Frequency within PD-1+ CD8 T (%) ✱ ✱✱ (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Mrpl58 1700021F05Rik Mrpl57 Uqcc2 Mrpl16 Slc25a33 Mrpl43 Mrpl51 Mrps2 Mrpl23 Mrps34 Mrpl12 Coa3 Mrps25 Mrpl52 Tsfm Mrps12 Mrps7 Tefm Chchd1 Mrps21 2 1 0 -1 -2 Putative targets Non-targets a 0 2 4 6 8 10 -Log10 P n=29 n=363 n=18 n=51 n=44 Mitochondrial respiratory chain complex assembly Metabolic process Ribosome biogenesis Translation Mitochondrion organization c b g e Fig. 3 YTHDF2 prevents mitochondrial stress and T cell exhaustion -log10P adj log2 (Ythdf2CKO/Ythdf2F/F) Downregulated genes (n=455) Upregulated genes (n=611) YTHDF2-target genes (n=511) i 0 10 20 30 40 Frequency within CD8 T (%) ✱ ✱✱✱PD-1+TIM3+ NACVeh NACVeh Ythdf2F/F Ythdf2CKO PD-1 TIM3 9.9922.4 14.033.3 15.1 7.6 11.5 6.61 Zombie NIR Count 0 5 10 15 20 Frequency within CD8 T (%) ✱ ✱✱✱ Viability Dye+ NAC − + − + NAC − + − + h Ythdf2 F/F Ythdf2 CKO 0 2 4 6 8MFI of mitoSOX red (104) ✱ Ythdf2 F/F Ythdf2 CKO 0.0 0.1 0.2 0.3 0.4 MO/MG (relative fold) ✱ d MO/MGlo MO/MGhi 9.4 87.9 20.8 74.8 Ythdf2F/F Ythdf2CKO f Ythdf2 F/F Ythdf2 CKO 0 1 2 3 4 5MFI of MG (103) ✱ Ythdf2 F/F Ythdf2 CKO 0 1 2 3MFI of mitoSOX red (103) ✱ MitoSOX Ythdf2F/F Ythdf2CKO MG Ythdf2F/F Ythdf2CKO MG MO MitoSOX CD8 Ythdf2F/F Ythdf2CKO NACVeh NACVeh Ythdf2F/F Ythdf2CKO Ythdf2CKOYthdf2F/F (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 0 2 4 6 8 10 -Log10 P n=81 n=64 n=39 n=17 n=5 n=13 a Positive regulation of transcription from RNA polymerase II promoter Response to hypoxia Histone H3-K4 methylation Transcription, DNA-templated Regulation of transcription, DNA-templated Covalent chromatin modification d e h CKO vs F/F Jurkat-KD vs Vec -log10FDR Log2FC 0 20 40 60 80 0-2.5-5.0 2.5 5.0 0 10 20 30 Log2FC 0-2.5-5.0 2.5 5.0 -log10FDR Up Down F/F CKO 0.0 0.5 1.0 1.5 Relative mRNA expression ✱✱✱ Rasgrp1 f g Center-2.0 2.0kb 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 IKZF1-bound loci Center-2.0 2.0kb 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00F/F1 F/F2 CKO1 CKO2 IKZF3-bound loci TSS-2.0 2.0kb TSS-2.0 2.0kb IKZF3-associated promotersIKZF1-associated promoters F/F CKO 0.15 0.20 0.25 0.35 0.30 0.15 0.20 0.25 0.35 0.30 Normalized density (CPM) Normalized density (CPM) i Fig. 4 YTHDF2 segregates IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T cells c b 70 70 IgG YTHDF2 IP Input IB: IKZF1 IP Input 70 70 IB: IKZF3 IgG YTHDF2 70 70IP Input IB: YTHDF2 IgG YTHDF2 70 70 IgG IKZF1 IP Input IB: YTHDF2 j Ythdf2CKOYthdf2F/F 210-1-2 Kdm5b Kmt2e Kat2b Kdm5a Tet2 Kmt2a Ash1l Kmt2d Ezh1 Sox4 Klf7 Stat5a Kdm7a Setd1a Kdm3a Foxo3 Nfat5 Il12a Cd226 Ptprf Map3k8 Ptpn4 Hivep2 Rasgrp1 Tnfaip3 Epigenetic regulators Transcriptional factors TCR signal engagers Stat5a F/F CKO 0.0 0.5 1.0 1.5 Relative mRNA expression ✱✱✱ Stat5a ATAC-seq CKO1 CKO2 F/F1 F/F2 CKO F/F H3K4me- CUT&RUN Rasgrp1 ATAC-seq CKO1 CKO2 F/F1 F/F2 CKO F/F H3K4me- CUT&RUN IgG IKZF3 70 70 IP Input IB: YTHDF2 Flow 70 70 70 YTHDF2 IKZF1 IKZF3 IgG YTHDF2 YTHDF2/IKZF1 WT CD8 T OT-1 CD8 T YTHDF2/IKZF3 Human CD8 T (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 0 5 8 11 13 16 19 0 500 1000 1500 2000 Days after tumor inoculation Mean tumor size (mm3) Ythdf2F/F+anti-PD-1+veh Ythdf2CKO+anti-PD-1+veh Ythdf2F/F+anti-PD-1+ len Ythdf2CKO+anti-PD-1+ len ******** **** 0 20 40 60 80 Fluorescence intensity ✱✱✱ ✱✱✱ NS F/F;OT-1 CKO;OT-1 EU DAPI c 0 1 2 3Ki-67 MFI (104) ✱✱ ✱✱ NS d Veh Len Hepa1-6 g F/F;OT-1 CKO;OT-1 a b f F/F CKO EU 0 20 40 60 80 100 Fluorescence intensity ✱✱✱ ✱✱✱ NS 0 1 2 3 4Ki-67 MFI (104) ✱✱ ✱✱✱ NS F/F CKO DAPI 0 6 9 12 15 17 21 24 27 0 500 1000 1500 2000 Days after tumor inoculation Mean tumor size (mm3) Ythdf2F/F+anti-PD-1+veh Ythdf2CKO+anti-PD-1+veh Ythdf2F/F+anti-PD-1+len Ythdf2CKO+anti-PD-1+len **** Ythdf2F/F+len Ythdf2CKO+len **** **** **** Len − + − + Len − + − + Len − + − + Len − + − + MC38 Fig. 5 Lenalidomide retrieves the anti-tumor function of YTHDF2-deficient CD8 T cells Veh Len e h Len − + − + 0 20 40 60 80 Frequency within CD8 T (%) ✱✱✱ ✱✱ ✱ Len − + − + Gzm B 0 20 40 60 Frequency within CD8 T (%) ✱✱✱ ✱✱ NSIFN-γ Ki-67 Veh Len Len Veh CKO;OT-1 Ki-67 F/F CKO IFN-γ CD8 50.840.1 47.015.4 LenVeh LenVeh Ythdf2F/F Ythdf2CKO 50.4 66.9 52.114.0 Gzm B CD8 Veh Len Len Veh F/F;OT-1 27.810.1 28.1 anti-PD-1+lenanti-PD-1+veh Ythdf2CKO Gzm B CD8 Ythdf2F/F anti-PD-1+len 24.6 anti-PD-1+veh 13.6 37.0 48.5 20.5 IFN-γ CD8 anti-PD-1 Combo anti-PD-1 Combo 0 10 20 30 Frequency within PD-1+ CD8 T (%) ✱✱✱ ✱✱ Gzm B anti-PD-1 Combo anti-PD-1 Combo 0 20 40 60 Frequency within PD-1+ CD8 T (%) ✱✱✱ ✱IFN-γ F/F CKO F/F CKO (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint a c YTHDF2/IKZF1 shCtrl shMETTL3 #1 shMETTL3 #2 e GAPDH YTHDF2 Cytoplasm Nucleus Lamin B 70 70 35 f 0 1 2 3 4 5Ki-67 MFI (104) ✱✱✱ ✱✱ ✱✱ Ctrl YTHDF2 OE shMETTL3 YTHDF2 OE + shMETTL3 Ki-67 YTHDF2 OE YTHDF2 OE +shMETTL3 shMETTL3 Ctrl EU DAPI GFP Merge Fig. 6 The m6A machinery regulates both YTHDF2 relocation and expression OE W432A W486A Flag/IKZF1 d 0 5 10 15 20 25 Fluorescence intensity ✱✱✱ ✱✱ ✱ j k 0 6h 0 2 4 6 8Relative mRNA expression NS✱✱✱ ✱✱✱ ✱✱Ythdf2 WT -249 WT -249 WT -249 WT -249 WT -249 0 24h 0 24h Cytoplasm Nucleus GAPDH YTHDF2 Lamin B l 70 70 35 0 1 2 3Relative mRNA expression ✱✱✱ ✱ CD3/28: − + − + ActD: − − + + b g ih Ythdf2 ATAC-seq 0 h_1 0 h_2 0 h_3 5 h_1 5 h_2 5 h_3 ActDVeh Mettl3F/F Mettl3CKO YTHDF2/IKZF1YTHDF2/IKZF1 Ythdf2 t1/2=2.1100 R2=0.8630 t1/2=1.2520 R2=0.9006 0 1 2 3 4 0.0 0.5 1.0 Ythdf2 mRNA remaining -249 WT P= 0.009 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint PR SD CR PD/SD PR/CR 0 2 4 6 8MFI of YTHDF2 in CD8 T cells ✱ CD8 DAPIYTHDF2 PD/SD PR/CR 0 5 10 15 20 CD8 T count / HPF (100×) P = 0.2272 PR CR SD PD/SD PR/CR 0 5 10 15 YTHDF2+/ CD8 T cells (%) ✱ PD/SD PR/CR 0 5 10 15 20 CD8 T count / HPF (100×) P =0.6518 e f d h i j Fig. 7 YTHDF2 expression are associated with T cell function in human cancers PD/SD PR/CR 0 10 20 30 40 YTHDF2+/ CD8 T cells (%) ✱✱ PD/SD PR/CR 0 2 4 6 8 10 MFI of YTHDF2 in CD8 T cells ✱ CD8 DAPIYTHDF2 g k a CRC (GSE146771) HCC (GSE206325)PDAC (GSE155698) Responder Non-responder 1.0 1.5 2.0 2.5 1.0 2.0 1.0 2.0 3.0 YTHDF2 expression level YTHDF2 expression level YTHDF2 expression level High Low P = 8.01e−4 P = 1.59e−11 P = 2.99e−04 0 3 1 2 4 YTHDF2 expression level P = 3.98e−24 P = 3.41e−291 0 3 1 2 4 YTHDF2 expression level P = 0.368 P = 1.45e−163 Melanoma (GSE120575) b High Low High Low Pre-treatment YTHDF2 expression level 1.0 1.5 2.0 2.5 0.5 Non-responder Responder P = 4.06e−1 P = 4.04e−4 Post-treatment 1.0 1.5 2.0 2.5 0.5 YTHDF2 expression level Non-responder Responder HCC (GSE206325) 1.0 2.0 3.0 YTHDF2 expression level Non-Responder Responder P = 8.58e−45 c (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint GSE29797 Supplementary Fig. 1 YTHDF2 is characteristically expressed upon T cell activation and reinvigoration a b c GSE132477 Mouse CD4 T α-CD3/28: 0 4hα-CD3/28: 0 4 8h Mouse CD8 T GSE70393 Wtap Rbm15 Alkbh5 Fto Ythdf1 Ythdf2 Ythdf3 Igf2bp1 Igf2bp2 Igf2bp3 Ythdc1 Ythdc2 Mettl16 Mettl14 Mettl3 Wtap Rbm15 Alkbh5 Fto Ythdf1 Ythdf2 Ythdf3 Igf2bp1 Igf2bp3 Ythdc1 Ythdc2 Mettl16 Mettl14 Mettl3 Wtap Rbm15 Alkbh5 Fto Ythdf1 Ythdf2 Ythdf3 Igf2bp1 Igf2bp2 Igf2bp3 Ythdc1 Ythdc2 Mettl14 Mettl3 Naïve Active 0.30 0.35 0.40 0.45 0.50 m6A/A in mRNA (%) ✱✱ g h Mouse CD4 T cell subsets f YTHDF2 β-Actin OVA: 0 6 12 24h β-Actin YTHDF2 α-CD3/28: 0 6 12 24h Mouse CD8 T cells e YTHDF2 β-Actin METTL14 FTO ALKBH5 METTL3 Human CD8 T cells α-CD3/28: 0 6 12 24h GSE110249 d Mettl3 Mettl14 Mettl16 Wtap Rbm15 Alkbh5 Fto Ythdf1 Ythdf3 Igf2bp1 Igf2bp2 Igf2bp3 Ythdc1 Ythdc2 0 20 40 60 mRNA (FKPM) OVA-specific CD8 TIL cIg Anti-PD-1 k l NucleusCytoplasm PHA: 0 24 0 24 h β-actin Lamin B YTHDF2 70 70 45 Naive Active 5’UTR 3’UTRCDS Region 0.0 0.5 1.0Density Naï ve P = 1×10-620 (n = 8166) Active P = 1×10-870 (n = 10302) m n B16F10 B16F10-OVA 0 5 10 15 20 25 YTHDF2+/ CD8 T (%) ✱ B16F10 B16F10-OVA 0.0 0.1 0.2 0.3 0.4 N/C ratio ✱ anti-CD3/28: 0 12 24 48 hi j 0 12 24 48 72 96h 0 10 20 30 40 Nuclear YTHDF2+ (%) ✱✱ ✱✱✱ 0 12 24 48 72 96h 0 1 2 3Fluorescence intensity ✱ ✱✱ ✱✱ cIg anti-PD-1 0 10 20 30 40 YTHDF2+/ CD8 T cells (%) ✱ cIg anti-PD-1 0.0 0.1 0.2 0.3 0.4 0.5 N/C ratio B16F10-OVA + cIg B16F10-OVA + anti-PD-1 CD8 DAPIYTHDF2 DAPIYTHDF2 CD8 DAPIYTHDF2 B16F10 B16F10-OVA (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint F/F CKO F/F CKO F/F CKO F/F CKO F/F CKO F/F CKO F/F CKO Heart Lung Liver Stomach Pancreas Intestine Thymus YTHDF2 β-Actin Ythdf2F/F;dLckcre (T cell CKO) mice YTHDF2 β-actin Splenic CD8 T cells 0 24h F/F CKO F/F CKO a b c Spleen Peripheral blood d Supplementary Fig. 2 Phenotyping for the YTHDF2 conditional knockout mice Ythdf2F/F Ythdf2CKO dLckCre CD3 + T CD8 + T CD4 + T Treg 0 100 200 300 400 Cell counts (103) CD3 + T CD8 + T CD4 + T Treg 0 20 40 60 Cell counts (105) CD3 + T CD8 + T CD4 + T Treg 0 1000 2000 3000 4000 5000 Cell counts ✱ ✱ Thymus 4.51% 100% YTHDF2 Gating on CD8 T cells B16-OVA tumor D13 Ythdf2 F/F Ythdf2 CKO 0 50 100 150 YTHDF2+ (%) ✱✱✱ Ythdf2F/F Ythdf2CKO e MC38 0 5 10 15 20 25 0 500 1000 1500 Days after tumor inoculation Tumor size (mm3) dLckCre Ythdf2CKO **** **** **** **** (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint a b CD8 T CD4 T 0.0 0.2 0.4 0.6 0.8 1.0 Cell count (106) ✱ ✱ MC38 dLN CD8 T CD4 T 0 10 20 30 40 50 Frequency within CD45 cells (%) ✱ CD8 T CD4 T Treg 0.0 0.5 1.0 1.5 2.0 Cell count / mg tumor mass （ 103） ✱ ✱✱✱ c Hepa1-6 TIL MC38 TIL CD8 T CD4 T Treg 0 10 20 30 40 50 Frequency within CD45 cells (%) e IFN-γ CD8 50.0 70.1 87.3 92.5 Gzm B 21.2 30.1 Ki-67 MC38 TIL-CX3CR1+PD-1+TIM-3+ CD8 T cells Ythdf2CKO Ythdf2F/F CD8+ PD-1+ Dextramer+T cells d T cellsT cells Ythdf2 F/F Ythdf2 CKO 0 1 2 3Frequency within CD8 T (%) ✱✱ CD101 PD-1 5.20 11.2 Ythdf2 F/F Ythdf2 CKO 0.0 0.5 1.0 1.5 2.0 2.5 Frequency within CD8 T (%) ✱✱ Ythdf2F/F Ythdf2CKO PD-1+TIM-3+ CD8 T cells Hepa1-6 TIL- IFN-γ Gzm B Ki-67 0 10 20 30 40 50 Frequency within CD8 T (%) ✱✱✱ ✱✱✱ ✱ CD8 T CD4 T Treg 0 5 10 15 20 25 Frequency within CD45 cells (%) Supplementary Fig. 3 YTHDF2 deprivation hinders antitumor CD8 T cell response f 0 5 10 15 20 25 0 500 1000 1500 2000 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F+anti-CD8 Ythdf2CKO+anti-CD8 NS 0 5 10 15 20 25 0 500 1000 1500 Days after tumor inoculation Tumor size (mm3) Ythdf2F/F+anti-CD4 Ythdf2CKO+anti-CD4 **** (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint 24 72 120h 0 20 40 60 80 Frequency within CD8 T (%) ✱ ✱ ✱ 24 72 120h 0 5 10 15 20 25 Frequency within CD8 T (%) Annexin V PI Ythdf2CKO Ythdf2F/F α-CD3/28: 5 μg/ml, 24 72 120h 0 24 72 120h 0 50 100 150 Celltrackerlo CD8 T (%) ✱ ✱ Ythdf2CKO α-CD3/28: 2.5 μg/ml, 0 24 72 120h Ythdf2CKO IFN-γ Gzm B Ki-67 Annexin Ⅴ+PI+ Annexin Ⅴ+PI- Ythdf2F/F Ythdf2F/F 37.7 10.9 25.1 9.67 53.4 22.0 62.6 14.9 36.7 11.3 29.4 13.0 0.02 0.015 0.12 9.83 11.8 72.6 79.1 96.7 CTV IFN-γ CountCD8 Celltrackerlo 9.64 23.1 15.1 46.4 16.3 55.3 Gzm B Ki-67 a b c d Ythdf2F/F Ythdf2CKO Supplementary Fig. 4 YTHDF2 maintains CD8 T cell function and persistence in vitro Ythdf2CKO PD-1 TIM-3 CD101 Ythdf2F/F 54.418.4 TIM-3 CD101 Ythdf2 F/F Ythdf2 CKO 0 10 20 30 40 Frequency within CD8 T (%) ✱✱ 26.8 Ythdf2 F/F Ythdf2 CKO 0 20 40 60 80 100 Frequency within CD8 T (%) ✱✱ PD-1 CD8 79.3 91.6 Ythdf2 F/F Ythdf2 CKO 0 20 40 60 80 100 Frequency within CD8 T (%) ✱✱ 84.7 Ythdf2 F/F Ythdf2 CKO 0 10 20 30 Frequency within CD8 T (%) ✱✱ Ythdf2 F/F Ythdf2 CKO 0 20 40 60 Frequency within CD8 T (%) ✱✱ Ythdf2 F/F Ythdf2 CKO 0 20 40 60 Frequency within CD8 T (%) ✱✱ 22.2 22.0 CD122 86.9 87.1 CCR7 88.6 88.4 CD62L Ythdf2CKO Ythdf2F/F CD8 Ythdf2 F/F Ythdf2 CKO 0 20 40 60 80 100 Frequency within CD8 T (%) Ythdf2 F/F Ythdf2 CKO 0 20 40 60 80 100 Frequency within CD8 T (%) Ythdf2 F/F Ythdf2 CKO 0 10 20 30 40 Frequency within CD8 T (%) CD62L CCR7 CD122 e (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Supplementary Fig. 5 YTHDF2 deficiency impairs the mitochondria function of CD8 T cells b Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 80 100 120 140 Basal OCR (pmol/min) Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 0 50 100 150 200 Maxima OCR (pmol/min) Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 0 20 40 60 Spare Respiratory Capacity (maximal OCR - basal OCR) 0 20 40 60 80 0 50 100 150 200 Time (minutes) OCR (pmol/min) Ythdf2F/F;OT-1 Ythdf2CKO;OT-1 Oligo FCCP Rot a 0 20 40 60 80 0 50 100 150 Time (minutes) ECAR (mpH/min) Ythdf2F/F;OT-1 Ythdf2CKO;OT-1 Glucose Oligo 2-DG Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 0 20 40 60 Glycolysis (pmol/min) ✱ Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 0 20 40 60 80 100 Glycolytic Capacity (pmol/min) ✱ Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 0 10 20 30 40 50 Glycolytic Reserve (pmol/min) ✱ Ythdf2 F/F ;OT-1 Ythdf2 CKO ;OT-1 0 10 20 30 No. of mitochondria / cell ✱ Ythdf2F/F;OT-1 Ythdf2CKO;OT-1 c d IFN-γ CD8 NACVeh NACVeh Ythdf2F/F Ythdf2CKO 14.1 10,4 39.9 60.7 52.0 66.9 12.8 13.5 Gzm B CD8 0 20 40 60 80 Frequency within CD8 T (%) ✱ ✱ NAC − + − + IFN-γ 0 20 40 60 80 Frequency within CD8 T (%) ✱ ✱ NAC − + − + Gzm B 0 2 4 6 8MFI of Ki-67 (104) ✱ ✱ NAC − + − + Ki-67 Ki-67 F/F CKO Veh NAC NAC Veh e (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint c YTHDF2 Flag GAPDH Vec OE 70 35 70 k m YTHDF2 GAPDH 70 100 35 g 0 10 20 30 40 MFI of mitoSOX red (104) ✱✱✱ ✱ ✱✱✱ ✱✱ ✱ n 0.0 0.5 1.0 1.5 2.0 MO/MG (relative fold) ✱✱ ✱✱ ✱✱✱ Vec OE (WT) W432A W486A 0 1 2 3MO/MG (relative fold) ✱✱✱ ✱ ✱ Supplementary Fig. 6 The m6A machinery is necessary for YTHDF2-regulated mitochondrial fitness shMETTL3 shCtrl YTHDF2 OE Flag METTL3 GAPDH YTHDF2 70 70 35 75 YTHDF2 OE+ shMETTL3 l W486A W432A OE Vec 70Flag h GAPDH 35 d 0 5 10 15 -Log10 P rRNA processing n = 72 rRNA metabolic process n = 89 Regulation of T cell activation n = 69 Mitochondrial gene expression n = 43 Ribosome biogenesis n = 92 i j e f Ctrl OE Mitosox Vec ctrl YTHDF2 OE 0 5 10 15 20 MFI of mitoSOX red (104) ✱✱✱ shCtrl shYTHDF2 0 2 4 6 8MFI of mitoSOX red (104) ✱✱✱ shCtrl shYTHDF2 Mitosox Vec ctrl YTHDF2 OE 0 2 4 6 8 10 MFI of MitoTracker Green (104) ✱ MG shCtrl shYTHDF2 shCtrl shYTHDF2 0 1 2 3 4MFI of MitoTracker Green (105) ✱✱ MG Ctrl OE Coa3 Mrpl16 Mrps12 Tefm Input m6A-IP YTHDF2-RIP a b 0 1 2 3 4 0.0 0.5 1.0 Coa3 mRNA remaining P= 0.0304 Coa3 t1/2=2.456 R2=0.9456 t1/2=1.987 R2=0.9330 0 1 2 3 4 0.0 0.5 1.0 Mrps12 mRNA remaining P= 0.0092 Mrps12 t1/2=0.8354 R2=0.9847 t1/2=0.5350 R2=0.9933 0 1 2 3 4 0.0 0.5 1.0 Mrpl16 mRNA remaining P= 0.0086 Mrpl16 t1/2=0.6400 R2=0.9637 t1/2=0.3711 R2=0.9813 Tefm t1/2=0.4067 R2=0.9581 t1/2=0.2539 R2=0.9920 0 1 2 3 4 0.0 0.5 1.0 Tefm mRNA remaining Ythdf2CKO Ythdf2F/FP= 0.0017 (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint F/F CKO a b F/F CKO 0 20 40 60 80 Fluorescence intensity ✱✱✱ F/F;OT-1 CKO;OT-1 F/F;OT-1 CKO;OT-1 0 20 40 60 80 Fluorescence intensity ✱✱✱ EU DAPI EU DAPI d f e h i j Center-2.0 2.0kb 0.2 0.4 0.6 0.8 1.0 Vec1 Vec2 KD1 KD2 IKZF1-bound loci Normalized density (CPM) Supplementary Fig. 7 Acute T cell activation enlists nuclear functionality of YTHDF2 Ythdf2F/F Ythdf2CKO 0 10 20 30Fluorescence intensity ns ✱✱✱ ✱ α-AMN: − + − + c Cumulative fraction Log2(TE) Putative target genes 0.00 0.25 0.50 0.75 1.00 -5 0 5 CKO F/F IP IgG YTHDF2 IKZF1 IKZF3 DNase RNase - - + - + - YTHDF2 g F/F CKO EU Vehα-AMN DAPI YTHDF2/IKZF1 Jurkat YTHDF2/IKZF3 0 24h 0.0 0.5 1.0 1.5 Relative mRNA expression ✱✱ 0 24h 0.0 0.5 1.0 1.5 Relative mRNA expression ✱✱ Ikzf1 Ikzf3 (which was not certified by peer review) is the author/funder. All rights reserved. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint Vec shIKZF1/3 shYTHDF2 EU DAPI shYTHDF2/ IKZF1/3 d 0 10 20 30 40 Fluorescence intensity ✱ ✱ shCtrl shYTHDF2 Veh Len 0 10 20 30Fluorescence intensity ✱ ✱ NS f EU DAPI Len − + − + shCtrl shYTHDF2 LenVeh LenVeh Ythdf2F/F Ythdf2CKO PD-1 TIM3 23.0 22.7 31.7 30.2 0 10 20 30 40 Frequency within CD8 T (%) Len − + − + PD-1+TIM3+ i Supplementary Fig. 8 IKZF1/3-associated transcriptional repression in YTHDF2-deficient T cells HDAC1/IKZF1HDAC1/IKZF3 Ythdf2F/F Ythdf2CKO c e j IKZF1 Ythdf2 F/F Ythdf2 CKO 0 5 10 15 20 MFI of IKZF1 (104) NS IKZF3 Ythdf2 F/F Ythdf2 CKO 0 1 2 3 4 5MFI of IKZF3 (104) NS a b TSS-3.0 3.0kb IKZF1 CUT&RUN 15 20 10 IKZF3 CUT&RUN TSS-3.0 3.0kb 10 12 8 6 4 F/F CKO F/F CKO IKZF1 IKZF3 GAPDH Jurkat Len - 100 100 10 10 µ m 24 6 24 6 h 55 70 55 40 35 KD F/F CKO Veh Len IKZF3 DAPI F/F CKO Veh Len IKZF1 DAPI 0 2 4 6 8 10Fluorescence intensity of IKZF3 ✱✱✱ ✱✱✱ NS Len − + − + 0 5 10 15Fluorescence intensity of IKZF1 ✱✱✱ ✱✱✱ NSLen − + − + g h Normalized density Normalized density 0.0 0.5 1.0 1.5 Relative mRNA Experssion(fold) ✱✱✱ ✱✱✱ Len − + − + Stat5a Rasgrp1 0.0 0.5 1.0 1.5 Relative mRNA Experssion(fold) ✱✱✱ ✱✱✱ Len − + − + (which was not certified by peer review) is the author/funder. 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The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint b c W432A W486A OE (WT) Vec EU DAPI GFP Merge a Vec OE W432A W486A 0 10 20 30 40 Fluorescence intensity ✱ ✱✱ ✱ e IFN- Gzm B Ki-67 0 5 10 15 20 25 Frequency within CD8 T cells (%) Mettl3F/F Mettl3CKO ✱ ✱✱ ✱✱ 0 5 10 15 20 25 0 500 1000 1500 2000 Days after tumor inoculation Tumor size (mm3) Mettl3F/F Mettl3CKO **** **** **** * d METTL3 GAPDH 35 75 shCtrl shYTHDF2 Veh FB23-2 10 15 20 25 30 Fluorescence intensity NS NS EU DAPI FB23-2 − + − + shCtrl shYTHDF2 F/F CKO 0 20 40 60 80 Fluorescence intensity NS NS FB23-2 − + − + Veh FB23-2 EU DAPI F/F CKO f g ATAC-seq_Up IKZF1/3-target RNA-seq_Down m6A-Me-RIP YTHDF2-RIP 230 Supplementary Fig. 9 YTHDF2 distribution and expression are governed by the m6A machinery Ythdf2 m6A-seq IP1 IP2 Input1 Input2 RIP-seq IP1 IP2 Input1 Input2 i 0 6 12 24h 0.0 0.5 1.0 1.5 2.0 2.5 Relative mRNA expression **** **** * Ythdf2 h Ythdf2 j Ythdf2 CKO1 CKO2 F/F1 F/F2 Mouse CD8 T cells Jurkat cells (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint AAAAA Tn or Tpex Teff-like AAAAA Lenalidomide IKZF1/3 Mitochondrion- related genes Teff or Teff-like Ythdf2 mRNA AAAAA YTHDF2 m6A AAAAA Ythdf2 Nucleus AAAAA Mitochondrial fitness & T cell persistence Antigen or ICB AAAAA Stat5a Rasgrp1 IKZF1/3 IKZF1/3 Mitochondrial molecules YTHDF2-deficient YTHDF2-competent T cell polyfunctionality Mitochondrial stress & T cell exhaustion Active chromatin Inactive chromatin * * * m6A AAAAA T cell polyfunctionality T cell polyfunctionality Teff or Teff-like Active chromatin ICB + (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint