{"paper_id":"41f6eba4-a074-4660-b641-bc60f81cd87d","body_text":"YTHDF2 upregulation and relocation dictate CD8 T cell polyfunctionality in tumor immunity 1 \n 2 \nAuthors: Haiyan Zhang1,11, Xiaojing Luo 2,3,11, Wei Yang 2,4, Zhiying Wu 2,3, Zhicong Zhao 5,10, Xin Pei1, 3 \nXue Zhang1, Chonghao Chen1, Josh Haipeng Lei1, Qingxia Shi1, Qi Zhao2,3, Yanxing Chen2,3, Wenwei 4 \nWu2,3, Zhaolei Zeng2,3, Huai-Qiang Ju2,3, Miaozhen Qiu2,3, Jun Liu6, Bin Shen7, Minshan Chen2,8, Jianjun 5 \nChen5, Chu-Xia Deng1,4, Rui-Hua Xu2,3,9*, Jiajie Hou1,4,8* 6 \n 7 \nAffiliations: 8 \n1Cancer Center, Faculty of Health Sciences, University of Macau, Macau SAR, China ; MOE Frontier 9 \nScience Center for Precision Oncology, University of Macau, Macau SAR, China  10 \n2State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, 11 \nSun Yat-sen University Cancer Center, Guangzhou 510060, China 12 \n3Research Unit of Precision Diagnosis and Treatment for Gastrointestinal Cancer, Chinese Aca demy 13 \nof Medical Sciences, Guangzhou 510060, China 14 \n4Translational Research Center, Zhuhai UM Science & Technology Research Institute, Zhuhai 519031, 15 \nChina 16 \n5Department of Systems Biology, The Beckman Research Institute of City of Hope, Duarte, CA91010, 17 \nUSA 18 \n6Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China  19 \n7State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166, China  20 \n8Department of Liver Surgery, Sun Yat-sen University Cancer Center, Guangzhou 510060, China  21 \n9Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou 510060, China 22 \n10Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 23 \nShanghai 200127, China 24 \n11Co-first authors 25 \n*Correspondence: xurh@sysucc.org.cn (R.-H.X.); jiajiehou@um.edu.mo (J.H.) 26 \n 27 \n 28 \n 29 \n 30 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nAbstract 31 \nEpigenetic traits impact the antitumor function of CD8 T cells, yet whether and how RNA methylation 32 \nprograms engage in T cell immunity is poorly understood. Here we show that  the N6-methyladenosine 33 \n(m6A) RNA reader YTHDF2 is highly expressed in early effector or effector -like CD8 T  cells and is 34 \npartially distributed in the nucleus. YTHDF2 loss in T cells exacerbates tumor progression and confers 35 \nunresponsiveness to PD -1 blockade in mice and humans. In addition to  initiating RNA decay for 36 \nmitochondrial fitness, YTHDF2 can orchestrate chromatin regulation to promote T cell polyfunctionality. 37 \nYTHDF2-mediatd preservation of gene transcription arises from the interaction of YTHDF2 with IKZF1/3. 38 \nAccordingly, immunotherapy-induced efficacy could be largely restored in YTHDF2 -deficient T cells 39 \nthrough combinational use of lenalidomide. Moreover, m 6A recognition is fundamental for YTHDF2 40 \ntranslocation to the nucleus and autoregulation at the RNA level. Thus, YTHDF2 coordinates 41 \nepitranscriptional and transcriptional networks to potentiate T cell immunity. 42 \n 43 \nKeywords: YTHDF2; Teff and Teff-like cells; Immunotherapy; Lenalidomide 44 \n 45 \nHighlights: 46 \n• YTHDF2 expression and distribution underpin the threshold for bona fide CD8 T cell effector 47 \nresponse 48 \n• Canonical YTHDF2 -mRNA decay pathway alleviates mitochondrial stress and CD8 T cell 49 \nexhaustion 50 \n• Nuclear YTHDF2 sequesters IKZF1/3-mediated transcriptional repression to safeguard CD8 T 51 \ncell polyfunctionality 52 \n• The tumoricidal activity of YTHDF2-deficient CD8 T cells could be repaired through the synergy 53 \nof anti-PD-1 and lenalidomide 54 \n 55 \n 56 \n 57 \n 58 \n 59 \n 60 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nIntroduction 61 \nAmong more than 170 types of RNA modifications, N6-methyladenosine (m 6A) represents the most 62 \nprevalent and abundant modification in eukaryotic mRNA. By enlisting the “writer” (methyltransferase), 63 \n“eraser” (demethylase) and “reader” proteins, dynamic m 6A modification regulates nearly every step of 64 \nmRNA metabolism and interferes with various biological processes 1. Emerging evidence shows that 65 \nm6A RNA modifiers within tumor cells or surrounding myeloid cells largely affect tumor immunity and 66 \nimmunotherapy efficacy2,3. For instance, the tumor-intrinsic m6A demethylase FTO can either subvert 67 \nthe host immune reaction by facilitating the expres sion of the immune checkpoint gene LILRB41 or 68 \nrestrict T cell activation by altering the metabolic microenvironment 4. The methyltransferase complex 69 \ncomponent METTL14 disrupts the interferon-γ signalling within microsatellite instability-low tumors and 70 \nlimits the response to immune checkpoint blockade (ICB) therapy 5; however, METTL14 governs m6A 71 \nRNA stabilization in a subpopulation of tumor-associated macrophages to prevent T cell dysfu nction6. 72 \nThe m 6A reader YTH domain family 1 (YTHDF1) in classic dendritic cells impedes the cross -73 \npresentation of tumor antigens and the cross-priming of CD8 T cells 7. Two recent reports showed that 74 \nmyeloid cell YTHDF2 is also associated with tumor immunosuppression8,9. These findings indicate that 75 \nthe m 6A machinery posttranscriptionally controls cancer -immune set points, which may be useful  76 \ntargets for overcoming immunotherapy resistance. 77 \n  Tumor-reactive CD8 T cells are key to both natural and therapy-induced antitumor immunity. However, 78 \nchronic exposure to tumor antigens or environmental stimuli may render CD8 T cells dysfunctional and 79 \nlimit the outcomes of cancer therapy 10,11. Remarkably, T cell activatio n and differentiation are often 80 \nconcomitant with epigenetic processes, many of which account for the molecular rewiring of T cell 81 \ndysfunction imposed by the tumor microenvironment (TME)12. Therefore, manipulating T cell epigenetic 82 \nprograms may foster cancer therapeutic efficacy owing to the acquisition of long -term T cell 83 \npersistence13,14. Although epigenetic changes  accompanying differentiation  have been extensively 84 \nstudied15, how T cell effector polyfunctionality is epigenetically safeguarded in the early phase remains 85 \nelusive. In addition, recent insights have revealed that a progenitor exhausted T (T pex) cell population 86 \ncan partially differentiate into effector-like transitory exhausted T ( Tex) cells, which serve as a cardinal 87 \nforce of T cell immunity when responding to anti-PD-1 therapy16,17, highlighting the need for knowledge 88 \nassimilation to better understand and harness this function al process. Given both distinct and shared 89 \nepigenetic circuits between different T cell subsets 18, we embark on identifying a novel regulator that 90 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\ngoverns early epigenetic events exclusively for tumoricidal effector and effector -like T cells. m 6A-91 \nmediated RNA methylation and destabilization have been recognized as an ingenious mechanism for 92 \nT cell homeostasis 19 and survival 20, but the implication of m 6A RNA modifiers in antitumor T cells 93 \nremains an enigma. Therefore, it is imperative to illustrate the m 6A machinery underlying T cell 94 \nactivation and therapy-induced rejuvenation. 95 \n  RNA m 6A is cotranscriptionally installed by the methyltransferase complex in the nucleus 21. Recent 96 \nstudies have suggested that m 6A modification has an interplay with and an impact on the chromatin 97 \nstate. In mouse embryonic stem cells, METTL14 can recognize transcription elongation mark histone 98 \nH3 trimethylation at Lys36 (H3K36me3), which guides m 6A deposition on actively transcribed nascent 99 \nRNAs22. Conversely, m 6A-modified nuclea r RNAs can direct chromatin organization and gene 100 \nexpression. The nuclear m6A reader YTHDC1 plays a sophisticated role in such a context; it can either 101 \nrecruit the histone demethylase KDM3B to erase the repressive histone mark H3K9me2 , or dictate 102 \nnuclear R NA decay to restrict the chromatin activity and downstream transcriptio n 23-25. These 103 \ndiscoveries raise questions about whether the m 6A machinery adapts the binding interface for core 104 \ntranscription factors and whether T cell activation necessitates the crosstalk between m6A modification 105 \nand chromatin organization. 106 \n  YTHDF2, a highly effective m6A reader, specifically recognizes and degrades m6A-containing mRNAs 107 \nin the cytoplasm, where it primarily resides 26. Otherwise, under heat shock stress, nucle us-localized 108 \nYTHDF2 protects the 5’ untranslated region (5’UTR) of stress -induced transcripts from FTO-mediated 109 \ndemethylation, resulting in cap-independent translation initiation27. In the present study, we reveal that 110 \nYTHDF2 is uniquely express ed and distributed during early T cell activation and therapy -induced 111 \nrejuvenation. Despite its short -term upregulation and nuclear localization, YTHDF2 ensures the 112 \nlongevity and tumoricidal activity of CD8 T effector (T eff) and T eff-like cells. In quiescent T cells, 113 \ncytoplasmic YTHDF2 potentially destabilizes its cognate coding mRNA via m6A recognition, self -114 \nmaintaining a low expression level under nonpathological conditions. When encountering robust 115 \nantigen stimuli or ICB therapy, a portion of YTHDF2 switches to a non-autoregulating state followed by 116 \nnuclear translocation , allowing cognate mRNA translation  in early polyfunctional T cells . The 117 \naccumulation of YTHDF2 in the cytoplasm results in the degradation of the redundant mitochondrial 118 \ncomponent-encoding mRNAs , withstanding mitochondrial stress and T cell exhaustion . In a more 119 \ntumor-rejecting manner, the nuclear import of YTHDF2 directs chromatin organization and effector 120 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\npolyfunctionality by limiting IKZF1 and IKZF3 from the transcriptional inhibition of T cell receptor (TCR) 121 \nsignalling. Conversely, YTHDF2 deficiency in T cells thwarts both endogenous and ICB-induced tumor 122 \nimmunity in mice and is correlated with a poor therapeutic response in cancer patients. Nevertheless, 123 \nowing to the dominant transcriptional repression  by IKZF1/3 in YTHDF2-null T cells, lenalidomide, a 124 \nclinically available IKZF1/3 inhibitor, could achieve a compensatory immune response in synergy with 125 \nICB. Collectively, these data provide proof-of-concept evidence that YTHDF2 integrates RNA and DNA 126 \nepigenetics to potentiate T cell antitumor immunity. 127 \n 128 \nResults 129 \nYTHDF2 is selectively upregulated and redistributed in early Teff and Teff-like cells 130 \nTo begin, we interrogated several transcriptomic datasets and assessed the expression of m 6A 131 \nmachinery components in T cells that undergo diverse signals. When stimulated with anti -CD3/CD28 132 \nantibodies, both CD4 and CD8 T cells showed a predominant increase in Ythdf2 expression during 133 \nearly activation (Supplementary Fig. 1 a, b). In an in vitro system mimicking different states of human 134 \nCD8 T cells 28, a high Ythdf2 mRNA level was observed during 3 –48 h of anti -CD3/CD28 stimulation, 135 \nbut its expression decreased at later time points of activation as well as toward s exhaustion-like and 136 \nmemory-like stages (Fig. 1a). Similarly, among  the in vitro-generated CD4 T cell subsets , a group of 137 \ninducible tolerant T cells exhibited the lowest YTHDF2 level (Supplementary Fig. 1c). In immunotherapy 138 \nsettings, PD-1 blockade induced the upregulation of YTHDF2 in CD8 T cells from responding tumors; 139 \nhowever, this upregulation occurred only during early tumor regression but not in the late regression or 140 \nprogression stage (Fig. 1b). As shown in these datasets, the expression of the gene encoding another 141 \nimportant m6A reader, YTHDF1, could also be upregulated upon T cell activation and rejuvenation, but 142 \nits expression level was relatively low in tumor-infiltrating CD8 T cells (Supplementary Fig. 1d). A recent 143 \nreport indicated that the loss of YTHDF1 in CD8 T cells does not affect antitumor immunity7. Therefore, 144 \nthe potential function of YTHDF2 in T cell-mediated tumor immunity was the focus of this study. 145 \n  We obtained both human peripheral and mouse splenic CD8 T cells for in vitro activation and validated 146 \nthe inducible upregulation of YTHDF2 expression at the protein level ( Supplementary Fig. 1e, f ). By 147 \nperforming liquid chromatography -tandem mass spectrometry (LC–MS/MS) analysis, we observed a 148 \nsignificant loss of m6A modification in the transcriptome after CD8 T cell activation (Supplementary Fig. 149 \n1g). We then conducted antibody -based m 6A sequencing (m 6A-seq) and noted that m 6A-150 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nhypomethylated peaks were mostly distributed in 3’ untranslated regions (3’UTRs) (Supplementary Fig. 151 \n1h). In addition to the possibility of mRNA 3′UTR shortening pending T cell activation 29, this observed 152 \nhypomethylation might also be explained by  YTHDF2-induced destabilization of m 6A-containing 153 \nmRNAs26. 154 \n  As shown by flow cytometry analysis, in vitro-generated early-phase effector CD8 T cells manifested 155 \na much higher YTHDF2 level than did exhausted CD8 T cells (Fig. 1c). In C57BL/6 mice subcutaneously 156 \nchallenged with ovalbumin (OVA) -expressing B16F10 cells, tumor-infiltrating T cells expressed more 157 \nYTHDF2 than did splenic T cells, among which CD44 +KLRG1+ terminal effector CD8 T cells were 158 \npredominant (Fig. 1d). Consistent with the transcriptomic data, PD -1+TIM3+TCF1- terminal exhausted 159 \nCD8 T cells exhibited a much lower YTHDF2 level than did PD-1+TCF1+SLAMF6+TIM3- Tpex and 160 \nCD44+PD-1-TCF1+CD127+ memory T cell (T mem) subsets ( Fig. 1e ). In addition, early anti-PD-1 161 \ntreatment led to a 3-fold increase in YTHDF2 expression in Tpex and their progeny PD-1+TCF1-KLRG1+ 162 \ntransitory Tex cells (also known as terminal T eff-like cells30, which are believed to expan d upon ICB for 163 \ntumor killing16,17,31 (Fig. 1f ). These observations imply that YTHDF2 may widely impact tumor-164 \nexperienced CD8 T cells, particularly the early effector and effector-like subsets. 165 \n  Whereas it is well-accepted that YTHDF2 primarily resides in the cytosol, where mRNA decay occurs, 166 \nprevious work has shown that heat shock stress c an lead to the relocation of YTHDF2 to the nucleus 167 \nthrough an unknown mechanism 27. In the present study, we as sessed whether T cel l activation or 168 \nreinvigoration could alter the subcellular localization of YTHDF2. Surprisingly, wild-type and OT-1 CD8 169 \nT cells accumulat ed nuclear YTHDF2 when stimulated with anti -CD3/CD28 antibodies and OVA 170 \npeptides, respectively, for 12 –48 h (Fig. 1g, h and Supplementary Fig. 1i, j ). In the case of  the Jurkat 171 \nhuman T lymphoma cell line , we detect ed an inherent nuclear fraction of YTHDF2, which modestly 172 \nincreased after phytohaemagglutinin (PHA) treatment (Supplementary Fig. 1k, l). Akin to the temporary 173 \nYTHDF2 relocation observed in vitro , tumor-infiltrating CD8 T cells showed nuclear expression of 174 \nYTHDF2 in regressing but not progressed lesions (Fig. 1i). We then implanted OT-1 (expressing a TCR 175 \nspecific to MHC -I-restricted OVA residues) transgenic mice with B16F10 or B16F10 -OVA cells but 176 \ndiscovered YTHDF2-redistributed CD8 T cells only within tumors formed by the latter ( Supplementary 177 \nFig. 1m), showing that such a phenotype depends on antigen -specific T cell reaction s. As expected, 178 \nearly anti-PD-1 treatment triggered the overexpression and nuclear relocation of YTHDF2 within a small 179 \nportion of CD8 + tumor-infiltrating lymphocytes ( TILs) (Supplementary Fig. 1n ). Specifically, PD-180 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n1+SLAMF6+TIM3- CD8 Tpex sorted from B16 -OVA tumors exhibited YTHDF2 overexpression and 181 \nnuclear relocation when cultured in the presence of anti-PD-1 (Fig. 1j ). Mirroring the selective 182 \noverexpression pattern, these data highlight subcellular YTHDF2 distribution as an acute T cell 183 \nphenotype underlying natural or therapy-induced tumor eradication, which prompted us to explore the 184 \nmultiple functions of YTHDF2 in T cell immunity. 185 \n 186 \nYTHDF2 is essential for the antitumor effects of CD8 T cells 187 \nWe crossed Ythdf2Flox/Flox (hereafter Ythdf2F/F) mice 32 with dLckCre transgenic mice (expressing Cre 188 \nrecombinase under the distal Lck promoter)33 to conditionally knockout Ythdf2 in T cel ls (Ythdf2CKO) 189 \n(Supplementary Fig. 2a, b). First, we compared the thymuses, spleens and peripheral blood from 6 -190 \nweek old Ythdf2F/F, dLckCre and Ythdf2CKO mice and found no obvious difference s in their T cell 191 \ncompositions (Supplementary Fig. 2c). Gene knockout efficiency was demonstrated by comparing the 192 \nintratumoral CD8 T cells from Ythdf2F/F and Ythdf2CKO mice (Supplementary Fig. 2d). When inoculated 193 \nwith hepatocellular carcinoma Hepa1 -6 cells, melanoma B16F10 cells, or colorectal carcinoma MC38 194 \ncells, Ythdf2CKO mice exhibited much faster tumor growth than did Ythdf2F/F or dLckCre mice (Fig. 2a–c 195 \nand Supplementary Fig. 2 e). Correspondingly, Ythdf2 deficiency led to lower numbers of tumor-196 \ninfiltrating CD8 T cells but did not affect CD4 T cells or regulatory T cells ( Fig. 2d and Supplementary 197 \nFig. 3a, c). Dextramer staining further indicated that the frequency of the tumor-specific CD8 T cell 198 \nsubpopulation was substantially decreased in the absence of YTHDF2 ( Supplementary Fig. 3a). 199 \nReduced CD8 T cell  numbers and percentages were also seen in the tumor-draining lymph nodes 200 \n(dLNs) of Ythdf2 CKO mice (Supplementary Fig. 3b). Moreover, the percentage of terminal effector CD8 201 \nT cells was lower in Ythdf2CKO mice at day 12 after MC38 tumor inoculation, consistent with increased 202 \napoptosis and impaired cytokine production and proliferative capacity of a broader CD8 T cell population 203 \n(Fig. 2e, f). In contrast, PD -1+TIM3+CD101+ terminally exhausted CD8 T cells were more frequently 204 \nfound in the Ythdf2CKO group at a later time point (Fig. 2g and Supplementary Fig. 3d). In addition to the 205 \ncrucial role of CD8 T cell immunity, we were also curious about whether CD4 T cells were affected in 206 \nYthdf2CKO mice. H owever, a ntibody-mediated neutralization confirmed that YTHDF2 loss mainly 207 \njeopardizes CD8 (but not CD4) T cell-mediated antitumor immunity (Supplementary Fig. 3e), possibly 208 \nbecause YTHDF2 expression by regulatory T cells is conductive to tumor growth34. 209 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n  To determine whether YTHDF2 engages in regulating tumor-specific CD8 T cells, we further bred 210 \nYthdf2CKO (or Ythdf2F/F) mice with OT-1 transgenic mice to generate a Ythdf2CKO;OT-1 (or Ythdf2F/F;OT-211 \n1) line. Strikingly, OVA-expressing B16F10 cells were resisted in Ythdf2F/F;OT-1 mice but rapidly grown 212 \nup in the Ythdf2CKO;OT-1 counterparts (Fig. 2h). To align the initial immune state, an equivalent number 213 \nof in vitro-activated CD8 T cells of Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1 origin were transferred into mice 214 \ninoculated with B16F10-OVA cells. As anticipated, YTHDF2-deficient T cells exhibited inferior antitumor 215 \nefficacy in this setting (Fig. 2i). 216 \n  Further, to substantiate the importance of YTHDF2 in ICB -induced T cell immunity, we subjected the 217 \nabove mice to grow MC38 or Hepa1 -6 cells, both of which  are thought to respond vigorously to anti -218 \nPD-1 monotherapy. Nonetheless, unlike Ythdf2F/F mice, the  Ythdf2CKO littermates produced a 219 \ncompromised response to PD-1 blockade in the MC38 model and gained no aid of killing effect toward 220 \nthe Hepa1-6 tumors (Fig. 2j, k). In keeping with this, anti -PD-1 therapy led to a much lower frequency 221 \nof tumor-specific or CX3CR1+Tim3+CD101- Teff-like CD8 T cells as well as less cytokine production by 222 \nthese cells in Ythdf2CKO mice (Fig. 2l and Supplementary Fig. 3f), indicating an indispensable role for 223 \nYTHDF2 in implementing ICB-elicited T cell functionality. 224 \n  Together, these observations have indicated that YTHDF2 expression is fundamental for antitumor 225 \neffector and effector-like CD8 T cells, which constitute natural and ICB-induced immunity, respectively. 226 \n 227 \nYTHDF2 prevents T cell mitochondrial dysfunction through m6A-dependent RNA decay 228 \nTo understand how YTHDF2 de pletion results in T cell dysfunction and to minimize the disturbance 229 \nposed by the TME, we assessed in vitro-activated Ythdf2F/F and Ythdf2CKO CD8 T cells under different 230 \nconditions. Consistent with our in vivo results, Ythdf2-deficient CD8 T cells yielded a decline in survival 231 \nand cytokine production as well as a susceptibility to exhaustion, but did not differ in memory T cell 232 \ndifferentiation (Supplementary Fig. 4). 233 \n  In terms of a selective expression pattern, by performing  RNA sequencing, we asked whether the 234 \nabnormalities caused by YTHDF2 loss were rooted in early effector CD8 T cells while doing RNA 235 \nsequencing. Among the 611 genes upregulated upon YTHDF2 ablation, gene ontology (GO)  analysis 236 \nrevealed dominant enrichment for gene sets related to mitochondrial organization and mRNA 237 \ntranslation ( Supplementary Table 1 and Fig. 3a, b ). Ythdf2CKO CD8 T cells exhibited perturbed 238 \nmitochondrial membrane potential and accumulated mitochondrial mass and reactive oxygen species 239 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n(ROS) both in vitro and in vivo (Fig. 3c–f). We further probed the metabolic phenotype of OVA-activated 240 \nYthdf2CKO;OT-1 CD8 T cells. Ythdf2CKO;OT-1 CD8 T cells exhibited decreased extracellular acidification 241 \nrate (ECAR) and oxygen consumption rate (OCR)  (Supplementary Fig. 5a, b) during a mitochon drial 242 \nstress test. Morphologically, activated Ythdf2CKO;OT-1 CD8 T cells had swollen and fewer mitochondria 243 \nwith disorganized crist ae, while Ythdf2F/F;OT-1 CD8 T cells had compact mitochondria with tightly 244 \npacked cristae  (Supplementary Fig. 5c). These observations suggest that YTHDF2 loss -associated 245 \nredundant mRNA translation and mitochondrial mass resulted in T cell stress, which can explain the 246 \nsusceptibility to exhaustion during chronic TCR stimulation 35 (Supplementary Fig. 4d). Given the 247 \nestablished causal relationship between mitochondrial malfunction and T cell exhaustion 24,36, YTHDF2 248 \nmight be critical for ensuring mitochondrial fitness and T cell persistence . SinceYthdf2CKO CD8 T cells 249 \ndid not preferentially express genes related to programmed cell death, we reasoned that the perturbed 250 \ncell survival might also be a result of mitochondrial stress ( Supplementary Table 1 and Fig. 3b). We 251 \nthen employed N -acetylcysteine (NAC) to s cavenge mitochondrial ROS in in vitro stimulated CD8 T 252 \ncells. Nonetheless, no effect on T cell proliferation or cytokine production was observed, but NAC was 253 \nsufficient to prevent excessive exhaustion and cell death caused by YTHDF2 loss (Supplementary Fig. 254 \n5d, e, g and Fig. 3g, h). 255 \n  To elucidate the underlying molecular mechanism, we performed RNA -immunoprecipitation 256 \nsequencing (RIP -seq) to map the target transcripts bound by YTHDF2 in CD8 T cells. Integrative 257 \nanalyses of RIP-seq, m6A-seq and RNA-seq data indicated that 47.9% of the differentially expressed 258 \ngenes caused by YTHDF2 deficiency were eligible for YTHDF2 recognition and m 6A modification (Fig. 259 \n3a and Supplementary Table 2). As potential RNA decay targets, the aforementioned mitochondr ia-260 \nrelated genes (including mitochondrial ribosomal protein-encoding genes) were frequently found among 261 \nthe YTHDF2- and m6A-bound transcripts (Fig. 3i). Noticeably, YTHDF2-RIP-seq and m6A-seq identified 262 \noverlapping peaks on the downstream coding regions  of Coa3, Mrpl16, Mrps12 and Tefm mRNAs 263 \n(Supplementary Fig. 6a ). Consistent with the increased mitochondrial stress , the half -lives of these 264 \ntranscripts were significantly prolonged in the absence of YTHDF2 (Supplementary Fig. 6b). 265 \n  Similarly, the knockdown of YTHDF2 in Jurkat cells also led to excessive mitochondri a-related gene 266 \nexpression and ROS accumulation ( Supplementary Fig. 6c–f and Supplementary Table 3). To 267 \ndetermine the dependency of this regulatory process on the m6A machinery, we exogenously 268 \nreconstituted YTHDF2 in Jurkat-shYTHDF2 cells. The overexpression of wild-type YTHDF2, but not its 269 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\ninactive mutants that fail in recognition of m 6A37, preserved mitochondrial fitness ( Supplementary Fig. 270 \n6g–k). However, dampening METTL3, which constructs the m 6A methylome in T cells 19, rekindled 271 \nmitochondrial malfunction despite the presence of abundant YTHDF2 expression (Supplementary Fig. 272 \n6l–n). These results demonstrate that the m6A machinery i s essential for  YTHDF2-regulated 273 \nmitochondrial fitness in T cells. 274 \n 275 \nYTHDF2 sequesters IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T cells  276 \nDespite identifying the YTHDF2-mediated regulation of  CD8 T cell persistence, the mechanism by 277 \nwhich YTHDF2 promotes CD8 T cell polyfunctionality remains to be addressed. Interestingly, YTHDF2 278 \ndepletion was thought to mainly stabilize its target genes, but RNA profiling revealed a comparable 279 \nnumber of downregulated genes, which integrally reflected an inactive chromatin state in Ythdf2CKO 280 \nCD8 T cells (Supplementary Table 1 and Fig. 4a). Provided that recent studies have uncovered a m6A-281 \nresponsible crosstalk between RNA modification and chromatin regulation 22,23,25, we tested whether 282 \ngene transcription was affected by YTHDF2 in early effe ctor CD8 T cells. Consistent with the 283 \ndistinguishing gene signature, compared with Ythdf2F/F (or Ythdf2F/F;OT-1) CD8 T cells  when shortly 284 \nactivated in vitro, Ythdf2CKO (or Ythdf2CKO;OT-1) CD8 T cells manifested a remarkable decrease in the 285 \nabundance of nascent transcripts (Supplementary Fig. 7a, b), hinting that YTHDF2 plays an important 286 \nrole in chromatin remodelling. No genes encoding general epigenetic regulators or transcription factors 287 \nwere found among those putative decay targets ( Supplementary Table 2), excluding the possibility of 288 \nindirect chromatin regulation rooted in the canonical function of YTHDF2. Interestingly, in the presence 289 \nof an RNA polymerase II-selective inhibitor α-Amanitin38, nascent transcripts were significantly reduced 290 \nin Ythdf2F/F but not Ythdf2CKO CD8 T cells  (Supplementary Fig. 7c ), suggesting that YTHDF2 mainly 291 \npromotes RNA polymerase II-dependent transcription. 292 \n  Nuclear YTHDF2 was known to promote translation initiation of stress-inducible transcripts27. However, 293 \nribosome profiling indicated no difference in translation efficiency between activated Ythdf2F/F and 294 \nYthdf2CKO CD8 T cells, even for YTHDF2 -targeted and m 6A-marked transcripts ( Supplementary Fig.  295 \n7d). To investigate the mechanism underlying YTHD F2-directed transcriptional adap tation, we 296 \nperformed immunoprecipitation followed by mass spectrometry (IP –MS) and identified proteins that 297 \nwere bound to YTHDF2  in the scenario of early T cell activation. Mirroring its potential nuclear 298 \nfunctionality, YTHDF2-binding partners included the lymphoid transcription factor Ikaros (IKZF1) and 299 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nAiolos (IKZF3) 39 (Supplementary Table 4). Fitting their roles as transcription repressors, IK ZF1 and 300 \nIKZF3 were expressed at lower levels in activated CD8 T cells than in naï ve CD8 T cells (Supplementary 301 \nFig. 7e). The results of a proximity ligation assay (PLA) showed that YTHDF2 interacted with IKZF1 and 302 \nIKZF3 in short -term primed  mouse or human  CD8 T cells and untreated Jurkat cells ( Fig. 4b  and 303 \nSupplementary Fig.  7f). Subsequent coimmunoprecipitation (CO-IP) assays further confirmed these 304 \nresults (Fig. 4c). Of note, upon early CD8 T cell activation, a majority of IKZF1 or IKZF3 protein could 305 \nbe bound to YTHDF2. To probe whether RNA species facilitates the interaction between YTHDF2 and 306 \nIKZF1/3, endogenous YTHDF2 immunoprecipitants from acutely stimulated CD8 T cells were incubated 307 \nwith RNase  or DNase.  As shown by  Supplementary Fig. 7g, neither RNase nor DNase repressed 308 \nYTHDF2 binding with IKZF1/3. 309 \n  To assess whether YTHDF2 affects chromatin openness, we performed an assay for transposase -310 \naccessible chromatin with high-throughput sequencing (ATAC-seq). Unexpectedly, YTHDF2 deficiency 311 \nresulted in enhanced chromatin accessibility in both activated CD8 T cells and Jurkat cells (Fig. 4d, e). 312 \nNotably, among the ‘ATAC gain’ regions conditioned by YTHDF2 depletion, an analysis of transcription 313 \nfactor (TF) motifs revealed that the ACAGGAAG element, which is capable of binding IKZF1 or IKZF3, 314 \nwas the top hit (Supplementary Fig. 7h, i), which is capable of binding IKZF1 or IKZF3. Using existing 315 \nchromatin immunoprecipitation sequencing (ChIP -seq) datasets 39,40, we determined that the specific 316 \nloci for these two transcription factors selectively displayed greater chromatin accessibility in YTHDF2-317 \ndeficient T cells than in YTHDF2 -competent T cells ( Fig. 4f and Supplementary Fig. 7j). Epigenomic 318 \nmapping through cleavage under targets and release using nuclease (CUT&RUN), however, 319 \nunderscored that the transcription start sites (TSS s) of IKZF1/3-regulated genes in YTHDF2 -deficient 320 \nT cells were marked by much lower amounts of histone H3 lysine 4 methylation (H3K4Me ) (Fig. 4g), 321 \nwhich is an active form of chromatin modification.  These unusual chromatin changes were profoundly 322 \nobserved for  genes responsible for TCR signalling, such as Stat5a9 and Rasgrp141, which were 323 \nconfirmed to undergo transcriptional silencing in early activated Ythdf2CKO T cells ( Fig. 4h–j). Genes 324 \nencoding epigenetic modulators were also widely affected, many of which were simultaneously found  325 \nto have ‘ATAC gain’ regions predicted for IKZF1/3 binding (Fig. 4j). 326 \n  Since the protein levels of IKZF1/3 were comparable between Ythdf2F/F and Ythdf2CKO CD8 T cells  327 \n(Supplementary Fig. 8a), we asked whether YTHDF2 deficiency had an impact on IKZF-DNA binding 328 \nactivity. As expected , CUT&RUN assay detected stronger IKZF1/3 binding signals at the promoter 329 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nregions of  genes expressed under a YTHDF2 -deficient condition  (Supplementary Fig. 8b ). As 330 \nsuggested by a recent report 42, nucleosome occupancy at IKZF motifs could inactivate effector gene 331 \ntranscription without reducing T cell chromatin op enness. The molecular basis might be IKZF1/3-332 \nmediated recruitment of histone deacetylase (HDAC) complexes 43,44. Here i n an early activation 333 \nscenario, Ythdf2CKO but not Ythdf2F/F CD8 T cells  enabled strong IKZF-HDAC1 interaction  334 \n(Supplementary Fig. 8c), raising the possibility of nucleosome occupancy and chromatin inactivation  335 \nupon YTHDF2 loss. Together, these data highlight that YTHDF2 loss may result in IKZF1/3-associated 336 \ntranscriptional repression. 337 \nLenalidomide rescues the polyfunctionality of YTHDF2-deficient CD8 T cells  338 \nWe next sought to unravel the dependency of YTHDF2 loss-associated T cell malfunction  on IKZF1 339 \nand IKZF3. As shown in Supplementary Fig. 8d, the knockdown of IKZF1 and IKZF3 restored nascent 340 \ntranscription in Jurkat -shYTHDF2 cells but did not change that in  control cells.  The myeloma drug 341 \nlenalidomide has been shown to cause the proteasomal degradation of IKZF1 and IKZF345,46. We asked 342 \nwhether this clinically available drug could re store the effector function of YTHDF2 -deficient T cells by 343 \ntargeting IKZF1/3. When lenalidomide was added to short -term activated CD8 T cells or untreate d 344 \nJurkat cells, it did not obviously enforce T cell function under a YTHDF2-competent condition; in contrast, 345 \nit restored nascent RNA synthesis as well as the proliferation of Ythdf2CKO CD8 T cells (Supplementary 346 \nFig. 8e–h and Fig. 5a–d), manifesting a context-dependent mode of action. Although there was no effect 347 \non preventing T cell exhaustion , lenalidomide profoundly provoked cytokine production by Ythdf2CKO 348 \nCD8 T cells  (Supplementary Fig. 8i and Fig. 5e). Supporting a retrieved functional state, Stat5a and 349 \nRasgrp1 expression in Ythdf2CKO CD8 T cells  returned to normal in the presence of lenalidomide 350 \n(Supplementary Fig. 8j). 351 \n  Taking into consideration of ICB-induced YTHDF2 relocation in effector-like CD8 T cells, we evaluated 352 \nthe dependency of these cells on IKZF1/3 signalling and the therapeutic effect of lenalidomide in vivo. 353 \nAlthough lenalidomide monotherapy was less efficient than anti -PD-1 therapy when administered to 354 \nMC38-bearing Ythdf2F/F mice, it had a slightly better effect on Ythdf2CKO mice; promisingly, lenalidomide 355 \ncombined with PD-1 blockade largely rescued tumor-eradicating immunity in Ythdf2CKO mice (Fig. 5f, 356 \ng). In line with this, flow cytometry analysis showed that combination therapy increased  cytokine 357 \nproduction by PD-1+ Ythdf2CKO CD8+ TILs to a level equivalent to that in the Ythdf2F/F group receiving 358 \nanti-PD-1 or combination therapy (Fig. 5h). While it is true lenalidomide is more than an IKZF degrader, 359 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nthese data have supported the likelihood that the unresponsiveness of YTHDF2-deficient T cells was 360 \ncaused by IKZF1/3.  Together, we conclude that YTHDF2 may achieve polyfunctionality in effector or 361 \neffector-like CD8 T cells by preventing IKZF1/3-associated transcriptional repression. 362 \n 363 \nYTHDF2 relies on m6A-bound transcripts for nuclear translocation 364 \nRegarding YTHDF2 nuclear relocation in early (re)activated T cells ( Fig. 1e–h), we inferred that newly 365 \nsynthesized RNA substrates at this stage might be necessary for YTHDF2 trafficking or acting in the 366 \nnucleus. In support of this notion, the addition of the transcription inhibitor actinomycin D (ActD) largely 367 \nblocked the interaction between YTHDF2 and IKZF3 in the T cell nucleus (Fig. 6a). Further, the finding 368 \nthat YTHDF2 was recruited to the m6A sites within mRNAs transcribed from its DNA targets raised the 369 \npossibility of cotranscriptional regulation ( Supplementary Fig.  9a and Supplementary Table 5 ). To 370 \ndetermine whether the m 6A machinery is required for nuclear YTHDF2 distribution and function, we 371 \ndepleted METTL3 in Jurkat cells and mouse T cells. Compared with Mettl3F/F mice, Mettl3CKO littermates 372 \nexhibited accelerated tumor growth, accompanied by decreased proliferation and cytokine production 373 \nin tumor-infiltrating CD8 T cells ( Supplementary Fig. 9b–d). Of importance, YTHDF2 failed to localize 374 \nto the nucleus of METTL3 -deficient CD8 T cells following in vitro priming (Fig. 6b). In line with this, 375 \nMETTL3 loss interrupted the binding of YTHDF2 to IKZF3 in both Jurkat cells and activated mouse T 376 \ncells ( Fig. 6c, d ). Mutation of a catalytic residue ( W386A or W432A) in the hydrophobic pocket of 377 \nYTHDF2, which specifically recognize s m6A, had a similar effect as METTL3 depletion (Fig. 6e and 378 \nSupplementary Fig.  9e). In parallel, the disruption of METTL3 in YTHDF2 -overexpressing cells 379 \nabrogated its ability to facilitate nascent RNA synthesis and proliferation ( Fig. 6f, g). We then explored 380 \nwhether augmented m6A mRNA modification could conversely reinforce YTHDF2 nuclear function by 381 \nrepressing the m 6A demethylase FTO in activated T cells. However, treatment with an FTO inhibitor 382 \n(FB23-2)47 did not promote nascent RNA synthesis regardless of the YTHDF2 concentration 383 \n(Supplementary Fig. 9f, g), suggesting that increasing m 6A deposition per se is not sufficient to boost 384 \nthe nuclear function of YTHDF2. These observations suggest that m 6A recognition might be a 385 \nprerequisite for YTHDF2 trafficking to the nucleus. 386 \n 387 \nYTHDF2 expression is posttranscriptionally autoregulated via the m6A machinery 388 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nAlthough Ythdf2 mRNA expression was increased at early timepoints of CD8 T cell priming 389 \n(Supplementary Fig. 9h), its chromatin accessibility at TSS regions remained unchanged after 5 h of in 390 \nvitro priming (Fig. 6h). The addition of ActD to CD8 T cell s did not weaken the fold change in Ythdf2 391 \nmRNA expression, thus excluding a direct transcriptional regulation (Fig. 6i). By incorporating YTHDF2-392 \nRIP-seq with m 6A-seq data from both primed CD8 T cells and untreated Jurkat  cells, we noticed that 393 \nYTHDF2 directly bound to its own mRNA and identified overlapping peaks on its m 6A-occupied exons 394 \n(Supplementary Fig. 9i). Therefore, one interpretation of these findings is that YTHDF2 recognizes and 395 \ndestabilizes its cognate mRNA in  the cytoplasm; once a portion of YTHDF2 is dissociated and enters 396 \nthe nucleus in response to T cell activation, its mRNA translation can be partially unleashed. To test 397 \nthis hypothesis, we generated a mouse line ( Ythdf2-249) carrying a n N-terminal truncated form of 398 \nYTHDF2, which is unable to translocate to mRNA decay sites but retains the domain for the recognition 399 \nof methylated RNA 26. A dditionally, this gene engineering strategy d id not affect the internal mRNA 400 \nregions that are able to undergo m6A modification. Compared to the wild -type control, naï ve Ythdf2-249 401 \nCD8 T cells expressed a much higher level of Ythdf2 mRNA, which did not further increase following 6 402 \nh of  in vitro  priming ( Fig. 6j ). A prolonged Ythdf2 mRNA lifetime in Ythdf2-249 CD8 T cells further 403 \nconfirmed the preexistence of autoregulated RNA decay (Fig. 6k). Ythdf2-249 CD8 T cells also exhibited 404 \nincreased protein expression in both the cytosol and the nucleus (Fig. 6l). Similarly, at the Ythdf2 gene 405 \nlocus of Ythdf2CKO CD8 T cells, RNA-seq captured a higher level of pseudogene expression than that 406 \nof Ythdf2 mRNA in Ythdf2F/F CD8 T cells  (Supplementary Fig. 9k). Taken together, these data have 407 \nshown an unprecedented mode of YTHDF2 autoregulation in CD8 T cells. 408 \n 409 \nYTHDF2 expression and distribution in human tumor-infiltrating T cells  410 \nTo explore the clinical relevance of our findings, we reanalyzed  single-cell RNA-seq data from human 411 \ncolorectal carcinoma (CRC)48 (GSE146771), pancreatic ductal adenocarcinoma (PDA)49 (GSE155698), 412 \nor anti -PD-1-treated hepatocellular carcinoma (HCC) 50 (GSE206325). We divided the single-cell 413 \ntranscriptomes of CD8+ TILs into two groups using a customi zed polyfunctional ity signature score 414 \n(based on Ifng, Gzma, Gzmb, and Prf1 gene expression). As shown in Fig. 7a, Ythdf2 mRNA expression 415 \nwas much greater in CD8 T cells assigned with high er scores. Notably, post -ICB datasets of human 416 \nmelanoma (GSE12057551) or HCC (GSE206325) showed that Ythdf2 level was much greater in CD8+ 417 \nTILs of responders than that of nonresponders (Fig. 7b), suggesting that YTHDF2 is involved in ICB -418 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\ninduced antitumor immunity.  Moreover, among the 7 different CD8 T cell subsets  from HCC patients 419 \nwho responded to anti-PD-1 therapy, cytotoxic CD8 T cells exhibited the greatest  Ythdf2 expression 420 \nlevel (Fig. 7c), which is consistent with the results of  our mouse experiments. However, those 421 \nnonresponder-derived cytotoxic CD8 T cells exhibited a Ythdf2 level comparable to that of the terminally 422 \nexhausted population (Fig. 7c). 423 \n  We further investigated YTHDF2 protein expression using tissue samples from HCC or CRC patients. 424 \nFor those who had not received preoperative treatment, YTHDF2 protein was moderately expressed in 425 \ncancer cells 32 but undetectable in CD8 + TILs. Results from our  clinical trials demonstrate d that 426 \nchemoimmunotherapy significantly improved overall survival (OS) and progression-free survival (PFS) 427 \nof patients with advanced cancer 52,53. YTHDF2-expressing CD8 T cells were substantially more 428 \ncommon in patients who received  neoadjuvant chemoimmunotherapy ( Fig. 7d, h ), supporting its 429 \ninducible upregulation concomitant with therapy-induced CD8 T cell infiltration and activation. Although 430 \na greater number of CD8 + TILs could not distinguish responders from nonresponders to neoadjuvant 431 \ntherapy, YTHDF2 positivity and nuclear accumulation in CD8 T cells were more frequently found in 432 \npatients who achieved  a complete or partial response ( Fig. 7d–k). These observations suggest that 433 \nYTHDF2-expressing CD8 T cells confer therapy -induced antitumor immunity, potentially serving as a 434 \nclinical indicator of cancer prognosis. 435 \n 436 \nDiscussion 437 \nT cell -mediated tumor-eradicating immunity forms the basis of successful cancer treatments 10,20. 438 \nIntense efforts have now been invested in elucidating and breaking the T cell -intrinsic barriers to 439 \nrejuvenation. Differing from e ffector and memory T cells, exhausted T cells  display distinct functional 440 \nproperties, which are attributed greatly to epigenetic and transcriptional mechanisms implicated in T 441 \ncell differentiation 12,54. TOX transcriptionally induces exhaustion -associated gene expression and 442 \nmeanwhile recruits chromatin modifiers to repress gene expression involved in T eff differentiation55. 443 \nSimilarly, NR4A1 restrains effector gene transcription by shielding AP -1 from its binding chr omatin 444 \nregions and promotes acetylation of histone 3 at lysine 27 (H3K27ac) for activating genes related to T 445 \ncell dysfunction56. Depletion of either TOX or NR4A1 provisions CD8 T cells with an effector phenotype 446 \nand boosts antitumor immunity. Besides, CD8 T cells acquire DNMT3A -dependnet de novo DNA 447 \nmethylation events upon effector -to-exhaustion transition even when subjected to PD -1 blockade 448 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\ntherapy. Co-targeting of this epigenetic program during ICB yields a more effective antitumor response57. 449 \nAs characterized in chro nic infection and cancer, proliferation -competent T pex cells, which co -express 450 \nthe transcription factor TCF -1 and exhaustion markers, represent the major therapeutic targets of 451 \nimmune interventions 58, such that epigenetic imprints in this T cell compartment irrevocably affect 452 \nimmunotherapy efficacy. For instance, the SWI/SNF chromatin remodelling complex PBAF 59 and the 453 \nNFAT family transcription factor NFAT5 60 can specifically drive T pex transition to terminal T ex cells, 454 \ntherefore limiting the outcome of T cell -based immunotherapy. In the present work, we embark on 455 \ninvestigating RNA epigenetics  in terms of both endogenous and ICB -induced T cell immunity , thus 456 \nuncovering an indispensable role for the m 6A reader YTHDF2. Aligning with robust antitumor immune 457 \nresponses, YTHDF2 expression is selectively upregulated and redistributed within both terminal Teff and 458 \nTeff-like cells. In accordance, the loss of YTHDF2 in T cells dampens both endogen ous and therapy -459 \ninduced tumor immunity. Unlike previously reported epigenetic events, YTHDF2 depletion does not 460 \nnecessarily affect T cell differentiation, but functionally erodes T cell proliferation and cytokine 461 \nproduction. Longitudinal analyses of CD8 + TILs suggest that YTHDF2 deficiency can impair effector 462 \nfunctionality and durability, thus yielding hyporesponsiveness to anti -PD-1 therapy. Coupled with 463 \nphenotypic observation and multi-dimensional sequencing, our data further demonstrate that YTHDF2 464 \nexpression impacts both effector and exhaustion phases through dual mechanisms.  465 \n  YTHDF2 has been found to exert context-dependent functions in cancer. While verifying the oncogenic 466 \nroles of m6A methylation in some cancer types, several studies postulated the a ccompanying position 467 \nof YTHDF2 as an executer for the RNA decay of tumor suppressors61,62. In fact, YTHDF2 could also 468 \ndestabilize oncogene-coding mRNAs in a m6A-dependent manner63,64. As demonstrated in our previous 469 \nwork, YTHDF2 inhibits mouse and human HCC by processing the decay of Il11 and Serpine2 mRNAs, 470 \nwhich are  responsible for inflammation -related cancer progression and metastasis 32. Otherwise, 471 \nYTHDF2 overexpressed in leukemic stem cells can decrease the ha lf-life of apoptosis gene Tnfrsf2, 472 \nthereby descending to a cancer-promoting position65. With immunobiology appearing as one of the most 473 \npromising frontiers, the m6A machinery in finetuning tumor immunity has been explored2. YTHDF2 has 474 \nbeen found to dominate immunosuppressive myeloid cell function in both natural and therapy -475 \nexperienced cancer contexts 8,9. On the other hand, YTHDF2 promotes NK cell immunity partially by 476 \ninhibiting the mRNA stability of Tardbp, a negative regulator of cell division and proliferation66. Uniquely, 477 \nhere we reveal that YTHDF2 dictates both posttranscriptional and transcriptional programs to reinforce 478 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nthe antitumor function of CD8 T cells. In a naï ve state, low-level YTHDF2 limits the stability of its cognate 479 \nencoding mRNA, which harbors bona fide m 6A sites. Acute a ctivation or rejuvenation signals incite 480 \nYTHDF2 relocation to the nucleus, albeit temporarily, paving an individual way for its abundant 481 \nexpression in the early phase. In the circumstance of natural or ICB-induced tumor-eradicating immunity, 482 \nnuclear YTHDF2 combines and curbs the transcriptional repressor IKZF1 /3 to safeguard gene 483 \ntranscription and T cell function. Meanwhile, cytoplasmic YTHDF2 -mediated mRNA decay can help 484 \nimprove the mitochondrial fitness and persistence of tumor-reactive T cells, opposing their progressive 485 \ntrajectory toward exhaustion. Further, we’ve shown the necessity of m6A recognition for YTHDF2 486 \nrepositioning into the nucleus, which highlights the important crosstalk between RNA modification and 487 \nchromatin regulation within antitumor CD8 T cells.  488 \n  RNA-binding proteins have been proven to pe rvasively participate in transcriptional control 67. 489 \nConsistent with this concept, YTHDC1 was found to regulate gene transcription by recruiting a histone 490 \nmodifier or by processing regulatory RNA species in close proximity to active chromatin regions 24,25. 491 \nOur data show that YTHDF2 depletion renders T cell chromatin more accessible to IKZF1 and IKZF3 492 \nand trapped in an inactive state, which influences the downstream effector genes such as Stat5a and 493 \nRasgrp1. Instead of regulating chromatin modifier transcripts in the cytoplasm, nuclear -localized 494 \nYTHDF2 directly interacts with repressive transcription factors. Despite this, its relocation depends on 495 \nm6A-labelled nascent RNAs, reminiscent of the previous observation that m6A deposition was essential 496 \nfor partitioning the stress-conditioned mRNA -YTHDF2 complexes into phase -separated subcellul ar 497 \ncompartments68. Similar to the perturbed YTHDF2 trafficking in METTL14 -deficient mouse embryonic 498 \nstem cells68, nuclear YTHDF2 is hardly detectable when METTL3 is depleted in either activated CD8 T 499 \ncells or untreated Jurkat cells . Importantly , the genes encoding YTHDF2 -bound transcripts largely 500 \noverlap with the IKZF1/3 targets, suggesting that m 6A may cotranscriptionally enlist YTHDF2 in 501 \nchromatin remodelling. As such, m 6A participates in every step of this non -canonical YTHDF2 signal 502 \nundertaken in T cells committed to effector or effector-like function. 503 \n  We pre viously reported that YTHDF2 expression could be transcriptionally silenced in the hypoxic 504 \nTME32. Here we show its low expression level in human intratumoral T cells might also be explained by 505 \nthe insufficiency of immune response . Only in cancer patients with a better therapeutic response can 506 \nYTHDF2-expressing CD8 T cells be profoundly detected, whic h are supposed to address therapy -507 \ninduced immunity through a positive feedback loop . Otherwise, the paucity of YTHDF2 unmasks an 508 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\noverlooked T cell epigenetic mechanism, posing threat of immunotherapy resistance. Rationally,  the 509 \nimmunomodulatory drug lenali domide, which targets IKZF1 /3 for degradation, facilitates  ICB-induced 510 \nrejuvenation of YTHDF2-deficient T cells. Thus, our understanding of the YTHDF2-centered regulatory 511 \ncircuit in antitumor T cells may inspire novel paths for the development of immunotherapies. 512 \n 513 \nMethods 514 \nAnimals 515 \nWild-type (WT) C57BL/6 mice were purchased from Charles River (Beijing, China) for Medical 516 \nResearch. Ythdf2flox/flox (Ythdf2F/F) mice in C57BL/6 background were described previously 32. 517 \nMettl3flox/flox (Mettl3F/F) mice were kindly provided by Prof.  Z. Yin (Jin an University). dLckCre and OT-1 518 \nTCR transgenic mice were purchased from the Jackson Laboratory. Ythdf2F/F or Mettl3F/F mice were 519 \nthen crossed with dLckCre transgenic mice to obtain Ythdf2CKO or Mettl3CKO mice with Ythdf2 or Mettl3 520 \nconditionally knocked out in T cells. For animal experiments referring to Ythdf2CKO or Mettl3CKO mice, 521 \nlittermate controls with normal YTHDF2  (Ythdf2F/F) or METTL3 ( Mettl3F/F) expression were used. 522 \nYthdf2CKO mice were also crossed with OT -1 TCR transgenic  mice to obtain  Ythdf2CKO;OT-1 mice. 523 \nYthdf2-249 transgenic mice were generated by depleting a 249-amino acid fragment from the N-terminus 524 \nof Ythdf2 gene coding region. Correctly targeted mice were determined by PCR and gene sequence. 525 \nPrimers used for genotyping of Ythdf2-249: Forward–5’-TGTGAATGATGTGGAAGGAA-3’ and Reverse–526 \n5’-CAACAGCAGAGCCTACAA-3’. All mice were maintained under specific pathogen -free conditions. 527 \nMice with 8–12 weeks of age were used for all animal experiments. Animals were randomly allocate d 528 \nto experimental groups.  529 \nCell cultures  530 \nPeripheral naï ve CD8 T cells were isolated from the mouse spleen by negative selection magnetic 531 \nbeads (STEM CELL). CD8 T cells were cultured in complete RPMI medium (RPMI 1640, 10% FBS, 20 532 \nmM HEPES, 1 mM sodium pyruvate, 0.05 mM 2 -mercaptoethanol, 2 mM glu tamine, 100 μg/ml 533 \nstreptomycin and 100 units/ml penicillin) and stimulated with plate -bound anti -CD3/CD28 in the 534 \npresence of 10 ng/ml IL -2 (Peprotech) as indicated. To detect T cell proliferation, naï ve CD8 T cells 535 \nwere stained with 0.5 μM CellTracker Violet fluorescent dye (Thermo Fisher) in serum-free medium for 536 \n20 min at 37 ° C, and then washed three times in PBS. Stained cells were activated by plate-bound anti-537 \nCD3/CD28 (biolegend) for 24–120 h and detected in the BV421 channel by FACS. To detect T cell 538 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nactivation-induced apoptosis, naï ve CD8 T cells were activated by plate -bound anti -CD3/CD28 for 539 \ndifferent number of hours, then analyzed with an annexin V/propidium iodide kit (BD).  540 \n  For in vitro T cell exhaustion assay 69, CD8 T cells were seeded at a concentration of 1 million/ml on 541 \nplates coated with of anti -CD3 (5 μg/ml) and anti -CD28 (2 μg/ml). After 48 h of activation , chronic 542 \nstimulation was performed using plates coated with anti -CD3 (5 μg/ml). Cells were passaged onto a 543 \nfresh anti-CD3-coated plate every two days, maintained at 1 million/ml (in the continued presence of 10 544 \nng/mL IL-2), and analyzed via flow cytometry on Day 8. 545 \n  To induce CD8 T cells with a memory phenotype in vitro, CD8 T cells were activated in a ‘‘transient’’ 546 \nstimulation condition in which beads were removed after the initial 3-day incubation as reported28. CD8 547 \nT cells were seeded at a concentration of 1 million/ml in presence of mouse  T-activator CD3/CD28 548 \nDynabeads (Thermo Fisher Scientific) and IL-2 (10 ng/ml) at 1:1 beads-to-cells ratio. After 2 days, cells 549 \nwere split 1:2. Beads were removed on day 3 and cells were maintained in culture for 6 days (with fresh 550 \nmedia added every 2 days) in the presence of 10 ng/ml IL-2. Cells were analyzed via flow cytometry on 551 \nDay 9.  552 \nTo obtain mouse tumor-infiltrating CD8 T pex, B16-OVA tumor-derived single cell suspension s were 553 \nstained with Zombie NIR and then sorted for live CD45+CD8+PD1+Tim3-SLAMF6+ cells by the BD FACS 554 \nAria II Cell Sorter. Sorted Tpex were cultured in U-bottom plates and stimulation assays were performed 555 \nusing T-activator CD3/CD28 Dynabeads and IL -2 (10 ng/ml) at 1: 2 beads-to-cells ratio with 10 μg/ml 556 \nanti-PD-1 (RMP1-14, BioXCell) or cIg for 48 h. 557 \nHuman peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers by 558 \ngradient centrifugation with Lymphoprep (STEM CELL) . CD8 T cells  were then purified by EasySep 559 \nHuman CD8 T Cell Isolation Kit (STEM CELL) . For in vitro T cell activation experiments, cells were 560 \nplated at 1 million/ml in  the presence of Human T -Activator CD3/CD28 Dynabeads (Thermo  Fisher 561 \nScientific) at 1:1 beads-to-cells ratio supplemented with 30U/ml human IL-2 (Peprotech). 562 \n  The MC38 (mouse colon adenocarcinoma) cell line was originally from Prof. Y.-X. Fu laboratory 563 \n(University of Texas Southwestern Medical Center) . The B16F10 (mouse melanoma) cell line (ATCC, 564 \nCRL-6475) was purchased from the American Type Culture Collection and the B16F10 -OVA cell line 565 \nwas generated by EGFP -OVA (SIINFEKEL) lentivirus transduction. Mouse he pa1-6 (hepatoma cells) 566 \nand human Jurkat (Clone E6-1, T lymphoblast) cells were purchased from the Cell bank of the Chinese 567 \nAcademy of Sciences. 568 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n  MC38, Hepa1 -6, B16F10, B16F10-OVA, Jurkat cells w ere grown in Dulbecco’s modified Eagle’s 569 \nmedium (DMEM) (Invitrogen) or RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine 570 \nserum (FBS) (Gibco), 10 mM HEPES (Gibco) and 1% Penicillin/Streptomycin (Gibco). All cell lines were 571 \nmaintained at 37°C, 5% CO2 and routinely tested negative for Mycoplasma. 572 \n  Short hairpin RNAs (shRNAs)  targeting human Mettl3, Ikzf1 and Ikzf3 were used to generate gene 573 \nknockdown in Jurkat cells. Target sequences are listed in Supplementary Table 6. Relevant shRNA-574 \nexpressing lentiviruses were produced by Obio technology or Gene Chem. YTHDF2-overexpressing or 575 \n-mutant (the m 6A recognition sites W432 and W486 were mutated into A) lentiviruses were designed 576 \nand synthesized as previously described32. Briefly, Jurkat cells were seeded 1 ×  10 6/ml in a 12 well -577 \nplate. HitransG P transfection reagent (GENE) and the corresponding lentiviruses were added. After 578 \ncentrifugation at 1,200 rpm, 37° C for 60 min, cells were incubated at 37° C, 5% CO 2 overnight then 579 \nculture medium was changed. To obtain stably transfected clones, these cell s were treated with 580 \npuromycin (3 μg/ml) for 1 week and maintained in 1 μg/ml. The knockdown or overexpression efficiency 581 \nwas confirmed by quantitative PCR and western blot analysis before the cells were used for subsequent 582 \nexperiments. 583 \n  In some settings, Jurkat cells were treated with 100 μΜ lenalidomide (Selleck) or vehicle for 24 h. 584 \nPrimed CD8 T cells were treated with 10 μΜ lenalidomide or vehicle for 24, 48 or 72 h as indicated. For 585 \nFTO inhibition, Jurkat cells or primed CD8 T cells were treated with10 μΜ FB23-2 (Selleck) or vehicle 586 \nfor 72 h. To neutralize ROS, primed CD8 T cells  were treated with 10 mΜ NAC(Sigma) or vehicle for 587 \n48 or 72 h as indicated. To selectively inhibit Pol II, primed CD8 T cells  were treated with 2 μg/mL α-588 \namanitin (MCE) or vehicle for 12 h as indicated. 589 \nTumor growth and treatments 590 \nMC38 (1 ×  106), Hepa1-6 (1 ×  106), B16F10 (5 ×  105) or B16-OVA (1 ×  106) tumor cells were injected 591 \nsubcutaneously (s.c.) into the right flank of mice. Tumor growth was monitored every 2 or 3 days. Tumor 592 \nvolumes were measured by length (a) and width (b) and calculated as tumor volume = ab2/2. 593 \n  For anti-PD-1 treatment, MC38 or Hepa1 -6 tumors were allowed to grow for five or six days then 594 \nintraperitoneally (i.p.) injected with 250 μg /dose anti-PD-1 (RMP1-14, BioXCell) or control IgG (cIg). 595 \nAnti-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 \ntumors. For adoptive cell transfer therapy, B16F10-OVA (1 ×  106) tumor cells were s.c. injected into the 597 \nright flank of C57BL/6 WT mice (female, 8 weeks). On day 6, tumor-bearing mice were randomly divided 598 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\ninto three groups (n = 5) and intravenously injected  with either PBS or 1 ×  106 OVA-primed (72 h) OT-599 \n1 CD8 T cells from Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1. Tumor growth was monitored every 2 or 3 days 600 \nfrom day 6. For in vivo lenalidomide treatment, tumor-bearing mice were i.p. injected once daily with 10 601 \nmg/kg lenalidomide70 (Selleck) dissolved in DMSO and diluted in 100 μl PBS or with DMSO in 100 μl 602 \nPBS. 603 \n  For antibody-mediated T cell depletion, 200 µ g anti-CD4 (BioXCell, BE0003-1, clone GK1.5) or anti -604 \nCD8 (BioXCell, BE0061, clone 2.43) was given by i.p. 3 days before MC38 tumor inoculation and on 605 \nday 1, 2, 4, 8, 12, 16 and 19, relative to tumor injection (day 0). 606 \nPatient samples 607 \nHuman HCC tissue specimens were collected from 45 patients receiving surgery with informed consent 608 \nat Sun Yat-sen University Cancer Center  (SYSUCC) from 2019 to 2021, who had been administrated 609 \nwith neo -adjuvant chemo -(immuno-) therapy (regional chemotherapy using a FOLFOX (oxaliplatin, 610 \nleucovorin, and fluorouracil) regimen, supplemented with or without anti -PD-1 therapy). Human CRC 611 \ntissue specimens were collected from 30 patients receiving surgery at SYSUCC from 2016 to 2019, 612 \nwho had been administrated with neo -adjuvant chemotherapy (systemic chemotherapy using a 613 \nFOLFIRI (irinotecan, leucovorin, and fluorouracil) regimen). All patients were followed up on a regular 614 \nbasis. The study was approved by the Medical Ethics Committee of SYSUCC. Written informed consent 615 \nwas obtained from the patients who provided samples. All patients had a histological diagnosis of HCC 616 \nor CRC. 617 \nFlow cytometry 618 \nTumors, tumor draining lymph nodes, livers, peripheral blood and spleens were harvested from mice 619 \nas indicated in figure legends. Tumors were sliced into small pieces and put into a gentleMACS C Tube 620 \n(Miltenyi) containing 100 ml Enzyme D, 50 ml Enzyme R, 12.5 ml E nzyme A (Miltenyi) and 2.35 ml 621 \nRPMI 1640. The C tube was then processed on a gentleMACS Octo Dissociator with Heaters (Miltenyi) 622 \nfor 30 min. The resulting cell suspension was passed through a 70 -mm cells strainer (Miltenyi), then 623 \nwashed with PBS buffer con taining 0.04% BSA. Single cell suspensions of spleens were depleted of 624 \nerythrocytes. Cells were re-suspended in staining buffer (PBS with 2% FBS and 1 mM EDTA). To block 625 \nmouse Fc receptors, cells were incubated with anti-CD16/CD32 antibody (BD) for 10 min. Subsequently, 626 \nspecific antibodies for cell surface epitope staining were added and staining was continued for 30 min 627 \nat 4° C in the dark. For mitochondrial staining, cells were incubated with 25 nM MitoTracker Orange 628 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n(ThermoFisher), 50nM MitoTracker Green (ThermoFisher) and 5 uM MitoSOX red (ThermoFisher) in 629 \nRPMI with 2% FBS for 30 min at 37 ° C after staining surface markers. For intracellular staining, cells 630 \nwere stimulated ex vivo with Cell Stimulation Cocktail plus protein transport inhibitors (eBioscience) for 631 \n4 h before surface staining 71. Following incub ation, cells were washed twice with buffer before 632 \nproceeding to intracellular staining. Cells were then fixed and permeabilized using the Foxp3/ 633 \ntranscription Factor Staining Buffer Set (eBioscience) according to the manufacturer's protocol and 634 \nstained with intracellular antibodies or respective isotype antibodies. Cells were analyzed by the Cytek 635 \nAurora (Cytek) or CytoFLEX (Beckman) machine. Analysis of flow cytometry data was performed using 636 \nFlowjo 10.7.1 (Treestar). Dead cells stained by live dead blue ( eBioscience) or Zombie Aqua 637 \n(BioLegend) were excluded from analysis.  Gating was confirmed with fluorescence -minus-one (FMO) 638 \ncontrols for low-density antigens. 639 \nRIP-seq 640 \nRIP-seq was conducted in accordance with a previously reported protocol with minor modifications26. 641 \nMouse activated CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) or Jurkat-YTHDF2 OE cells were collected 642 \nthen the pellet was  treated with cell lysis buffer. The 10% lysis sample  was saved as input, 80% was 643 \nused in immunoprecipitation reactions with anti-YTHDF2 (Abcam) or anti-Flag (CST) antibody, and 10% 644 \nwas incubated with rabbit IgG (CST) as a negative control. The RIP step was performed by using Epi 645 \nRNA immunoprecipitation kit  (Epibiotek) following the manufacturer’s protocols. RNA was then 646 \nextracted using TRIzol reagent (Invitrogen) . Input and immunoprecipitated RNA of each sample were 647 \nused to generate the library using a TruSeq stranded mRNA sample preparation kit (Illumina). Libraries 648 \nquality were determined on Qseq100 Bio -Fragment Analyzer (Bioptic). The strand -specific libraries 649 \nwere sequenced on Illumina Novaseq 6000 system with paired-end 2 x 150 bp read length. 650 \nm6A-seq 651 \nTotal RNA in CD8 T cells or Jurkat  cells was extracted by using TRIzol Reagent. DNase I (Invitrogen) 652 \ntreatment was adopted to remove DNA contamination. Additional phenol -chloroform isolation and 653 \nethanol precipitation treatments were performed to remove enzyme contamination. Following meRIP -654 \nSeq was carried out as previously described 72. Briefly, 20 μg purified RNA was fragmented into ~200 655 \nnucleotide-long fragments by incubating in magnesium RNA fragmentation buffer for 6 min at 70° C. 656 \nThe fragmentation was stopped by adding EDTA. Then, RNA clean and concentrator-5 kit (Zymo) was 657 \nused to purify fragmented total RNA. Next, m 6A immunoprecipitation was performed by using Epi m 6A 658 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nimmunoprecipitation kit (Epibiotek). Fragmented total RNA (Input) and immunoprecipitated RNA (IP) 659 \nwere subjected to library construction by using Epi mini longRNA -seq kit (Epib iotek) according to the 660 \nmanufacturer’s protocols. Briefly, reverse transcription was performed using random primers and the 661 \nribosome cDNA was removed after cDNA synthesis using probes specific to mammalian rRNA. The 662 \ndirectionality of the template-switching reaction not only preserves the 5’ end sequence information of 663 \nRNA but the strand orientation of the original RNA. Libraries for immunoprecipitated RNA were PCR 664 \namplified for 18 cycles. The quality of libraries was determined on Qseq100 Bio -Fragment Analyzer 665 \n(Bioptic). The strand-specific libraries were sequenced on Illumina Novaseq 6000 system with paired -666 \nend 2 x 150 bp read length. 667 \nLC–MS/MS quantification of m6A 668 \nTotal RNA of naï ve or activated CD8 T cells (anti-CD3/CD28, 5 μg /ml, 24 h) was extracted by using 669 \nTRIzol Reagent. Quantification of m 6A in mRNAs was carried out as previously described 32.100 ng of 670 \nmRNA was digested by nuclease P1 (NEB) in 25 μl of buffer containing 25 mM NaCl, and 2.5 mM ZnCl2 671 \nat 42 ° C for 2 h, followed by the addition of NH4HCO3  and alkaline phosphatase and incubation at 37 ° C 672 \nfor 2 h. The sample was then fi ltered (0.22 μm, Millipore) and injected into the LC -MS/MS. The  673 \nnucleosides were separated by reverse -phase ultraperformance liquid chromatography on a C18 674 \ncolumn using an Agilent  6410 QQQ triple -quadrupole LC mass spectrometer in  positive electrospray 675 \nionization mode. The nucleosides were quantified by using the nucleoside-to-base ion mass transitions 676 \nof 282 to 150 (m 6A) and 268 to 136 (A). Quantification was carried out by comparison with a standard  677 \ncurve obtained from pure nucleoside standards run with  the same batch of samples. The m 6A/A ratio 678 \nwas calculated based on the calibrated concentrations. 679 \nBulk RNA-seq 680 \nRNA-seq was performed as previously described32. Briefly, RNA was isolated from CD8 T cells or Jurkat 681 \ncells using TRIzol for subsequent RNA library construction. The libraries were sequenced on Illumina 682 \nnova 6000 in a 150-bp pair-end run (PE150). 683 \nATAC-seq 684 \nATAC libraries were generated as described with minor modifications 73. In brief, mouse CD8 T cells or 685 \nJurkat cells were harvested and c ounted. Nuclei from 50,000 cells were isolated using a lysis solution 686 \ncomposed of 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% IGEPAL CA-630. Immediately after 687 \ncell lysis, nuclei were pelleted in low-bind 1.5-ml tubes and resuspended in transposition mix (10 µ l 5 x 688 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nTD buffer, 5 µ l Tn5 transposase, 35 µ l nuclease-free water). The transposition reaction was performed 689 \nat 37° C for 45 min. DNA fragments were purified from enzyme solution using Zymo DNA Clean and 690 \nConcentrator TM -5 kit (Zymo). Libraries were barcoded (Nextera Index Kit, Illumina) and amplified with 691 \nNEBNext High Fidelity PCR Mix (New England Biolabs). Size selection of the PCR product were 692 \nperformed by using DNA clean beads (Epibiotek). The quality of libraries was determined on Qseq100 693 \nBio-Fragment Analyzer (Bioptic) and sequenced on Illumina Novaseq 6000 system with paired-end 2 x 694 \n150 bp read length. 695 \nRibo-seq 696 \nRibosome profiling libraries were prepared as described with minor changes74. Briefly, CD8 T cells were 697 \nexposed to cycloheximide (CHX, 100 μg/ml) for 15 min, washed twice with 5 ml cold PBS with CHX 698 \n(100 μg/ml), pelleted, and lysed in Lysis Buffer (20 mM Tris -HCl, pH 7.8, 100 mM KCl, 10 mM MgCl 2, 699 \n1% Triton X-100, 2 mM DTT, 100 μg/ml cycloheximide, 1:100 protease inhibitor, 40 U/ml SUPERasin). 700 \nThe following Ribo-Seq experiment were performed by using Epi TM Ribosome Profiling Kit (Epibiotek) 701 \naccording to the manufacturer’s protocols. Released RNA fragments were purified using Zymo RNA 702 \nClean&ConcentratorTM-5 kit (Zymo) an d ribosomal RNA was deleted by using RiboRNA Depletion Kit 703 \n(Epibiotek). Ribosome protected fragments (RPF) were recovered by using Zymo RNA 704 \nClean&ConcentratorTM-5 kit (Zymo). RNA fragments were prepared into libraries using a QIAseq miRNA 705 \nLibrary kit (Qiag en). Size selection of the library products were performed by using Native -PAGE 706 \nelectrophoresis to capture fragments range from 178 -180bp. Libraries quality were determined on 707 \nQseq100 Bio -Fragment Analyzer (Bioptic) and sequenced on Illumina Novaseq 6000 s ystem with 708 \nsingle-end 1 x 75 bp read length. 709 \nImmunoprecipitation (IP) 710 \nCells were washed with 2 ml of phosphate -buffered saline twice and then lysed with IP lysis buffer 711 \n(Beyotime). After incubation on ice for 15 min and centrifugation at 12,000 rpm at 4° C for 20 min, the 712 \nsupernatant was saved and the protein concentration was determined with the BCA assay 713 \n(ThermoFisher). Proteins were either directly analyzed by immunoblotting as input or used for 714 \nimmunoprecipitation analysis. Briefly, the proteins were fir st incubated with the corresponding 715 \nantibodies (anti-YTHDF2 (Abcam, ab246514), anti-Ikaros (CST, 14859) or anti-Aiolos (CST, 15103)) 716 \novernight and then mixed with Protein G beads (MCE) and incubated for 2 more hours. The beads were 717 \ncollected with a magnetic stand (ThermoFisher) and washed five times with Wash Buffer. After the final 718 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nwash, the beads were resuspended and heated in loading buffer and the supernatant was 719 \nelectrophoresed through SDS-PAGE.  720 \nMass spectrometry (MS) 721 \nProtein lysates from naï ve and ac tivated CD8 T cells were immunoprecipitated with anti -YTHDF2 722 \n(Abcam, ab246514) antibody, separated by SDS-PAGE, and finally visualized with Coomassie brilliant 723 \nblue staining. Protein in-gel digestion and nano-HPLC MS/MS were carried out as described75. 724 \nCUT&RUN 725 \nCUT&RUN was carried out as previously described 76. Briefly,105 primed Ythdf2F/F and Ythdf2CKO CD8 726 \nT cells (anti -CD3/CD28, 5 μg/ml, 24 h) were washed and bound to concanavalin A -coated magnetic 727 \nbeads, then permeabilized with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine 728 \nand protease in hibitor cocktails from Sigma -Aldrich) containing 0.05% digitonin (Dig Wash), and 729 \nincubated with anti-H3K4Me (Active Motif, 39635), anti-Ikaros (CST, 14859) or anti-Aiolos (CST, 15103) 730 \novernight at 4° C. Cell-bead slurry was washed twice with 1 ml Dig Wash, incubated with Protein A -731 \nMNase (pA-MN) for 1h at 4C, then washed twice more with Dig Wash to remove unbound PA/G-MNase 732 \nprotein. Slurry was then placed on a pre -cooled metal block and incubated with cold Dig Wash 733 \ncontaining 2 mM CaCl 2 to activate pA-MN digestion. After 30 min incubation, one volume of 2 x Stop 734 \nBuffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 50 μg/ml glycogen, 50 μg/ml RNase 735 \nA, 4 pg/ml heterologous spike -in DNA) was added to stop the reaction, and fragments were released 736 \nby incubating the tubes on a heat block at 37C for 30 min. Samples were centrifuged 5 minutes at 737 \n16000xg at 4° C, and supernatant was recovered and DNA extracted via phenol-chloroform extraction 738 \nand ethanol precipitation. Extract DNA was processed for library generation using the QIAseq Ultralow 739 \nInput Library Kit (QIAGEN) following the manufacturer’s protocol. Libraries quality were determined on 740 \nQseq100 Bio -Fragment Analyzer (Bioptic) and sequenced on Illumina Novaseq 6000 system with 741 \npaired-end 2× 150 bp read length. 742 \nProximity Ligation Assays (PLA) Fluorescence Assay 743 \nThe DuoLink In Situ Red Starter Kit Mouse/Rabbit (Sigma -Aldrich) was used to detect interacting 744 \nproteins. The assay was performed according to the manufacturer’s instructions. Glass bottom cell 745 \nculture dishes were treated with poly-lysine (Sigma-Aldrich) at 37° C for 4 hours. Then cells were seeded 746 \nto the culture dishes and settled for 15 min. Cells were fixed with 4% paraformaldehyde solution for 20 747 \nmin. Then the dishes were permeabilized with 0.05% Trit on X-100 and blocked with Duolink Blocking 748 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nSolution in a pre-heated humidified chamber for 60 min at 37° C. The primary antibodies (anti-YTHDF2 749 \n(Abcam, ab246514), anti-DYKDDDDK Tag antibody  (Cell Signaling Technology , 14793), anti-Ikaros 750 \n(Proteintech, 66966), anti-Aiolos ( Lesding Biology , AMM16470VCF), and anti -HDAC1 (Proteintech, 751 \n66085)) were added to the dishes and incubated overnight at 4° C. Then the dishes were washed with 752 \nWash Buffer A and subsequently incubated with the PLA probes for 60 min, the Ligation-Ligase solution 753 \nfor 30 min, and the Amplification-Polymerase solution for 100 min in a pre -heated humidified chamber 754 \nat 37° C. Before imaging, the dishes were mounted with a cover slip using Duolink In Situ Mounting 755 \nMedium with DAPI. Fluorescence images  were acquired using a ZEISS LSM880 with fast airyscan 756 \nconfocal microscope. 757 \nNuclear and cytoplasmic protein extraction 758 \nMouse CD8 T cells or Jurkat cells were harvested and washed twice in PBS. Cytoplasmic and nuclear 759 \nfractions were separated using the Minute Cytoplasmic and Nuclear Fractionation kit (Invent) according 760 \nto the manufacturer’s instructions. Briefly, cells were lysed in the cytoplasmic l ysis buffer on ice for 5 761 \nmin and then centrifuged at 12000 rpm for 5 min at 4 °C. The supernatant containing the cytoplasmic 762 \nproteins were harvested. The pellet containing the cell nucleus were wash with ice-cold PBS for 5 times. 763 \nThen the pellet was lysed with the nuclear lysis buffer on ice for 40 min and with violent vortex for 15s 764 \nevery 10 min. The nuclear lysate was centrifuged at 12,000 rpm for 5 min and the supernatant 765 \ncontaining the nuclear proteins were harvested. The concentrations of both fractions  were normalized 766 \nwith BCA assay and subjected to western blot analysis.  767 \nWestern blot analysis 768 \nCells were lysed on ice for 15 min using lysis buffer (Beyotime) supplemented with a protease inhibitor 769 \ncocktail (ThermoFisher). The cell lysate was centrifuged at 12,000 rpm at 4° C for 20 min. The protein 770 \nconcentrations were normalized with a BCA assay kit (ThermoFisher). Equivalent proteins were loaded 771 \ninto 10% SDS-PAGE Gel and transferred to PVDF membranes (Life Technologies). Membranes were 772 \nblocked for 1 h in TBST buffer with 5% skim milk and then incubated with primary antibodies in the 773 \nblocking buffer at 4° C overnight. After being washed three times in TBST, membranes were incubated 774 \nwith secondary antibodies for 1 h at room temperature. The quantitative densi tometry of immunoblots 775 \nwas analyzed by using Image J software. Relevant antibodies are listed in Supplementary Table 6. 776 \nNascent RNA labeling assay 777 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nCD8 T cells or Jurkat were cultured on pre -coated glass over slides. A nascent RNA synthesis assay 778 \nwas conduc ted using Click -It RNA Imaging Kits (Invitrogen) following the manufacturer’s protocols. 779 \nImages were captured with LSM 880 (Zeiss) confocal and intensity of signal was quantified using Zen 780 \n2.6 software (Zeiss). 781 \nQuantitative PCR (qPCR) 782 \nRNAs were extracted f rom primary CD8 T cells and Jurkat cells by using RNA -Quick Purification Kit 783 \n(ESscience), according to the manufacturer’ protocol, and were reverse transcri bed using the 784 \nPrimeScript RT Master Mix (Takara). The GoTaq qPCR Master Mix (Promega) was used to pe rform 785 \nquantitative real-time PCR on the LightCycler 480 System (Roche). The primer sequences are listed in 786 \nSupplementary Table 6. 787 \nMeasurement of RNA lifetime 788 \nCells were treated with Act D (500 μg/ml, MCE) for 1, 2, or 4 h. Untreated cells were used as 0 h. Cells 789 \nwere collected at the indicated timepoints. The total RNA was purified by EasySep™ Total Nucleic Acid 790 \nExtraction Kit (STEM CELL) with an additional DNase -I digestion step (Invitrogen). The quality of the 791 \ntotal RNA w as assessed using a Bioanalyzer 2100 instrument and the RNA 6000 Nano Assay Kit 792 \n(Agilent). RNA quantities were determined using qPCR. 793 \nImmunofluorescent Staining 794 \nYTHDF2 and CD8 in mouse samples or human specimen were detected by Tyramide SuperBoost kits 795 \n(Alexa Fluor 488 -labeled tyramide, Cy3 -labeled tyramide) (ThermoFisher), according to the 796 \nmanufacturers’ protocol. Briefly, paraffin -embedded tissue specimen was first dewaxed at 70° C for 20 797 \nmin. After antigen retrieval and blocking, tissue sections were incuba ted with corresponding primary 798 \nantibodies overnight at 4° C and then incubated with poly -HRP-conjugated secondary antibody and 799 \nAlexa Fluor tyramide reagent. Finally, HRP reaction was stopped and the tissue sections were 800 \nmultiplexed for second and third sign al detection. Nucleus was counterstained with DAPI. Whole slide 801 \noverview images at 40x magnification were obtained using Pannoramic MIDI (3DHISTECH).   802 \nSeahorse metabolic assay 803 \nSeahorse assay was performed to measure OCR and ECAR of primed Ythdf2F/F;OT-1 or Ythdf2CKO;OT-804 \n1 CD8 T cells . CD8 T cells were washed in assay media (XF RPMI medium pH 7.4 (Agilent)) and 805 \nseeded in a 96-well Seahorse Cells Culture Plate (Agilent) in a non-CO2 incubator at 37 ° C for 40 min. 806 \nOCR and ECAR were measured by a Seahorse XFe96 Extracellular Flux Analyzer (Agilent) following 807 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nthe manufacturer’s instructions. During a mito-stress assay, cells were treated with oligomycin (1.5μM, 808 \nSigma-Aldrich), carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1.5 μM, Sigma-Aldrich), 809 \nrotenone (0.5 μM, Sigma-Aldrich) and antimycin A (0.5 μM, Sigma-Aldrich). During a glycolysis assay, 810 \ncells were treated with glucose (10 mM, Sigma -Aldrich), oligomycin (1 μM, Sigma -Aldrich) and 2 -DG 811 \n(50 mM, Sigma -Aldrich). Each condition was performed with 3 –6 replicates in a single experiment. 812 \nOXPHOS and glycolysis were calculated according to the previous report 77. 813 \nTransmission electron microscopy 814 \nCell pellets were fixed in 2.5% glutaraldehyde for 4h at 22 °C. Following pre -fixation, samples were  815 \nwashed in PBS and post fixed in 1% osmium tetroxide for 1h at 22°C. After several washes in PBS and 816 \ndehydration in acetone, samples were embedded in Epon. Ultrathin sections of 100 nm were prepared 817 \non a Leica EM UC7 Ultramicrotome (Leica Microsystems) and stained with uranyl acetate and lead 818 \ncitrate. Images of mitochondria morphology were captured using a Tecnai G2 Spirit transmission 819 \nelectron microscope (FEI Company). 820 \nProcessing of single-cell RNA sequencing data 821 \nHuman single-cell RNA sequencing  datasets (GSE206325 50, GSE146771 48, GSE155698 49, 822 \nGSE12057551) were pretreated by R (v4.2.2). The metadata was loaded and pre -processed using the 823 \nR package Matrix (v1.4 -1). For the analysis of tumor-infiltrating CD8 T cells in the violin plot, we 824 \nexcluded cells that met the criteria as reported 78. Single-cell data processing was carried out using the 825 \nR package Seurat (version 4.3.0). The defining gene sets for the polyfunctionality score of CD8 T cells 826 \ninclude Ifng, Gzma, Gzmb, and Prf1. The scRNA-seq gene set functional score was generated using 827 \nthe R package Seurat (version 4.3.0) function 'AddModuleScore'79-82. We then categorized intratumoral 828 \nCD8 T cells based on the median of the polyfunctionality score a nd compared the Ythdf2 expression 829 \nlevel between the high and low polyfunctionality groups. The resulting graphs were plotted using the R 830 \npackage ggplot2 (version 3.3.6). In the GSE206325 dataset, the cells were subjected to quality control, 831 \nstandardization, clustering, and dime nsionality reduction, resulting in 34 cell clusters. Using hallmark 832 \ngenes, these clusters were further categorized into seven subpopulations: Terminal 1, Progenitor 2, 833 \nProliferating 3, Effector 4, Effector 5, Memory 6, and Cytotoxic 7. We examined the diff erences in 834 \nYTHDF2 expression levels among the seven subpopulations or based on the computed 835 \npolyfunctionality score. 836 \nStatistical analysis 837 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nHistological analyses of both mouse and human tissue were performed in a blinded fashion. Immunoblot 838 \nand immunofluorescence images are representative of experiments that have been repeated at least 839 \nthree times with similar results. Data from cell culture -based flow cytometry and qPCR assays were 840 \ngenerated from two or three independent experiments. Data are presented as me an ±  standard error 841 \nof the mean (SEM). The statistical significance of differences was evaluated by two -tailed unpaired 842 \nStudent’s t test, two -sided Wilcoxon tests or one(two) -way ANOVA. P values of less than 0.05 were 843 \nconsidered statistically significant. All statistical analyses were carried out using R (v4.2.2) or Graphpad 844 \nPrism 8 (GraphPad Software). 845 \nStudy approval 846 \nAll patient samples were obtained from Sun Yat -sen University Cancer Center (SYSUCC). T he 847 \ncollection of tissue specimens was approved by the internal review and ethics boards of SYSUCC. All 848 \nanimal care and handling procedures were performed in accordance with the NIH’s Guide for the Care 849 \nand Use of Laboratory Animals (National Academies Press, 2011) and were approved by the ethics 850 \ncommittees of Sun Yat-sen University and University of Macau. 851 \nData availability 852 \nData for bulk RNA -seq, RIP -seq, meRIP -seq, ATAC -seq, Ribo -seq and CUT& RUN are available 853 \nthrough the BioProject portal (BioProject ID: PRJNA748842).  All other data are available from the 854 \ncorresponding author on reasonable request. 855 \n 856 \nReferences 857 \n1 Su, R. et al. 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This work was supported by 1071 \nGuangdong Provincial Science Fund for Distinguished Young Scholars (2021B1515020007, to J.H.), 1072 \nGeneral Program of National Natural Science Foundation of China (8271881 & 81871970, to J.H.), 1073 \nMacau Science and T echnology Development Fund (FDCT)  (0071/2023/RIA2, to J.H. ), CAMS 1074 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nInnovation Fund for Medical Sciences (CIFMS) (2019-I2M-5-036, to R.-H. X.), Science and Technology 1075 \nProgram of Guangdong (2019B020227002 , to R. -H. X. ), Hundred Talent s Program of Sun Yat -sen 1076 \nUniversity (2019079, to J.H.) and Ministry of Education Frontiers Science Centre for Precision Oncology, 1077 \nUniversity of Macau (SP2023-00001-FSCPO, to J.H.). 1078 \n 1079 \nAuthor contributions  1080 \nH.Z performed the animal and immunological experiments, interpreted the data and prepare the 1081 \nmanuscript. X.L., Z.W., Zhicong Zhao, X.P. , J.H.L., and W.W. performed the cellular and molecular 1082 \nexperiments. W.Y., X.Z., and Q.S. performed the animal experiments. C.C., Q.Z. and Y.C. analysed the 1083 \nmultiple sequencing data. M.Q., Zhaolei Zeng and M.C. collected the clinical samples and patient 1084 \ninformation. C.D., J.C., B.S., J.L. and H.-Q.J. provided key suggestions. R.-H.X. and J.H. co-supervised 1085 \nthe study. J.H. conceived the project, designed the study, interpreted the data and wrote the manuscript. 1086 \nThe order of the co–first authors was determined according to the time spent on this project.  1087 \n 1088 \nCompeting interests   1089 \nJ.C. is a scientific founder of Genovel Biotech Corp. and holds equities with the company, and is also a 1090 \nScientific Advisor for Race Oncology. Other authors declare no conflict of interests.  1091 \n 1092 \nFigure Legends 1093 \nFig. 1 | YTHDF2 is selectively upregulated and redistributed in early Teff and Teff-like cells.  1094 \na–b Transcriptomic data were mined from existing datasets. mRNA expression of m 6A modifiers in 1095 \nCD3/CD28 bead -based stimulation of human CD8 T cells at various time points indicating 1096 \nnaive/memory, activated, and exhausted populations (GSE212357) (a). mRNA expression of m 6A 1097 \nmodifiers in total tumor-infiltrating CD8 T cells from HKP1 lung cancer-bearing mice upon anti-PD-1 or 1098 \ncIg treatment (GSE114300) (b). c Quantification of YTHDF2 MFI between in vitro-generated effector 1099 \nCD8 T cells (n = 5) and exhausted CD8 T cells (n = 5). d–e Quantification of YTHDF2 MFI in various 1100 \ncell populations from late time point (Day 13) B16F10 -OVA tumor (n = 5)  and spleen (left panels) or 1101 \nfrom early time point (D6) B16F10 -OVA tumor (n = 5)  by flow cytometry. f Quantification of YTHDF2 1102 \nMFI in Tpex cells and transitory Tex cells from B16F10-OVA tumor treated with anti-PD-1 (250μg/mouse) 1103 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n(n = 5) or control IgG (cIg) (n = 5). g Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of 1104 \nmouse (WT or OT-1) and human CD8 T cells stimulated with anti-CD3/CD28 (5 μg/ml) or OVA (10 nM).  1105 \nh Representative confocal immunofluorescence images of YTHDF2 (red) and DAPI (blue) in naï ve (n 1106 \n= 5)  or activated (anti -CD3/CD28, 5 μg/ml, 12 –96h) CD8 T cells (n = 5) . Scale bar, 10 μm. i 1107 \nRepresentative immunofluorescent staining of CD8 (red) and YTHDF2 (green) in regressing (n = 5) or 1108 \nprogressed (n = 5)  B16-OVA tumors. A dashed box represents the 4 × enlarged area shown in the 1109 \nbottom panels with separate channels. White arrows point to cells positive for YTHDF2 and CD8. Scale 1110 \nbar, 10 μm. Middle panel, frequencies of YTHDF2 -positive CD8 T cells. Right panel, quantification of 1111 \nthe nuclear to cytoplasmic ratios of YTHDF2 intensity in YTHDF2-positive CD8 T cells. j Representative 1112 \nconfocal immunofluorescence images of YTHDF2 (red) and DAPI (blue) in PD -1+SLAMF6+TIM3- CD8 1113 \nTpex cells from B16-OVA tumors (Day 16) after in vitro anti-PD-1 (10 μg/ml, 48 h) or cIg stimulation (n = 1114 \n6). Scale bar, 10 μm. Error bars, mean ±  s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. One-way analysis 1115 \nof variance (ANOVA) (d, e) or two-tailed unpaired Student’s t-test (c, f, i, j). 1116 \nFig. 2 | YTHDF2 is essential for the antitumor effects of CD8 T cells.  1117 \na Male Ythdf2F/F (n = 6) or Ythdf2CKO (n = 6) mice were injected subcutaneously with 106 Hepa1-6 cells. 1118 \nTumor growth was monitored ever 2 or 3 days. b–c FemaleYthdf2F/F (n = 6) and Ythdf2CKO (n = 5–6) 1119 \nmice were injected subcutaneously with 5 ×  105 B16F10 (b) or 106 MC38 (c) cells. Tumor growth was 1120 \nmonitored ever 2 or 3 days. d–f Tumor-infiltrating lymphocytes (TILs) were isolated from Ythdf2F/F (n = 1121 \n6) and Ythdf2CKO (n = 6) mice 12 days after MC38 tumor inoculation. Numbers of immune subsets (CD8 1122 \nT, CD4 T and T reg cells) within TILs (d) and frequencies of CD8 T cell subpopulations positive for 1123 \nCD44+KLRG1+ (e) active caspase-3 (Casp-3), granzyme B (Gzm B), IFN -γ or Ki-67 (f) were assessed 1124 \nby flow cytometry. g Frequencies of PD-1+TIM3+CD101+CD8 T cell subpopulations from MC38 tumor -1125 \nbearing Ythdf2F/F (n = 6) and Ythdf2CKO (n = 6) mice (Day 18). h FemaleYthdf2F/F;OT-1 (n = 8)  or 1126 \nYthdf2CKO;OT-1 (n = 6) mice were injected subcutaneously with 10 6 B16F10-OVA cells and monitored 1127 \nfor tumor growth. i Adoptive transfer therapy using PBS control or OVA-primed (72 h) Ythdf2F/F;OT-1 or 1128 \nYthdf2CKO;OT-1  CD8 T cells against B16F10 -OVA melanoma (n = 5 per group). j–k Female Ythdf2F/F 1129 \n(n = 12) or Ythdf2CKO (n = 10) mice were injected subcutaneously with 106 MC38 cells (j). Male Ythdf2F/F 1130 \n(n = 16) or Ythdf2CKO (n = 12) mice were injected subcutaneously with 10 6 Hepa1-6 cells (k). Tumor-1131 \nbearing mice were treated with anti-PD-1 (250 μg/mouse) or cIg. l TILs were isolated from Ythdf2F/F (n 1132 \n= 6)  and Ythdf2CKO (n = 6)  mice 12 days after MC38 tumor inoculation with a nti-PD-1 treatment. 1133 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nFrequencies of CD8 T cell subpopulations positive for PD-1+Dextramer+, CX3CR1+Tim3+CD101-PD-1+ 1134 \nwere assessed by flow cytometry. Error bars, mean ±  s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P 1135 \n< 0.0001. Two-way ANOVA (a–c, h–k) or two-tailed unpaired Student’s t-test (d-g, l). 1136 \nFig. 3 | YTHDF2 prevents mitochondrial stress and T cell exhaustion.  1137 \na Volcano plots of genes with differential expression in  activated Ythdf2F/F or Ythdf2CKO CD8 T cells 1138 \n(anti-CD3/CD28, 5 μg/ml, 24 h) (n = 2 per group), putative YTHDF2 targets that were enriched in both 1139 \nRIP-seq and m6A-seq are marked with yellow circles. b GO enrichment analysis of upregulated genes 1140 \nin Ythdf2CKO compared with Ythdf2F/F CD8 T cells  after priming (anti -CD3/CD28, 5 μg /ml, 24 h). c 1141 \nMitochondrial membrane potential and mitochondrial mass was measured by MitoTracker Orange (MO) 1142 \nand MitoTracker Green (MG) staining in activated Ythdf2F/F and Ythdf2CKO CD8 T cells (n = 5 per group). 1143 \nMitochondrial fitness was evaluated according to the MO/MG ratio. d Mitochondrial ROS was measured 1144 \nby MitoSOX staining in activated CD8 T cells from Ythdf2F/F or Ythdf2CKO mice (n = 5 per group) . e 1145 \nMitoSOX staining in MC38 tumor-infiltrating CD8 T cells from Ythdf2F/F and Ythdf2CKO mice (n = 5 per  1146 \ngroup). f Quantification of the MG MFI in MC38 tumor-infiltrating CD8 T cells from Ythdf2F/F or Ythdf2CKO 1147 \nmice (n = 5 per group) . g–h Quantification of Tim3 + PD-1+ (g), Zombie NIR + (h) f requencies among 1148 \nprimed Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 48 h)  in the presence of 10 mΜ 1149 \nNAC or veh (n = 5 per group) . i Heatmap showing the relative expression of representative genes 1150 \n(mitochondrion-related and up-regulated in Ythdf2CKO) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells 1151 \nfrom RNA-seq data. Putative YTHDF2 targets are depicted by filled circles . Error bars, mean ±  s.e.m. 1152 \n*P < 0.05; **P < 0.01. Two-way ANOVA (g, h) or two-tailed unpaired Student’s t-test (c–f). 1153 \nFig. 4 | YTHDF2 segregates IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T 1154 \ncells.  1155 \na GO enrichment analysis of downregulated genes in Ythdf2CKO compared with Ythdf2F/F CD8 T cells 1156 \nafter priming (anti -CD3/CD28, 5 μg/ml, 24 h). b Proximity ligation assay (PLA) analysis of YTHDF2 1157 \nassociated with IKZF1 or IKZF3 in primed mouse (WT or OT-1) and human CD8 T cells stimulated with 1158 \nanti-CD3/CD28 (5 μg/ml, 24 h) or OVA (10 nM, 24 h). Scale bar, 10 μm. c Coimmunoprecipitation 1159 \nassays of YTHDF2 associated with IKZF1 or IKZF3 in primed WT CD8 T cells (anti-CD3/CD28, 5 μg/ml, 1160 \n24 h). d Volcano plot of genes with differential chromatin accessibility between activated Ythdf2F/F and 1161 \nYthdf2CKO CD8 T cells (anti -CD3/CD28, 5 μg/ml, 24 h). e Volcano plot of genes with differential 1162 \nchromatin accessibility bet ween Jurkat -shCtrl (Vec) and Jurkat -shYTHDF2 (KD) cells. f–g ChIP-seq 1163 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\ndatasets for IKZF1 (GSM1296538) and IKZF3 (GSM803106) in mouse T cells were obtained using 1164 \nCistrome Data Browser. ATAC -seq (f) or H3K4me CUT&RUN (g) profiles of activated  Ythdf2F/F and 1165 \nYthdf2CKO CD8 T cells were represented on IKZF1/3 -bound loci or IKZF1/3 -associated promoters. h 1166 \nStat5a (left) and Rasgrp1 (right) mRNA levels detected by RT -qPCR in activated Ythdf2F/F and 1167 \nYthdf2CKO CD8 T cells (anti -CD3/CD28, 5 μg/ml, 24 h)  (n = 3 per group). i ATAC-seq and H3K4me 1168 \nCUT&RUN tracks on the gene loci of Stat5a (top) and Rasgrp1 (bottom) in activated Ythdf2F/F and 1169 \nYthdf2CKO CD8 T cells. j Heatmap showing the relative expression of representative genes (down -1170 \nregulated in Ythdf2CKO) in activated Ythdf2F/F and Ythdf2CKO CD8 T cells from RNA-seq data. Enhanced 1171 \nchromatin accessibility or putative IKZF1/3 binding is depicted by filled circle. Error bars, mean ±  s.e.m.  1172 \n***P < 0.001. Two-tailed unpaired Student’s t-test (h). 1173 \nFig. 5 | Lenalidomide retrieves the antitumor function of YTHDF2-deficient CD8 T cells.  1174 \na Click-it RNA imaging and analysis of nascent RNA synthesis (green) in Ythdf2F/F and Ythdf2CKO CD8 1175 \nT cells primed ( anti-CD3/CD28, 5 μg/ml, 24 h ) in the presence of 10 μΜ lenalidomide (len) or vehicle 1176 \n(veh) (n = 5 per group). Scale bar, 10 μm. b Quantification of Ki-67 MFI among Ythdf2F/F and Ythdf2CKO 1177 \nCD8 T cells primed in the presence of 10 μΜ len or veh (n =4 per group). c Click-it RNA imaging and 1178 \nanalysis of nascent RNA synthesis (green) in Ythdf2F/F;OT-1 and Ythdf2CKO;OT-1 CD8 T cells primed 1179 \n(OVA, 10 nM, 24 h)  in the presence of 10 μΜ len or veh (n = 5 per group) . Scale bar, 10 μm. d 1180 \nQuantification of Ki -67 MFI among Ythdf2F/F;OT-1 and Ythdf2CKO;OT-1 CD8 T cells primed (OVA, 10 1181 \nnM, 72 h) (n = 5 per group) in the presence of 10 μΜ len or veh. e Quantification of Gzm B, IFN-γ MFI 1182 \namong Ythdf2F/F and Ythdf2CKO CD8 T cells primed (anti -CD3/CD28, 5 μg/ml, 48 h)  in the presence of 1183 \n10 μΜ len or veh (n = 4 per group). f MC38-bearing Ythdf2F/F (n = 21) or Ythdf2CKO (n = 18) mice were 1184 \ntreated with anti-PD-1 (250 μg/mouse) and/or len (10 mg/kg) and monitored for tumor growth. g Hepa1-1185 \n6 tumor-bearing Ythdf2F/F (n = 12) or Ythdf2CKO (n = 12) mice were treated with anti-PD-1 (250 μg/mouse) 1186 \nand/or len (10 mg/kg) and monitored for tumor growth. h Quantification of Gzm B+ CD8 T or IFN-γ+ CD8 1187 \nT frequencies within TILs from Hepa1-6-bearing Ythdf2F/F (n = 6) or Ythdf2CKO (n = 6) mice treated with 1188 \nanti-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 \n***P < 0.001. Two-way ANOVA (a–h). 1190 \nFig. 6 | The m6A machinery regulates both YTHDF2 relocation and expression. 1191 \na PLA analysis of YTHDF2 associated with IKZF3 in Jurkat cells treated with or without ActD (500ug/ml, 1192 \n4h). Scale bar, 10 μm. b Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of Mettl3F/F or 1193 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nMettl3CKO CD8 T cells stimulated with anti -CD3/CD28 (5 μg/ml, 24 h). c PLA analysis of YTHDF2 1194 \nassociated with IKZF3 in Jurkat-shCtrl and Jurkat-shMETTL3 cells. Scale bar, 5 μm. d PLA analysis of 1195 \nYTHDF2 associated with IKZF3 in primed Mettl3F/F or Mettl3CKO CD8 T cells (5 μg/ml, 24 h). Scale bar, 1196 \n10 μm. e PLA analysis of Flag associated with IKZF3 in Jurkat cells introduced with Flag-tagged WT or 1197 \nmutant YTHDF2. Scale bar, 10 μm. f Quantification of Ki -67 MFI among METTL3 -knockdown and 1198 \ncontrol Jurkat cells with or without YTHDF2 overexpression (OE) (n = 5 per group) . g Click-it RNA 1199 \nimaging and analysis of nascent RNA synthesis in METTL3-knockdown and control Jurkat cells with or 1200 \nwithout YTHDF2 overexpression (OE) (n = 5 per group). Scale bar, 10 μm. h ATAC-seq tracks of Ythdf2 1201 \nloci on naï ve or activated human T cells (anti-CD3/CD28, 5 h) (GSE116696). i Ythdf2 mRNA levels 1202 \ndetected by qPCR in CD8 T cells stimulated with or without anti -CD3/CD28 (5 μg/ml) and ActD (500 1203 \nug/ml) for 24 h (n = 5 per group). j Ythdf2 mRNA levels detected by qPCR in naï ve (0 h) or activated (6 1204 \nh) WT (n = 5) and Ythdf2-249 (n = 5) CD8 T cells. K Naï ve Ythdf2-249 and WT CD8 T cells were treated 1205 \nwith ActD (500 μg/ml) and RNAs were collected at different time points after ActD treatment. Ythdf2 1206 \nmRNA levels were measured using qPCR and represented as mRNA remaining after transcription 1207 \ninhibition (TI) (n = 3 per group) . l Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of 1208 \nnaï ve (0 h) or activated (24 h) WT compared with Ythdf2-249 CD8 T cells. Error bars, mean ±  s.e.m. NS, 1209 \nno significance; * P < 0.05; ** P < 0.01; *** P < 0.001. One -way (f, i, g) or two -way ANOVA (j) or non-1210 \nlinear regression (k). 1211 \nFig. 7 | YTHDF2 expression and distribution are associated with T cell function in human cancers.  1212 \na Violin plot comparing Ythdf2 gene expression levels of CD8 T cells assigned with high or low 1213 \npolyfunctionality signature scores  that were derived from single-cell RNA -seq datasets . L eft, CRC. 1214 \nMiddle, PDAC. Right, HCC.  b Violin plot comparing pre - or post -treatment Ythdf2 gene expression 1215 \nlevels of CD8 T cells between responders and nonresponders. Left, melanoma. Right, HCC. c Violin 1216 \nplot comparingYthdf2 gene expression levels among CD8 T cell clusters generated from neoadj uvant 1217 \nanti-PD-1-treated HCC. d–i Tissue sections from patients with hepatocellular carcinoma (HCC) (n = 45) 1218 \nor colorectal carcinoma (CRC) (n = 30) were stained for CD8 (red), YTHDF2 (green) and DAPI (blue). 1219 \nd–h Representative immunofluorescent images of sections from HCC (d) and CRC (h) patients showing 1220 \ndifferent responses to neo -adjuvant chemo -(immuno-) therapy. A dashed box represents the 4 × 1221 \nenlarged area shown in the bottom panels with separate channels. Whit e arrows point to cells positive 1222 \nfor YTHDF2 and CD8. Quantification of CD8 T cells (e, i), frequencies of YTHDF2-positive CD8 T cells 1223 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n(f, j) and quantification of YTHDF2 intensity of CD8 T cells (g, k). CR, complete response; PR, partial 1224 \nresponse; SD, stable disease; PD, progressed disease. Scale bar, 10 μm. Error bars, mean ± s.e.m. *P 1225 \n< 0.05; **P < 0.01. Two-tailed unpaired Student’s t-test (e–g, i–k). 1226 \n 1227 \nSupplementary Figure Legends 1228 \nSupplementary Fig. 1 | YTHDF2 is characteristically expressed upon T cell activation and 1229 \nreinvigoration. 1230 \na–d Transcriptomic data were mined from existing datasets. mRNA expression of m 6A modifiers in 1231 \nmouse naï ve or activated WT CD8 (a) and CD4 (b) T cells. mRNA expression of m 6A modifiers in T 1232 \ntolerant (Ttol), T helper (T H1, TH2 and TH17), natural regulatory T (nT reg) and naï ve T cells (c). mRNA 1233 \nexpression of m6A modifiers in OVA-specific CD8 TILs from anti-PD-1- or cIg-treated mouse tumors (d). 1234 \ne–f Protein expression of m 6A modifiers in CD8 T cells primed for the indicated amoun t of time. One 1235 \nrepresentative of three independent experiments is shown. Human CD8 T cells from peripheral blood 1236 \nwere stimulated with 5 μg/ml anti -CD3/CD28 (e). Wild -type (WT) (top) or OT -1 mouse CD8 T cells 1237 \n(bottom) were stimulated with 5 μg/ml anti -CD3/CD28 or 10 nM OVA (f). g LC-MS/MS-based m 6A 1238 \nquantification in mRNAs from naï ve and activated CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h)  (n = 3 1239 \nper group). h Metagene distribution of the m6A peaks of naï ve and active CD8 T cells (anti-CD3/CD28, 1240 \n5 μg/ml, 24 h) along the whole transcriptome. The enriched consensus motifs were detected within m6A 1241 \npeaks. i Representative confocal Z-stack images of YTHDF2 (red), and DAPI (blue) in naï ve or activated 1242 \n(anti-CD3/CD28, 5 μg/ml, 0–48 h) CD8 T cells. j Quantification of YTHDF2 intensity of CD8 T cells (left) 1243 \nand frequencies of nuclear YTHDF2 + cells from naï ve or activated (anti-CD3/CD28, 5 μg/ml, 0 –96 h) 1244 \n(right) CD8 T cells (n = 5 per group). k Immunoblotting analysis of YTHDF2 in the cytosol and nucleus 1245 \nof Jurkat cells stimulated with or without PHA (150 ng/ml, 24 h). respectively. Relative YTHDF2 levels 1246 \nwere calculated using densitometry values for β -actin or Lamin B as calibrators. l Representative 1247 \nconfocal Z-stack images of YTHDF2 (red) and DAPI (blue) in unstimulated  Jurkat cells. Scale bar, 10 1248 \nμm. m Representative multiplexable immunofluorescent staining of CD8 (red) and YTHDF2 (green) 1249 \nwithin B16F10 or B16F10-OVA tumors grown in OT-1 mice (n = 5 per group). A dashed box represents 1250 \nthe 4× enlarged area shown in the bottom panels with separate channels. White arrows point to cells 1251 \npositive for YTHDF2 and CD8. Scale bar, 10 μm. Middle panel, frequencies of YTHDF2 -positive CD8 1252 \nT cells. Right panel, quantification of the nuclear to cytoplasmic ratios of YTHDF2 intensity in YTHDF2-1253 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\npositive CD8 T cells. n Representative multiplexable immunofluorescent staining of CD8 (red) and 1254 \nYTHDF2 (green) within B16F10-OVA tumors treated with cIg or anti -PD-1(n = 5 per group) . A dashed 1255 \nbox represents the 4× enlarged area shown in the bottom panels with separate channels. White arrows 1256 \npoint to cells positive for YTHDF2 and CD8. Scale bar, 10 μm. Middle panel, frequencies of YTHDF2 -1257 \npositive CD8 T cells. Right panel, quantification of the nuclear to cytoplasmic ratios of YTHDF2 intensity 1258 \nin YTHDF2-positive CD8 T cells. Error bars, mean ±  s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-1259 \ntailed unpaired Student’s t-test (j, m, n). 1260 \nSupplementary Fig. 2 | Phenotyping for the YTHDF2 conditional knockout miceYTHDF2 1261 \ndeprivation hinders antitumor T cell immune response.  1262 \na Immunoblotting analysis of YTHDF2 expression in heart, lung, liver, stomach, pancreas, intestine and 1263 \nthymus tissues from Ythdf2F/F and Ythdf2CKO mice. b Protein expression of YTHDF2 in naï ve and 1264 \nactivated CD8 T cells (anti -CD3/CD28, 5 μg/ml, 24 h) derived from Ythdf2F/F and Ythdf2CKO mouse 1265 \nspleens. c Flow cytometry analysis of immune cells within the thymuses, spleens, peripheral blood from 1266 \nYthdf2F/F, dLckCre and Ythdf2CKO mice (n = 6 per group). d Quantification of YTHDF2 expression in B16-1267 \nOVA tumor-infiltrating CD8 T cells from Ythdf2F/F and Ythdf2CKO mice (n = 5 per group). e Female dLckCre 1268 \n(n = 5) and Ythdf2CKO (n = 5) mice were injected subcutaneously with 10 6 MC38 cells. Tumor growth 1269 \nwas monitored ever 2 or 3 days.  Error bars, mean ±  s.e.m. *P < 0.05; *** P < 0.001; ****P < 0.0001. 1270 \nOne-way (c) or two-way ANOVA (e) or two-tailed unpaired Student’s t-test (d). 1271 \nSupplementary Fig. 3 | YTHDF2 deprivation hinders antitumor T cell immune response.  1272 \na TILs were isolated from Ythdf2F/F (n = 5) and Ythdf2CKO (n = 5) mice 12 days after MC38 tumor 1273 \ninoculation. Frequencies of immune subsets (CD8 T, CD4 T and Treg cells) within TILs and frequencies 1274 \nof PD -1+KILTFDRL-Dextramer+ CD8 T cell subpopulations were assessed by flow cytometry. b 1275 \nNumbers and frequencies of immune subsets within tumor -draining lymph nodes (dLN) from MC38 1276 \ntumor-bearing Ythdf2F/F (n = 6) and Ythdf2CKO (n = 6) mice. c TILs were isolated from Ythdf2F/F (n = 6) 1277 \nand Ythdf2CKO (n = 6) mice 10 days after Hepa1 -6 tumor inoculation. Frequencies of immune subsets 1278 \n(CD8 T, CD4 T and T reg cells) within TILs and frequencies of PD -1+KILTFDRL-Dextramer+ CD8 T cell 1279 \nsubpopulations were assessed by flow cytometry. d Frequencies of PD -1+TIM3+CD101+ CD8 T cell 1280 \nsubpopulations from Hepa1 -6 tumor-bearing Ythdf2F/F (n = 6) and Ythdf2CKO (n = 6) mice (Day 14). e 1281 \nMC38-bearing Ythdf2F/F (n = 5) and Ythdf2CKO (n = 5) mice were treated with anti-CD8 (200 μg/mouse) 1282 \n(left) or anti -CD4 antibody (200 μg/mouse) (right) and monitored for tumor growth.  f Frequencies of 1283 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nCX3CR1+Tim3+CD101- CD8 T cell subpopulations positive for Gzm B, IFN-γ or Ki-67 from MC38 tumor-1284 \nbearing Ythdf2F/F (n = 6) and Ythdf2CKO (n =6) mice with anti-PD-1 treatment (D12). Error bars, mean ±  1285 \ns.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Two-way ANOVA (e) or two-tailed unpaired 1286 \nStudent’s t-test (a–d, f). 1287 \nSupplementary Fig. 4 | YTHDF2 maintains CD8 T cell expansion and activation in vitro.  1288 \na–c Naï ve CD8 T cells isolated from Ythdf2F/F (n = 5) or Ythdf2CKO (n = 5) mice were stimulated with 1289 \nanti-CD3/CD28 (2.5 or 5 μg/ml as indicated) for the indicated time. Apoptosis of Ythdf2F/F or Ythdf2CKO 1290 \nCD8 T cells was measured by Annexin V and propidium iodide (PI) staining (a). Proliferation of Ythdf2F/F 1291 \nor Ythdf2CKO CD8 T cells was assessed by Celltracker V (CTV) dilution (b). Quantification of frequencies 1292 \nof IFN-γ+, GZM B+, and Ki-67+ subpopulations within activated Ythdf2F/F or Ythdf2CKO CD8 T cells (c). d 1293 \nNaï ve CD8 T cells isolated from Ythdf2F/F (n = 5) or Ythdf2CKO (n = 5) mice to induce T cell exhaustion 1294 \nby chronic stimulation (plates coated with anti-CD3, 5 mg/mL, 8 days). Quantification of frequencies of 1295 \nIFN-γ+, GZM B+, and Ki-67+ subpopulations within Ythdf2F/F or Ythdf2CKO CD8 T cells from in vitro T cell 1296 \nexhaustion assay. e Quantification of frequencies of CD62L +, CCR7+, and CD122 + subpopulations at 1297 \nDay 9 of transient stimulation (CD3/CD28 beads,1:1 beads-to-cells ratio, 72h) in Ythdf2F/F or Ythdf2CKO 1298 \nCD8 T cells from in vitro memory-like T cell induction assay (n = 6 per group). Error bars, mean ±  s.e.m. 1299 \n*P < 0.05; **P < 0.01. Two-tailed unpaired Student’s t-test (a–e).  1300 \nSupplementary Fig. 5 | YTHDF2 deficiency impairs the mitochondria function of CD8 T cells.  1301 \na–b Extracellular acidification rate (ECAR)  levels (a) and oxygen consumption rate ( OCAR) levels of 1302 \nprimed Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1 CD8 T cells (OVA, 10 nM, 72 h)  were measured in real-time  1303 \nwith Seahorse assay (n = 3 per group). c Transmission electron microscopic (TEM) analysis of 1304 \nmitochondrial morphology in activated Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1 CD8 T cells (OVA, 10 nM, 72 1305 \nh). Red arrows in the left panels show the position of mitochondria in respective higher magnification in 1306 \nthe right panels. Scale bar, 200 nm. d–e Quantification of Ki-67 MFI (d), IFN-γ+, GZM B+ (e) frequencies 1307 \namong Ythdf2F/F and Ythdf2CKO CD8 T cells primed in the T cell exhaustion assay (plates coated with 1308 \nanti-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 \n*P < 0.05. Two-tailed unpaired Student’s t-test (a–c). Two-way ANOVA (d, e). 1310 \nSupplementary Fig. 6 | The m 6A machinery is necessary for YTHDF2 -regulated mitochondrial 1311 \nfitness.  1312 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\na m6A-seq and RIP-seq tracks of Coa3, Mrpl16, Mrps12 and Tefm mRNA loci on primedYthdf2F/F and 1313 \nYthdf2CKO CD8 T cells. b Activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) 1314 \nwere treated with ActD (500 μg/ml) and RNAs were collected at different time points. Coa3, Mrpl16, 1315 \nMrps12 and Tefm mRNA levels were measured using qPCR and represented as mRNA remaining after 1316 \nActD treatment (n = 3 per group) . c Immunoblotting analysis of YTHDF2 expression in Jurkat -shCtrl 1317 \nand Jurkat -shYTHDF2 cells. d GO enrichment analysis of upregulated genes in Jurkat-shCtrl and 1318 \nJurkat-shYTHDF2 cells. e–f Quantification of MitoSOX (e) and MG (f) MFI in Jurkat-shCtrl and Jurkat-1319 \nshYTHDF2 cells (n = 4 per group). g Immunoblotting analysis of YTHDF2 and Flag expression in Jurkat 1320 \ncells with or without YTHDF2 overexpression (OE) (n = 4 per group). h Immunoblotting analysis of Flag 1321 \nexpression in Jurkat cells transduced with  wild-type (OE) or mutant YTHDF2 (W432A and W486A) or 1322 \nempty vector lentiviruses. i–j Quantification of MitoSOX (i) and MG (j) MFI in Jurkat cells with or without 1323 \nYTHDF2 OE (n = 4 per group). k Quantification of the MO/MG ratio in Jurkat cells transduced with wild-1324 \ntype or mutant YTHDF2 or empty vector lentiviruses (n = 3 per group). l Immunoblotting analysis of 1325 \nMETTL3, YTHDF2 and Flag expression in YTHDF2 -OE Jurkat cells with or without knockdown of 1326 \nMETTL3. m MitoSOX staining among METTL3 -knockdown and contr ol Jurkat cells with or without 1327 \nYTHDF2 overexpression (n = 3 per group). n Quantification of the MO/MG ratio among METTL3 -1328 \nknockdown and control Jurkat cells with or without YTHDF2 overexpression (n = 3 per group).  Error 1329 \nbars, 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 \none-way ANOVA (k, m, n) or non-linear regression (b). 1331 \nSupplementary Fig. 7 | Acute T cell activation enlists nuclear functionality of YTHDF2.  1332 \na–b Click-it RNA imaging and analysis of nascent RNA (green) synthesis in activated  Ythdf2F/F or 1333 \nYthdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) (a) and activated Ythdf2F/F;OT-1 or Ythdf2CKO;OT-1334 \n1 CD8 T cells (OVA, 10 nM, 24 h) (b). Scale bar, 10 μm. c Click-it RNA imaging and analysis of nascent 1335 \nRNA (green) synthesis in activated Ythdf2F/F or Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h)  1336 \nin the presence of α -amanitin (2μg/mL, 12 h)  or vehicle (n = 5 per group) . Scale bar, 10 μm. d 1337 \nCumulative distribution of the fold change in translational efficiency of YTHDF2 -targeted and m 6A-1338 \nmarked transcripts between activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 1339 \nh). e Ikzf1 and Ikzf3 mRNA levels detected by qPCR in naï ve or activated (anti-CD3/CD28, 5 μg/ml, 24 1340 \nh) CD8 T cells (n = 3 per group). f PLA analysis of YTHDF2 associated with IKZF1 or IKZF3 in 1341 \nunstimulated Jurkat cells. Scale bar, 10 μm. g Whole-cell lysates of activated WT CD8 T cells (anti -1342 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nCD3/CD28, 5 μg/ml,  24 h) were subjected to  immunoprecipitation using anti -YTHDF2 antibody. The 1343 \nimmunoprecipitants were incubated with RNase  A (1 ug/ul) or DNase I (0.4 U/ul) DNase followed by 1344 \nimmunoblot analysis. h–i Motif discovery analysis of the genomic sequences under A TAC-seq peaks 1345 \nconditioned by YTHDF2 depletion using HOMER. Red rectangle shows the IKZF1/3-binding motif within 1346 \nATAC-sequenced Ythdf2F/F and Ythdf2CKO CD8 T cells (h) or Jurkat-shCtrl and Jurkat-shYTHDF2 cells 1347 \n(i). j ChIP-seq datasets for IKZF1 (GSM935442) in human T cells were obtained using Cistrome Data 1348 \nBrowser. ATAC -seq profiles of Jurkat -shCtrl and Jurkat -shYthdf2 cells were represented on IKZF1 -1349 \nbound loci. Error bars, mean ±  s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed unpaired Student’s 1350 \nt-test (a, b, e) or two-way ANOVA (c). 1351 \nSupplementary Fig. 8 | IKZF1/3 -associated transcriptional repression in YTHDF2 -deficient T 1352 \ncells 1353 \na Quantitative flow cytometry analysis of IKZF1 (top) or IKZF3 (bottom) expression in activated Ythdf2F/F 1354 \nand Ythdf2CKO CD8 T cells  (anti-CD3/CD28, 5 μg/ml, 24 h) (n = 3). b IKZF1 (top) or IKZF3 (bottom) 1355 \nCUT&RUN profiles of activated  Ythdf2F/F and Ythdf2CKO CD8 T cells  (anti-CD3/CD28, 5 μg/ml, 24 h)  1356 \nwere represented at the gene promoter regions. c PLA analysis of HDAC1 associated with IKZF1 or 1357 \nIKZF3 in activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h). Scale bar, 10 1358 \nμm. d Click-it RNA imaging and analysis of nascent RNA synthesis (red) in Jurkat -shCtrl, Jurkat -1359 \nshIKZF1/3 cells, Jurkat -shYTHDF2 cells and Jurkat -shYTHDF2/IKZF1/3 cells (n = 5 per group). e 1360 \nImmunoblotting analyses of IKZF1 and IKZF3 in Jurkat cells treated with len (10 or 100 μΜ) or veh for 1361 \n6 or 24 h. f Click-it RNA imaging and analysis of n ascent RNA synthesis (red) in Jurkat -shCtrl and 1362 \nJurkat-shYTHDF2 cells in the presence of 100 μΜ len or veh for 24 h (n = 5 per group). g–h 1363 \nRepresentative confocal immunofluorescence images of IKZF1 (g) or IKZF3 (h) (green) and DAPI (blue) 1364 \nin activated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) in the presence of 10 1365 \nμΜ len or veh (n = 5 per group). Scale bar, 10 μm. i Quantification of Tim3+ PD-1+ frequencies among 1366 \nprimedYthdf2F/F and Ythdf2CKO CD8 T cells (anti -CD3/CD28, 5 μg/ml,  48 h) in the presence of 10 μΜ 1367 \nlen or veh (n = 4 per group). j Stat5a (left) and Rasgrp1 (right) mRNA levels detected by qPCR in 1368 \nactivated Ythdf2F/F and Ythdf2CKO CD8 T cells (anti-CD3/CD28, 5 μg/ml, 24 h) in the presence of 10 μΜ 1369 \nlen or veh (n = 3 per group). Error bars, mean ±  s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed 1370 \nunpaired Student’s t-test (a). One (d) or two-way (f–j) ANOVA. 1371 \nSupplementary Fig. 9 | YTHDF2 distribution and expression are governed by the m6A machinery.  1372 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\na Venn diagram showing the overlap of putative YTHDF2 -target genes and IKZF1/3-target genes with 1373 \nboth enhanced chromatin accessibility and reduced mRNA expression found in Ythdf2CKO CD8 T cells. 1374 \nb Immunoblotting analysis of METTL3 and GAPDH expression in ac tivated Mettl3F/F or Mettl3CKO CD8 1375 \nT cells (anti -CD3/CD28, 5 μg/ml, 24 h). c Female Mettl3F/F (n = 6) and Mettl3CKO (n = 6) mice were 1376 \ninjected subcutaneously with 10 6 MC38 cells. Tumor growth was monitored ever 2 or 3 days. d TILs 1377 \nwere isolated from Mettl3F/F (n = 5) and Mettl3CKO (n = 5) mice 12 days after MC38 tumor inoculation. 1378 \nFrequencies of CD8 T cell subpopulations positive for Gzm B, IFN -γ, or Ki-67 were assessed by flow 1379 \ncytometry. e Click-it RNA imaging and analysis of nascent RNA (red) synthesis in Jurkat cells introduced 1380 \nwith WT or mutant YTHDF2. Scale bar, 10 μm (n = 5 per group). f Click-it RNA imaging and analysis of 1381 \nnascent RNA synthesis (red) in Jurkat -shCtrl and Jurkat -shYTHDF2 cells in the presence of 10 μΜ 1382 \nFB23-2 or veh for 72 h (n = 5 per group). Scale bar, 10 μm. g Click-it RNA imaging and analysis of 1383 \nnascent RNA synthesis (green) in Ythdf2F/F and Ythdf2CKO CD8 T cells primed in the presence of 10 μΜ 1384 \nFB23-2 or veh for 72 h (n = 5 per group). Scale bar, 10 μm. h Ythdf2 mRNA levels detected by qPCR 1385 \nin CD8 T cells stimulated with anti -CD3/CD28 (5 μg/ml) for the indicated amount of time (n = 3 per 1386 \ngroup). i m6A-seq and RIP-seq tracks of Ythdf2 mRNA loci on mouse CD8 T cells (left) and Jurkat cells 1387 \n(right). j RNA-seq tracks of Ythdf2 mRNA loci on primedYthdf2F/F and Ythdf2CKO CD8 T cells. Error bars, 1388 \nmean ±  s.e.m. NS, no significance; *P < 0.05; ****P < 0.0001. Two-tailed unpaired Student’s t-test (d). 1389 \nOne-way (e, h) or two-way ANOVA (c, f, g). 1390 \nSupplementary Fig. 10 | Schematic diagra m of YTHDF2 functioning in antitumor CD8 T cells. 1391 \nContrary to the autoregulated Ythdf2 mRNA decay in a quiescent state,  YTHDF2 protein is partially 1392 \nrelocated and swiftly accumulated upon early CD8 T cell activation or reinvigoration. While cytoplasmic 1393 \nYTHDF2 degrades redundant mitochondrial component-encoding mRNAs to sustain T cell persistence, 1394 \nits nuclear translocation is likely to safeguard T cell effectiveness and ICB responsiveness by minimizing 1395 \nIKZF1/3-mediated transcriptional repression . Converse ly, YTHDF2 defect-associated ICB resistance 1396 \ncould be overcome by targeting IKZF1/3. 1397 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nFig. 1 YTHDF2 is selectively upregulated and redistributed in early Teff and Teff-like cells\nTumor-infiltrating CD8 T GSE114300\nb\nYTHDF2\nLamin B\nGAPDH\n0  24      0   24 hAnti-CD3/28\nCytosol   Nucleus\nWT CD8 T\n70\n70\n35\n0  24       0   24 h\ng h\nGSE212357In vitro-generated CD8 T cell subsets \na\nc d\nYTHDF2\nD2 Teff\nD8 Tex\nIsotype\nYTHDF2\nCD44+KLRG1+\nCD44+KLRG1-\nCD44-KLRG1-\nGating on CD8 T cells\nYTHDF2\nSpleen CD8\nTumor CD4\nTumor CD8\nGating on CD3+ T cells\nPD-1+TCF1-KLRG1+ CD8 T\ncIg\nPD-1\n0\n1\n2\n3\n4MFI of YTHDF2 (104)\n✱✱✱\nPD-1\ncIg\nPD-1+TCF1+TIM3- CD8 T\nPD-1\ncIg\ncIg\nPD-1\n0\n2\n4\n6\n8\n10\nMFI of YTHDF2 (104)\n✱✱\nGating on CD8 T cells\nYTHDF2\nB16-OVA tumor D6\nB16-OVA tumor D13\nTpex\nTmem\nTex \nWtap\nRbm15\nAlkbh5\nFto\nYthdf1\nYthdf2\nYthdf3\nIgf2bp1\nIgf2bp2\nIgf2bp3\nYthdc1\nYthdc2\nMettl16\nMettl14\nMettl3\n2\n1\n0\n-1\n-2\n0h\n3h\n24h\n48h\n72h\nD6\nD9 Exhaustion-like\nD9 Memory-like\nWtap\nRbm15\nAlkbh5\nFto\nYthdf1\nYthdf2\nYthdf3\nIgf2bp1\nIgf2bp2\nIgf2bp3\nYthdc1\nYthdc2\nMettl16\nMettl14\nMettl3\ncIg, progressing, early\nPD-1, regressing, early\nPD-1, progressing, early\nPD-1, progressing, late\nPD-1, partially regressing, late\nPD-1, progressing, late\n2\n1\n0\n-1\n-2\nYTHDF2YTHDF2\nSpleen CD8\nTumor CD4\nTumor CD8\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nMFI of YTHDF2 (104)\n✱✱✱\n✱✱\nTex\nTmem\nTpex\n0\n1\n2\n3MFI of YTHDF2 (104)\n✱✱✱\n✱✱\nCD44\n- KLRG1\n- \nCD44\n+ KLRG1\n- \nCD44\n+ KLRG1\n+  \n0\n5\n10\n15\n20\nMFI of YTHDF2 (104)\n✱✱✱\n✱✱\n✱✱\nTex\nTeff\n0\n5\n10\n15\nMFI of YTHDF2 (104)\n✱✱✱\ne f\nB16-OVA tumor D13\nj\n48\n240 12\nDAPIYTHDF2\n96h72\ni\nProgressed\nRegressed\n0.0\n0.1\n0.2\n0.3\n0.4\n N/C ratio\n✱\nProgressed\nRegressed\n0\n10\n20\n30\nYTHDF2+/ CD8 T (%)\n✱\nCD8 DAPIYTHDF2\nProgressed\nYTHDF2\nLamin B\nGAPDH\n0  24      0   24 hAnti-CD3/28\nCytosol   Nucleus\nHuman CD8 T\n70\n70\n35\nDAPIYTHDF2\n cIgGanti-PD-1\ncIgG\nanti-PD-1\n0\n10\n20\n30\n40\nFluorescence intensity of\nYTHDF2\n✱✱✱\ncIgG\nanti-PD-1\n0\n5\n10\n15\n20\n25\nNuclear YTHDF2+ (%)\n✱✱✱\nRegressing\nYTHDF2\nLamin B\nGAPDH\nOVA\nCytosol   Nucleus\nOT-1 CD8 T\n70\n70\n35\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nFig. 2 YTHDF2 is essential for the antitumor effects of CD8 T cells\nh\n0 10 20 30\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nTumor size (mm3) Ythdf2F/F;OT-1\nYthdf2CKO;OT-1\n***\n**\n****\nB16F10-OVA\n0 5 10 15 20 25\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nTumor size (mm3) Ythdf2F/F\nYthdf2CKO\n****\n****\n****\nMC38B16F10\n0 5 10 15 20 25\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nTumor size (mm3) Ythdf2F/F\nYthdf2CKO\n****\n****\n****\n****b c d\ne\nCD45 CD45\nKi-67\n22.6 12.3\n12.8 7.58\nIFN-γ\n21.1 12.7\nGzm B\n46.2\n32.6\nCasp-3\nYthdf2F/F                    Ythdf2CKO         Ythdf2F/F                    Ythdf2CKO         \nMC38 TIL-CD8 T cells\n46.2\n0 5 10 15 20 25\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nTumor size (mm3)\nPBS\nYthdf2F/F;OT-1\nYthdf2CKO;OT-1 ********\n****\nB16F10-OVAi\n0 5 10 15 20 25\n0\n500\n1000\n1500\n2000\n2500\nDays after tumor inoculation\nTumor size (mm3)\nYthdf2F/F+cIg\nYthdf2F/F+anti-PD-1\nYthdf2CKO+cIg\nYthdf2CKO+anti-PD-1 ****\n*******\nj\n0 5 10 15 20\n0\n500\n1000\n1500\n2000\n2500\nDays after tumor inoculation\nTumor size (mm3)\nYthdf2F/F+cIg\nYthdf2F/F+anti-PD-1\nYthdf2CKO+cIg\nYthdf2CKO+anti-PD-1\n****\n**** MC38 Hepa1-6k\nYthdf2F/F\nYthdf2CKO\nCD8 T \nCD4 T\nTreg\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nCell count / mg tumor mass （ 103）\n✱\ng\na\nl Ythdf2F/F                 Ythdf2CKO         \nDextramer\nCD8\n18.3\n 6.63\nMC38 TIL-PD-1+ CD8 T cells\nTIM-3\nCX3CR1\n69.5\n 32.6\n0 5 10 15 20\n0\n500\n1000\n1500\n2000\n2500\nDays after tumor inoculation\nTumor size (mm3) Ythdf2F/F\nYthdf2CKO\n****\n****\n****\nHepa1-6\nf\nCasp-3\nIFN-\nGzm B\nKi-67\n0\n10\n20\n30\n40\n50\nFrequency within CD8 T cells (%)\nYthdf2F/F\nYthdf2CKO\n✱✱✱\n✱\n✱✱\n✱✱\n30.1 18.2\nCD44\nKLRG1\nYthdf2\nF/F\nYthdf2\nCKO\n0\n10\n20\n30\n40\nFrequency within CD8 T (%) ✱✱\nYthdf2F/F                 Ythdf2CKO         \nMC38 TIL-CD8 T cells\nCD44+KLRG1+\nCD101\nTIM-3 4.79 10.1\nYthdf2\nF/F\nYthdf2\nCKO\n0\n2\n4\n6\n8\n10\nFrequency within CD8 T (%) ✱✱\nYthdf2F/F                 Ythdf2CKO         \nPD-1+TIM-3+CD101+\nMC38 TIL-PD-1+ CD8 T cells\nDextramer\nCX3CR1\n+ TIM-3\n+\n0\n20\n40\n60\nFrequency within PD-1+ CD8 T (%)\n✱\n✱✱\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nMrpl58\n1700021F05Rik\nMrpl57\nUqcc2\nMrpl16\nSlc25a33\nMrpl43\nMrpl51\nMrps2\nMrpl23\nMrps34\nMrpl12\nCoa3\nMrps25\nMrpl52\nTsfm\nMrps12\nMrps7\nTefm\nChchd1\nMrps21\n2\n1\n0\n-1\n-2\nPutative targets\nNon-targets\na\n0 2 4 6 8 10\n-Log10 P\nn=29\nn=363\nn=18\nn=51\nn=44\nMitochondrial respiratory \nchain complex assembly\nMetabolic process\nRibosome biogenesis\nTranslation\nMitochondrion organization\nc\nb\ng\ne\nFig. 3 YTHDF2 prevents mitochondrial stress and T cell exhaustion\n-log10P adj\nlog2 (Ythdf2CKO/Ythdf2F/F)\nDownregulated\ngenes (n=455)\nUpregulated\ngenes (n=611)\nYTHDF2-target\ngenes (n=511)\ni\n0\n10\n20\n30\n40\nFrequency within CD8 T (%)\n✱\n✱✱✱PD-1+TIM3+\nNACVeh NACVeh\nYthdf2F/F Ythdf2CKO\nPD-1 \nTIM3\n9.9922.4\n 14.033.3\n15.1\n 7.6\n11.5\n 6.61\nZombie NIR \nCount\n0\n5\n10\n15\n20\nFrequency within CD8 T (%)\n✱\n✱✱✱\nViability Dye+\nNAC   −      +            −      +\nNAC   −      +            −      +\nh\nYthdf2\nF/F\nYthdf2\nCKO\n0\n2\n4\n6\n8MFI of mitoSOX red (104)\n✱\nYthdf2\nF/F\nYthdf2\nCKO\n0.0\n0.1\n0.2\n0.3\n0.4\nMO/MG (relative fold)\n✱\nd\nMO/MGlo\nMO/MGhi\n9.4\n87.9\n20.8\n74.8\nYthdf2F/F                         Ythdf2CKO         \nf\nYthdf2\nF/F\nYthdf2\nCKO\n0\n1\n2\n3\n4\n5MFI of MG (103)\n✱\nYthdf2\nF/F\nYthdf2\nCKO\n0\n1\n2\n3MFI of mitoSOX red (103)\n✱\nMitoSOX\nYthdf2F/F\nYthdf2CKO\nMG \nYthdf2F/F\nYthdf2CKO\nMG MO\nMitoSOX\nCD8\nYthdf2F/F                         Ythdf2CKO         \nNACVeh NACVeh\nYthdf2F/F Ythdf2CKO\nYthdf2CKOYthdf2F/F\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n0 2 4 6 8 10\n-Log10 P\nn=81\nn=64\nn=39\nn=17\nn=5\nn=13\na\nPositive regulation of transcription \nfrom RNA polymerase II promoter\nResponse to hypoxia\nHistone H3-K4 methylation\nTranscription, DNA-templated\nRegulation of transcription, DNA-templated\nCovalent chromatin modification \nd e\nh\nCKO vs F/F Jurkat-KD vs Vec\n-log10FDR\nLog2FC\n0\n20\n40\n60\n80\n0-2.5-5.0 2.5 5.0\n0\n10\n20\n30\nLog2FC\n0-2.5-5.0 2.5 5.0\n-log10FDR\nUp\nDown\nF/F CKO\n0.0\n0.5\n1.0\n1.5\nRelative mRNA expression\n✱✱✱\nRasgrp1\nf\ng\nCenter-2.0 2.0kb\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n1.4\n1.6\nIKZF1-bound loci\nCenter-2.0 2.0kb\n0.25\n0.50\n0.75\n1.00\n1.25\n1.50\n1.75\n2.00F/F1\nF/F2\nCKO1\nCKO2\nIKZF3-bound loci\nTSS-2.0 2.0kb TSS-2.0 2.0kb\nIKZF3-associated promotersIKZF1-associated promoters\nF/F\nCKO\n0.15\n0.20\n0.25\n0.35\n0.30\n0.15\n0.20\n0.25\n0.35\n0.30\nNormalized density (CPM)\nNormalized density (CPM)\ni\nFig. 4 YTHDF2 segregates IKZF1/3 to dictate an active chromatin state in polyfunctional CD8 T cells\nc\nb\n70\n70\nIgG\nYTHDF2\nIP\nInput\nIB: IKZF1\nIP\nInput\n70\n70\nIB: IKZF3\nIgG\nYTHDF2\n70\n70IP\nInput\nIB: YTHDF2\nIgG\nYTHDF2\n70\n70\nIgG\nIKZF1\nIP\nInput\nIB: YTHDF2\nj\nYthdf2CKOYthdf2F/F\n210-1-2\nKdm5b\nKmt2e\nKat2b\nKdm5a\nTet2\nKmt2a\nAsh1l\nKmt2d\nEzh1\nSox4\nKlf7\nStat5a\nKdm7a\nSetd1a\nKdm3a\nFoxo3\nNfat5\nIl12a\nCd226\nPtprf\nMap3k8\nPtpn4\nHivep2\nRasgrp1\nTnfaip3\nEpigenetic \nregulators\nTranscriptional \nfactors\nTCR signal \nengagers\nStat5a\nF/F CKO\n0.0\n0.5\n1.0\n1.5\nRelative mRNA expression\n✱✱✱\nStat5a\nATAC-seq\nCKO1\nCKO2\nF/F1\nF/F2\nCKO\nF/F\nH3K4me-\nCUT&RUN\nRasgrp1\nATAC-seq\nCKO1\nCKO2\nF/F1\nF/F2\nCKO\nF/F\nH3K4me-\nCUT&RUN\nIgG\nIKZF3\n70\n70\nIP\nInput\nIB: YTHDF2\nFlow\n70\n70\n70\nYTHDF2\nIKZF1\nIKZF3\nIgG\nYTHDF2\nYTHDF2/IKZF1\nWT CD8 T             OT-1 CD8 T               \nYTHDF2/IKZF3\nHuman CD8 T\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n0 5 8 11 13 16 19\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nMean tumor size (mm3) Ythdf2F/F+anti-PD-1+veh\nYthdf2CKO+anti-PD-1+veh\nYthdf2F/F+anti-PD-1+ len\nYthdf2CKO+anti-PD-1+ len\n********\n****\n0\n20\n40\n60\n80\nFluorescence intensity\n✱✱✱\n✱✱✱\nNS\nF/F;OT-1\nCKO;OT-1\nEU DAPI\nc\n0\n1\n2\n3Ki-67 MFI (104)\n✱✱\n✱✱\nNS d\nVeh\nLen\nHepa1-6\ng\nF/F;OT-1\nCKO;OT-1\na b\nf\nF/F CKO\nEU\n0\n20\n40\n60\n80\n100\nFluorescence intensity\n✱✱✱\n✱✱✱\nNS\n0\n1\n2\n3\n4Ki-67 MFI (104)\n✱✱\n✱✱✱\nNS\nF/F\nCKO\nDAPI\n0 6 9 12 15 17 21 24 27\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nMean tumor size (mm3) Ythdf2F/F+anti-PD-1+veh\nYthdf2CKO+anti-PD-1+veh\nYthdf2F/F+anti-PD-1+len\nYthdf2CKO+anti-PD-1+len\n****\nYthdf2F/F+len\nYthdf2CKO+len\n****\n****\n****\n Len    −        +               −        + Len    −        +               −        +\nLen    −        +               −        + Len    −        +               −        +\nMC38\nFig. 5 Lenalidomide retrieves the anti-tumor function of YTHDF2-deficient CD8 T cells \nVeh\nLen\ne\nh\nLen    −        +               −        +\n0\n20\n40\n60\n80\nFrequency within CD8 T (%)\n✱✱✱\n✱✱\n✱ Len    −        +               −        +\nGzm B \n0\n20\n40\n60\nFrequency within CD8 T (%)\n✱✱✱\n✱✱\nNSIFN-γ\nKi-67\nVeh\nLen\nLen\nVeh\nCKO;OT-1\nKi-67\nF/F\nCKO\nIFN-γ\nCD8\n50.840.1 47.015.4\nLenVeh LenVeh\nYthdf2F/F Ythdf2CKO\n50.4 66.9\n 52.114.0\nGzm B \nCD8\nVeh\nLen\nLen\nVeh\nF/F;OT-1\n27.810.1\n28.1\nanti-PD-1+lenanti-PD-1+veh\nYthdf2CKO\nGzm B \nCD8\nYthdf2F/F\nanti-PD-1+len\n24.6\nanti-PD-1+veh\n13.6\n 37.0\n48.5\n20.5\nIFN-γ\nCD8\nanti-PD-1\nCombo\nanti-PD-1\nCombo\n0\n10\n20\n30\nFrequency within PD-1+ CD8 T (%) ✱✱✱\n✱✱\nGzm B \nanti-PD-1\nCombo\nanti-PD-1\nCombo\n0\n20\n40\n60\nFrequency within PD-1+ CD8 T (%) ✱✱✱\n✱IFN-γ\nF/F\nCKO\nF/F\nCKO\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\na c\nYTHDF2/IKZF1\nshCtrl shMETTL3 #1 shMETTL3 #2\ne\nGAPDH\nYTHDF2\nCytoplasm          Nucleus  \nLamin B\n70\n70\n35\nf\n0\n1\n2\n3\n4\n5Ki-67 MFI (104)\n✱✱✱\n✱✱\n✱✱\nCtrl\nYTHDF2 OE\nshMETTL3 \nYTHDF2 OE\n+ shMETTL3\nKi-67\nYTHDF2 OE\nYTHDF2 OE\n+shMETTL3 shMETTL3 Ctrl\nEU DAPI GFP Merge \nFig. 6 The m6A machinery regulates both YTHDF2 relocation and expression\nOE W432A W486A\nFlag/IKZF1\nd\n0\n5\n10\n15\n20\n25\nFluorescence intensity\n✱✱✱\n✱✱\n✱\nj k\n0 6h\n0\n2\n4\n6\n8Relative  mRNA expression NS✱✱✱\n✱✱✱\n✱✱Ythdf2\nWT\n-249\nWT  -249         WT  -249            WT  -249           WT   -249 \n0                    24h                       0                       24h\nCytoplasm                                   Nucleus   \nGAPDH\nYTHDF2\nLamin B\nl\n70\n70\n35\n0\n1\n2\n3Relative mRNA expression\n✱✱✱\n✱\nCD3/28:       −          +         −          +\nActD:       −          −         +          +\nb\ng\nih\nYthdf2\nATAC-seq\n0 h_1\n0 h_2\n0 h_3\n5 h_1\n5 h_2\n5 h_3\nActDVeh\nMettl3F/F                          Mettl3CKO\nYTHDF2/IKZF1YTHDF2/IKZF1\nYthdf2\nt1/2=2.1100\nR2=0.8630\nt1/2=1.2520\nR2=0.9006\n0 1 2 3 4\n0.0\n0.5\n1.0\nYthdf2 mRNA remaining -249\nWT\nP= 0.009\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nPR\nSD\nCR\nPD/SD\nPR/CR\n0\n2\n4\n6\n8MFI of YTHDF2 in CD8 T cells\n✱\nCD8 DAPIYTHDF2\nPD/SD\nPR/CR\n0\n5\n10\n15\n20\n CD8 T count / HPF (100×) P = 0.2272\nPR\nCR\nSD\nPD/SD\nPR/CR\n0\n5\n10\n15\nYTHDF2+/ CD8 T cells (%) ✱\nPD/SD\nPR/CR\n0\n5\n10\n15\n20\n CD8 T count / HPF (100×) P =0.6518\ne\nf\nd h i\nj\nFig. 7 YTHDF2 expression are associated with T cell function in human cancers\nPD/SD\nPR/CR\n0\n10\n20\n30\n40\nYTHDF2+/ CD8 T cells (%) ✱✱\nPD/SD\nPR/CR\n0\n2\n4\n6\n8\n10\nMFI of YTHDF2 in CD8 T cells ✱\nCD8 DAPIYTHDF2\ng k\na CRC (GSE146771) HCC (GSE206325)PDAC (GSE155698)\nResponder Non-responder\n1.0\n1.5\n2.0\n2.5\n1.0\n2.0\n1.0\n2.0\n3.0\nYTHDF2 expression level\nYTHDF2 expression level\nYTHDF2 expression level\nHigh Low\nP = 8.01e−4 P = 1.59e−11 P = 2.99e−04\n0\n3\n1\n2\n4\nYTHDF2 expression level\nP = 3.98e−24\nP = 3.41e−291\n0\n3\n1\n2\n4\nYTHDF2 expression level\nP = 0.368\nP = 1.45e−163\nMelanoma (GSE120575)\nb\nHigh Low High Low\nPre-treatment\nYTHDF2 expression level\n1.0\n1.5\n2.0\n2.5\n0.5\nNon-responder Responder\nP = 4.06e−1 P = 4.04e−4\nPost-treatment\n1.0\n1.5\n2.0\n2.5\n0.5\nYTHDF2 expression level\nNon-responder Responder\nHCC (GSE206325)\n1.0\n2.0\n3.0\nYTHDF2 expression level\nNon-Responder Responder\nP = 8.58e−45\nc\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nGSE29797\nSupplementary Fig. 1 YTHDF2 is characteristically expressed upon T cell activation and reinvigoration\na b c\nGSE132477\nMouse CD4 T\nα-CD3/28:           0                           4hα-CD3/28:      0         4       8h\nMouse CD8 T GSE70393\nWtap\nRbm15\nAlkbh5\nFto\nYthdf1\nYthdf2\nYthdf3\nIgf2bp1\nIgf2bp2\nIgf2bp3\nYthdc1\nYthdc2\nMettl16\nMettl14\nMettl3\nWtap\nRbm15\nAlkbh5\nFto\nYthdf1\nYthdf2\nYthdf3\nIgf2bp1\nIgf2bp3\nYthdc1\nYthdc2\nMettl16\nMettl14\nMettl3\nWtap\nRbm15\nAlkbh5\nFto\nYthdf1\nYthdf2\nYthdf3\nIgf2bp1\nIgf2bp2\nIgf2bp3\nYthdc1\nYthdc2\nMettl14\nMettl3\nNaïve\nActive\n0.30\n0.35\n0.40\n0.45\n0.50\nm6A/A in mRNA (%)\n✱✱\ng h\nMouse CD4 T cell subsets\nf\nYTHDF2\nβ-Actin\nOVA: 0 6 12 24h\nβ-Actin\nYTHDF2\nα-CD3/28: 0 6 12 24h\nMouse CD8 T cells\ne\nYTHDF2\nβ-Actin\nMETTL14\nFTO\nALKBH5\nMETTL3\nHuman CD8 T cells\nα-CD3/28: 0 6 12 24h\nGSE110249\nd\nMettl3\nMettl14\nMettl16\nWtap\nRbm15\nAlkbh5 Fto\nYthdf1\nYthdf3\nIgf2bp1\nIgf2bp2\nIgf2bp3\nYthdc1\nYthdc2\n0\n20\n40\n60\nmRNA (FKPM)\nOVA-specific CD8 TIL\ncIg\nAnti-PD-1\nk\nl\nNucleusCytoplasm\nPHA: 0     24             0     24 h\nβ-actin\nLamin B\nYTHDF2 70\n70\n45\nNaive\nActive\n5’UTR 3’UTRCDS\nRegion\n0.0\n0.5\n1.0Density\nNaï ve\nP = 1×10-620 \n(n = 8166)\nActive\nP = 1×10-870 \n(n = 10302)\nm\nn\nB16F10\nB16F10-OVA\n0\n5\n10\n15\n20\n25\nYTHDF2+/ CD8 T (%)\n✱\nB16F10\nB16F10-OVA\n0.0\n0.1\n0.2\n0.3\n0.4\n N/C ratio\n✱\nanti-CD3/28: 0                            12                            24                         48 hi\nj\n0 12 24 48 72 96h\n0\n10\n20\n30\n40\nNuclear YTHDF2+ (%) ✱✱\n✱✱✱\n0 12 24 48 72 96h\n0\n1\n2\n3Fluorescence intensity\n✱\n✱✱\n✱✱\ncIg\nanti-PD-1\n0\n10\n20\n30\n40\nYTHDF2+/ CD8 T cells (%) ✱\ncIg\nanti-PD-1\n0.0\n0.1\n0.2\n0.3\n0.4\n0.5\n N/C ratio\nB16F10-OVA\n+ cIg\nB16F10-OVA\n+ anti-PD-1\nCD8 DAPIYTHDF2\nDAPIYTHDF2\nCD8 DAPIYTHDF2\nB16F10 B16F10-OVA\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nF/F   CKO    F/F   CKO    F/F   CKO   F/F   CKO    F/F   CKO    F/F   CKO    F/F   CKO\nHeart Lung Liver Stomach Pancreas Intestine Thymus\nYTHDF2\nβ-Actin\nYthdf2F/F;dLckcre (T cell CKO) mice\nYTHDF2\nβ-actin\nSplenic CD8 T cells\n0 24h\nF/F CKO    F/F   CKO\na b\nc Spleen Peripheral blood\nd\nSupplementary Fig. 2 Phenotyping for the YTHDF2 conditional knockout mice\nYthdf2F/F\nYthdf2CKO\ndLckCre\nCD3\n+ T \nCD8\n+ T \nCD4\n+ T\nTreg\n0\n100\n200\n300\n400\nCell counts (103)\nCD3\n+ T \nCD8\n+ T \nCD4\n+ T\nTreg\n0\n20\n40\n60\nCell counts (105)\nCD3\n+ T \nCD8\n+ T \nCD4\n+ T\nTreg\n0\n1000\n2000\n3000\n4000\n5000\nCell counts\n✱\n✱\nThymus\n4.51%\n100%\nYTHDF2\nGating on CD8 T cells\nB16-OVA tumor D13\nYthdf2\nF/F\nYthdf2\nCKO\n0\n50\n100\n150\nYTHDF2+ (%)\n✱✱✱ Ythdf2F/F\nYthdf2CKO\ne\nMC38\n0 5 10 15 20 25\n0\n500\n1000\n1500\nDays after tumor inoculation\nTumor size (mm3) dLckCre\nYthdf2CKO\n****\n****\n****\n****\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\na b\nCD8 T \nCD4 T\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\nCell count (106)\n✱\n✱\nMC38 dLN\nCD8 T \nCD4 T\n0\n10\n20\n30\n40\n50\nFrequency within CD45 cells (%)\n✱\nCD8 T \nCD4 T\nTreg\n0.0\n0.5\n1.0\n1.5\n2.0\nCell count / mg tumor mass （ 103）\n✱\n✱✱✱\nc Hepa1-6 TIL\nMC38 TIL \nCD8 T \nCD4 T\nTreg\n0\n10\n20\n30\n40\n50\nFrequency within CD45 cells (%)\ne\nIFN-γ\nCD8\n50.0\n70.1\n87.3\n92.5\nGzm B \n21.2\n30.1\nKi-67\nMC38 TIL-CX3CR1+PD-1+TIM-3+ CD8 T cells\nYthdf2CKO Ythdf2F/F\nCD8+ PD-1+ Dextramer+T cells\nd\nT cellsT cells\nYthdf2\nF/F\nYthdf2\nCKO\n0\n1\n2\n3Frequency within CD8 T (%)\n✱✱\nCD101\nPD-1\n5.20\n 11.2\nYthdf2\nF/F\nYthdf2\nCKO\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nFrequency within CD8 T (%)\n✱✱\nYthdf2F/F                 Ythdf2CKO         \nPD-1+TIM-3+ CD8 T cells\nHepa1-6 TIL-\nIFN-γ\nGzm B\nKi-67\n0\n10\n20\n30\n40\n50\nFrequency within CD8 T (%) ✱✱✱\n✱✱✱\n✱\nCD8 T \nCD4 T\nTreg\n0\n5\n10\n15\n20\n25\nFrequency within CD45 cells (%)\nSupplementary Fig. 3 YTHDF2 deprivation hinders antitumor CD8 T cell response\nf\n0 5 10 15 20 25\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nTumor size (mm3) Ythdf2F/F+anti-CD8\nYthdf2CKO+anti-CD8\nNS\n0 5 10 15 20 25\n0\n500\n1000\n1500\nDays after tumor inoculation\nTumor size (mm3) Ythdf2F/F+anti-CD4\nYthdf2CKO+anti-CD4\n****\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\n24 72 120h\n0\n20\n40\n60\n80\nFrequency within CD8 T (%) ✱\n✱\n✱\n24 72 120h\n0\n5\n10\n15\n20\n25\nFrequency within CD8 T (%)\nAnnexin V \nPI\nYthdf2CKO\nYthdf2F/F\nα-CD3/28: 5 μg/ml,              24                               72                           120h\n0 24 72 120h\n0\n50\n100\n150\nCelltrackerlo CD8 T (%)\n✱\n✱\nYthdf2CKO\nα-CD3/28: 2.5 μg/ml,         0                         24                        72                       120h\nYthdf2CKO\nIFN-γ Gzm B Ki-67\nAnnexin Ⅴ+PI+ Annexin Ⅴ+PI-\nYthdf2F/F\nYthdf2F/F\n37.7\n10.9\n25.1\n9.67\n53.4\n22.0\n62.6\n14.9\n36.7\n11.3\n29.4\n13.0\n0.02\n0.015\n0.12\n9.83\n11.8\n72.6\n79.1\n96.7\nCTV \nIFN-γ\nCountCD8\nCelltrackerlo\n9.64\n23.1\n15.1\n46.4\n16.3\n55.3\nGzm B Ki-67 \na\nb\nc\nd\nYthdf2F/F\nYthdf2CKO\nSupplementary Fig. 4 YTHDF2 maintains CD8 T cell function and persistence in vitro\nYthdf2CKO\nPD-1 TIM-3 CD101\nYthdf2F/F\n54.418.4\nTIM-3 CD101 \nYthdf2\nF/F\nYthdf2\nCKO\n0\n10\n20\n30\n40\nFrequency within CD8 T (%)\n✱✱\n26.8\nYthdf2\nF/F\nYthdf2\nCKO\n0\n20\n40\n60\n80\n100\nFrequency within CD8 T (%)\n✱✱\nPD-1\nCD8\n79.3\n91.6\nYthdf2\nF/F\nYthdf2\nCKO\n0\n20\n40\n60\n80\n100\nFrequency within CD8 T (%)\n✱✱\n 84.7\nYthdf2\nF/F\nYthdf2\nCKO\n0\n10\n20\n30\nFrequency within CD8 T (%)\n✱✱\nYthdf2\nF/F\nYthdf2\nCKO\n0\n20\n40\n60\nFrequency within CD8 T (%)\n✱✱\nYthdf2\nF/F\nYthdf2\nCKO\n0\n20\n40\n60\nFrequency within CD8 T (%)\n✱✱\n22.2\n22.0\nCD122 \n86.9\n87.1\nCCR7\n88.6\n88.4\nCD62L\nYthdf2CKO\nYthdf2F/F\nCD8\nYthdf2\nF/F\nYthdf2\nCKO\n0\n20\n40\n60\n80\n100\nFrequency within CD8 T (%)\nYthdf2\nF/F\nYthdf2\nCKO\n0\n20\n40\n60\n80\n100\nFrequency within CD8 T (%)\nYthdf2\nF/F\nYthdf2\nCKO\n0\n10\n20\n30\n40\nFrequency within CD8 T (%)\nCD62L CCR7 CD122\ne\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nSupplementary Fig. 5 YTHDF2 deficiency impairs the mitochondria function of CD8 T cells\nb\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n80\n100\n120\n140\nBasal OCR\n(pmol/min)\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n0\n50\n100\n150\n200\nMaxima OCR\n(pmol/min)\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n0\n20\n40\n60\nSpare Respiratory Capacity\n(maximal OCR - basal OCR)\n0 20 40 60 80\n0\n50\n100\n150\n200\nTime (minutes)\nOCR (pmol/min)\nYthdf2F/F;OT-1\nYthdf2CKO;OT-1\nOligo FCCP Rot\na\n0 20 40 60 80\n0\n50\n100\n150\nTime (minutes)\nECAR (mpH/min)\nYthdf2F/F;OT-1\nYthdf2CKO;OT-1\nGlucose Oligo 2-DG\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n0\n20\n40\n60\nGlycolysis\n(pmol/min)\n✱\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n0\n20\n40\n60\n80\n100\nGlycolytic Capacity\n(pmol/min)\n✱\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n0\n10\n20\n30\n40\n50\nGlycolytic Reserve\n(pmol/min)\n✱\nYthdf2\nF/F ;OT-1\nYthdf2\nCKO ;OT-1\n0\n10\n20\n30\nNo. of mitochondria\n/ cell\n✱\nYthdf2F/F;OT-1 Ythdf2CKO;OT-1\nc d\nIFN-γ\nCD8\nNACVeh NACVeh\nYthdf2F/F Ythdf2CKO\n14.1\n 10,4\n39.9\n 60.7\n52.0\n 66.9\n 12.8\n 13.5\nGzm B \nCD8\n0\n20\n40\n60\n80\nFrequency within CD8 T (%)\n✱\n✱\nNAC   −      +            −      +\nIFN-γ\n0\n20\n40\n60\n80\nFrequency within CD8 T (%) ✱\n✱\nNAC   −      +            −      +\nGzm B \n0\n2\n4\n6\n8MFI of Ki-67 (104)\n✱\n✱\nNAC   −      +            −      +\nKi-67\nKi-67\nF/F\nCKO\nVeh\nNAC\nNAC\nVeh\ne\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nc\nYTHDF2\nFlag\nGAPDH\nVec OE\n70\n35\n70\nk m\nYTHDF2\nGAPDH\n70\n100\n35\ng\n0\n10\n20\n30\n40\nMFI of mitoSOX red (104)\n✱✱✱ ✱\n✱✱✱\n✱✱\n✱\nn\n0.0\n0.5\n1.0\n1.5\n2.0\nMO/MG (relative fold)\n✱✱\n✱✱\n✱✱✱\nVec\nOE (WT)\nW432A\nW486A\n0\n1\n2\n3MO/MG (relative fold) ✱✱✱\n✱\n✱\nSupplementary Fig. 6 The m6A machinery is necessary for YTHDF2-regulated mitochondrial fitness\nshMETTL3\nshCtrl\nYTHDF2 OE\nFlag\nMETTL3\nGAPDH\nYTHDF2 70\n70\n35\n75\nYTHDF2 OE+\nshMETTL3\nl\nW486A\nW432A\nOE\nVec\n70Flag\nh\nGAPDH 35\nd\n0 5 10 15\n-Log10 P\nrRNA processing n = 72\nrRNA metabolic process n = 89\nRegulation of T cell activation n = 69 \nMitochondrial gene expression n = 43\nRibosome biogenesis n = 92\ni j\ne f\nCtrl\nOE\nMitosox\nVec ctrl\nYTHDF2 OE  \n0\n5\n10\n15\n20\nMFI of mitoSOX red (104)\n✱✱✱\nshCtrl\nshYTHDF2\n0\n2\n4\n6\n8MFI of mitoSOX red (104) ✱✱✱\nshCtrl\nshYTHDF2\nMitosox\nVec ctrl\nYTHDF2 OE  \n0\n2\n4\n6\n8\n10\nMFI of MitoTracker Green (104) ✱\nMG\nshCtrl\nshYTHDF2\nshCtrl\nshYTHDF2\n0\n1\n2\n3\n4MFI of MitoTracker Green (105) ✱✱\nMG\nCtrl\nOE\nCoa3 Mrpl16 Mrps12 Tefm\nInput\nm6A-IP\nYTHDF2-RIP\na\nb\n0 1 2 3 4\n0.0\n0.5\n1.0\nCoa3 mRNA remaining\nP= 0.0304\nCoa3\nt1/2=2.456\nR2=0.9456\nt1/2=1.987\nR2=0.9330\n0 1 2 3 4\n0.0\n0.5\n1.0\nMrps12 mRNA remaining\nP= 0.0092\nMrps12\nt1/2=0.8354\nR2=0.9847\nt1/2=0.5350\nR2=0.9933\n0 1 2 3 4\n0.0\n0.5\n1.0\nMrpl16 mRNA remaining\nP= 0.0086\nMrpl16\nt1/2=0.6400\nR2=0.9637\nt1/2=0.3711\nR2=0.9813\nTefm\nt1/2=0.4067\nR2=0.9581\nt1/2=0.2539\nR2=0.9920\n0 1 2 3 4\n0.0\n0.5\n1.0\nTefm mRNA remaining Ythdf2CKO\nYthdf2F/FP= 0.0017\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nF/F CKO\na b\nF/F\n CKO\n0\n20\n40\n60\n80\nFluorescence intensity\n✱✱✱\nF/F;OT-1 CKO;OT-1\nF/F;OT-1\n CKO;OT-1\n0\n20\n40\n60\n80\nFluorescence intensity\n✱✱✱\nEU\nDAPI \nEU\nDAPI \nd\nf\ne\nh\ni j\nCenter-2.0 2.0kb\n0.2\n0.4\n0.6\n0.8\n1.0 Vec1\nVec2\nKD1\nKD2\nIKZF1-bound loci\nNormalized density (CPM)\nSupplementary Fig. 7 Acute T cell activation enlists nuclear functionality of YTHDF2\nYthdf2F/F\nYthdf2CKO\n0\n10\n20\n30Fluorescence intensity\nns\n✱✱✱\n✱\nα-AMN:   −        +               −        +\nc\nCumulative fraction\nLog2(TE)\nPutative target genes\n0.00\n0.25\n0.50\n0.75\n1.00\n-5 0 5\nCKO\nF/F\nIP       IgG\nYTHDF2\nIKZF1\nIKZF3\nDNase\nRNase - - + \n- +        -\nYTHDF2\ng\nF/F CKO\nEU\nVehα-AMN\nDAPI\nYTHDF2/IKZF1\nJurkat\nYTHDF2/IKZF3\n0 24h\n0.0\n0.5\n1.0\n1.5\nRelative mRNA expression ✱✱\n0 24h\n0.0\n0.5\n1.0\n1.5\nRelative mRNA expression ✱✱\nIkzf1 Ikzf3\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nVec\nshIKZF1/3\nshYTHDF2\nEU DAPI\nshYTHDF2/\nIKZF1/3\nd\n0\n10\n20\n30\n40\nFluorescence intensity ✱\n✱\nshCtrl shYTHDF2\nVeh\nLen\n0\n10\n20\n30Fluorescence intensity\n✱\n✱\nNS\nf\nEU DAPI\n Len    −        +               −        +\nshCtrl\nshYTHDF2\nLenVeh\nLenVeh\nYthdf2F/F\nYthdf2CKO\nPD-1 \nTIM3\n23.0 22.7\n31.7 30.2\n0\n10\n20\n30\n40\nFrequency within CD8 T (%)\nLen    −      +            −      +\nPD-1+TIM3+\ni\nSupplementary Fig. 8 IKZF1/3-associated transcriptional repression in YTHDF2-deficient T cells\nHDAC1/IKZF1HDAC1/IKZF3\nYthdf2F/F Ythdf2CKO\nc\ne\nj\nIKZF1\nYthdf2\nF/F\nYthdf2\nCKO\n0\n5\n10\n15\n20\nMFI of IKZF1 (104)\nNS\nIKZF3\nYthdf2\nF/F\nYthdf2\nCKO\n0\n1\n2\n3\n4\n5MFI of IKZF3 (104)\nNS\na b\nTSS-3.0 3.0kb\nIKZF1 CUT&RUN\n15\n20\n10\nIKZF3 CUT&RUN\nTSS-3.0 3.0kb\n10\n12\n8\n6\n4\nF/F\nCKO\nF/F\nCKO\nIKZF1\nIKZF3\nGAPDH\nJurkat\nLen - 100 100 10 10 µ m\n24 6 24 6 h\n55\n70\n55\n40\n35\nKD\nF/F CKO\nVeh\nLen\nIKZF3 DAPI\nF/F CKO\nVeh\nLen\nIKZF1 DAPI\n0\n2\n4\n6\n8\n10Fluorescence intensity of IKZF3\n✱✱✱\n✱✱✱\nNS Len    −        +               −        +\n0\n5\n10\n15Fluorescence intensity of IKZF1\n✱✱✱\n✱✱✱\nNSLen    −        +               −        +\ng h\nNormalized density Normalized density \n0.0\n0.5\n1.0\n1.5\nRelative mRNA Experssion(fold)\n✱✱✱\n✱✱✱\nLen    −        +               −        +\nStat5a Rasgrp1\n0.0\n0.5\n1.0\n1.5\nRelative mRNA Experssion(fold) ✱✱✱\n✱✱✱\nLen    −        +               −        +\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nb\nc\nW432A\nW486A OE (WT) Vec\nEU DAPI GFP Merge\na\nVec OE\nW432A\nW486A\n0\n10\n20\n30\n40\nFluorescence intensity\n✱\n✱✱\n✱ e\nIFN-\nGzm B\nKi-67\n0\n5\n10\n15\n20\n25\nFrequency within CD8 T cells (%)\nMettl3F/F\nMettl3CKO\n✱\n✱✱ ✱✱\n0 5 10 15 20 25\n0\n500\n1000\n1500\n2000\nDays after tumor inoculation\nTumor size (mm3) Mettl3F/F\nMettl3CKO\n****\n****\n****\n*\nd\nMETTL3\nGAPDH 35\n75\nshCtrl shYTHDF2\nVeh\nFB23-2\n10\n15\n20\n25\n30\nFluorescence intensity\nNS\nNS\nEU DAPI FB23-2    −        +               −        +\nshCtrl\nshYTHDF2\nF/F CKO\n0\n20\n40\n60\n80\nFluorescence intensity\nNS\nNS\nFB23-2    −        +               −        +\nVeh\nFB23-2\nEU DAPI\nF/F\nCKO\nf g\nATAC-seq_Up\nIKZF1/3-target\nRNA-seq_Down\nm6A-Me-RIP\nYTHDF2-RIP\n230\nSupplementary Fig. 9 YTHDF2 distribution and expression are governed by the m6A machinery \nYthdf2\nm6A-seq\nIP1\nIP2\nInput1\nInput2\nRIP-seq\nIP1\nIP2\nInput1\nInput2\ni\n0 6 12 24h\n0.0\n0.5\n1.0\n1.5\n2.0\n2.5\nRelative mRNA expression ****\n****\n*\nYthdf2\nh\nYthdf2\nj\nYthdf2\nCKO1\nCKO2\nF/F1\nF/F2\nMouse CD8 T cells Jurkat cells\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint \n\nAAAAA\nTn or Tpex \nTeff-like\nAAAAA\nLenalidomide \nIKZF1/3\nMitochondrion-\nrelated genes\nTeff or Teff-like \nYthdf2 mRNA\nAAAAA\nYTHDF2\nm6A\nAAAAA\nYthdf2 Nucleus\nAAAAA\nMitochondrial fitness\n& T cell persistence\nAntigen or ICB\nAAAAA\nStat5a\nRasgrp1\nIKZF1/3\nIKZF1/3\nMitochondrial\n molecules\nYTHDF2-deficient\nYTHDF2-competent\nT cell polyfunctionality \nMitochondrial stress\n& T cell exhaustion\nActive chromatin\nInactive chromatin\n* * *\nm6A\nAAAAA\nT cell polyfunctionality T cell polyfunctionality \nTeff or Teff-like \nActive chromatin\nICB\n+\n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted July 16, 2024. ; https://doi.org/10.1101/2024.07.11.603088doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}