Peptide-stimulation of autologous T cells reverts immune checkpoint inhibitor resistance of hypermutated colorectal cancer cells

21 Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating 22 T cells available were selected for detailed investigation of immunological response. 23 T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines 24 and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors 25 (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI -9) was detected in 26 tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, 27 only p re-stimulation with tumor -specific peptides (cryptic and neoantigenic) significantly increased 28 recognition and elimination of tumor cells by T cells independently of ICI or PI-9i. 29 We showed, that ICI resistant tumor cells can be targete d by tumor -primed T cells and also 30 demonstrated the superiority of tumor -naïve peripheral blood T cells compared to highly exhausted 31 tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal 32 strategies to successfully induce durable anti-tumor immune responses. 33 34 1. Introduction 35 The interaction of tumor with immune cells triggers dynamic adaption to evade immunosurveillance. 36 A prominent example is tumors’ genetic or epigenetic modifications to reduce or switch-off HLA class 37 I expression, thus preventing detection by T cells. Depending on the tumor entity, up to 80% of solid 38 tumors have completely lost HLA class I expression 1. Another tool to repress antitumor immune 39 responses is the expression of immunomodulatory proteins like programmed cell death protein 1 (PD-40 1), cytotoxic T lymphocyte associated protein 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3) or 41 their respective ligands in the tumor microenvironment 2. Via these immune checkpoint molecules , 42 tumor cells utilize the physiologicall y programmed down -modulation of activated immune cells. 43 However, therapeutic interfering with these immunosuppressive mechanisms via immune checkpoint 44 inhibitors (ICI) represents the latest revolution in cancer treatment. 45 The application of ICI is mostly limited to tumors with high mutational burden (TMB), since clinical 46 trials proved highest response rates in this patient group (reviewed in 3). However, despite preselection 47 of patients, studies with microsatellite instable colorectal cancer (CRC) revealed , that up to 70% of 48 patients do not benefit from pembrolizumab 4, nivolumab 5 or durvalumab 6 treatment. Even though 49 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 3 combining ipilimumab and nivolumab raised patients’ objective response rates to 55% 7, still a 50 substantial number of hyper-mutated tumors resist ICI treatment. 51 High TMB and constitutive HLA class I expression are b oth favorable prognostic markers for 52 immunotherapy 8,9, but ICI therapy response prediction remains unsatisfactory. High TMB of ≥10 53 mutations/Mb is insufficient to predict ICI success, neither across cancer entities nor within a specific 54 one 10,11. However, the combination of TMB and loss of heterozygosity in HLA class I improved 55 differentiation between ICI therapy responders and non -responders in lung cancer 12,13. Similarly, a 56 score retrospectively determining tumor immunogenicity by TMB combined with antigen processing 57 machinery gene expression improved ICI therapy response prediction in urothelial cancer and 58 melanoma datasets 14. 59 T cells interaction with tumors can further be impaired by several other immunosuppressive 60 mechanisms beyond dysfunctional antigen presentat ion. Malignant cells often secrete immune 61 regulatory molecules, e.g. cytokines, which exert immunosuppressive effects in the tumor 62 microenvironment 15,16. Likewise, enzymes and enzyme inhibitors with a role in regulating immune cell 63 activity have been identified as complementary tumor immune evasion strategies 17,18. 64 Here, we selected two microsatellite instable CRC cases exhibiting constitutive HLA expression and 65 investigated the interaction of T cells with autologous tumor cells ex vivo. Co-culture of tumor cells 66 with autologous peripheral (pTc) or tumor-infiltrating T cells (TiTc) was applied to test the antitumor 67 effect of isolated T cells alone or in combination with immune modulatory agents matching tumor 68 cells’ characteristics. Moreover, effects of individualized inhibition of immunosuppress ion were 69 directly compared to anti -tumor immune responses of autologous T cells recognizing tumor-specific 70 antigenic peptides. 71 2. Results 72 2.1 Selection of tumor cases 73 For a detailed analysis of tumor and T cell interactions, appropriate patient biomaterials were selected 74 from our comprehensive set of CRC models available at the BioBank Rostock 19. 75 Considering all decisive factors (biomaterial availability, frequent non-synonymous mutations, antigen 76 processing, HLA class I expression and HLA class II inducibility), the two CRC cases HROC113 and 77 HROC285 were selected. Both tumors showed high TMB (HROC113: 176 mutations/Mb; HROC285: 212 78 mutations/Mb) and microsatellite instability (MSI). Tumor cell lines were established directly from the 79 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 4 patient’s tumor (HROC113) or from a patient-derived xenograft (HROC285 T0 M2). TiTc and pTc as well 80 as B-LCLs were also available for both patients. 81 82 2.2 T cells do not get activated by co-culture with autologous tumor cells 83 Co-culture experiments of T cells and autologous tumor cells were performed to investigate if the 84 tumors with high TMB and HLA expression can induce sufficient immune responses. Tumor cells were 85 incubated with T cells, which were pre -treated as follows: 1) expanded T cells, 2) T cells, which were 86 expanded and co-cultured with autologous tumor cells for 14 days and 3) T cells, which were expanded 87 and cultured for 14 days in the same conditions (temperature, media, supplements, change of media) 88 but without tumor cells (= control culture with tumor-naïve T cells). In the degranulation assay (Figure 89 1A), the amount of T cells recognizing tumor cells (CD8+/CD107a+/IFNγ+ cells) was larger in co-cultured 90 compared to unspecifically expanded pTc HROC113 (p=0.04) . Similarly, more degranulating T cells 91 were detected among tumor-naïve pTc HROC113 from the control culture than among unspecifically 92 expanded pTc HROC113 (p=0.02). There was no significant difference observed between the reactivity 93 of co-cultured and control-cultured pTc HROC113 (p>0.05; Figure 1A). The TiTc HROC113 showed a 94 similar pattern (Figure 1B) and corroborated the previous results (pco-culture vs expanded Tc=0.02; pcontrol-culture 95 vs expanded Tc=0.03; pco-culture vs control culture>0.05). In contrast, the population of tumor -reactive cells among 96 pTc HROC285 was similar in all three tested conditions (unspecific expansion, co -culture, control 97 culture) and no distinct differences were observed (Figure 1C). TiTc HROC285 were also assessed using 98 this experimental approach but unfortunately the proportion of CD8+ cells, being as low as 27% in the 99 beginning, even decreased further during the cultivation period and no reliable measurements of 100 CD8+/CD107a+/IFNγ+ cells could be obtained. 101 102 2.3 ICI do not increase the recognition of autologous tumor cells by T cells 103 Since tumor cells failed to induce T cell responses, ICI (pembrolizumab (anti-PD-1), durvalumab (anti-104 PD-L1), and ipilimumab (anti-CTLA-4)) were added to the co-culture to improve anti-tumor reactions. 105 Contrasting expectations, pTc HROC113 recognized tumor cells better after control culture than after 106 ICI-supplemented co-culture, showing significantly deterioration after co-culture with PD-1 or CTLA-4 107 supplementation (p=0.048 and p=0.041, respectively) (Figure 1A). Moreover, the degranulation 108 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 5 capacity of co -cultured pTc HROC113 , with and without ICI treatment, was significantly lower 109 compared to tumor-naïve pTc HROC113 (p=0.004). 110 Due to the low quantity of available TiTc HROC113, single ICI testing was replaced by a combination 111 treatment (Figure 1B) but did also not improve tumor cell recognition . The results of pTc HROC285 112 (Figure 1C) resembled those of pTc HROC113: tumor-naïve pTc HROC285 showed high degranulation, 113 contrasting co-cultured pTc HROC285 with decreased tumor cell recognition. The addition of ICI was 114 insufficient to overcome the tumor cell-induced T cell inhibition. 115 116 2.4 HROC113 and HROC285 T0 M2 express genes of the antigen-processing 117 machinery 118 To investigate reasons for the failure of ICI treatment in vitro, we analyzed the expression of genes 119 involved in antigen processing and presentation. By determining the expression of 18 genes used in 120 the study of Wang et al 14, their immunological functionality was assessed in the established tumor cell 121 lines. Respective results were compared to datasets of normal colon and CRC samples from TCGA and 122 GTEX ( Colon: TCGA normal: n=51; TCGA tumor: n=484; GTEX normal: n=308). Surprisingly , when 123 compared to normal colon samples, gene expression was higher in both tumor cell lines as well as in 124 TMB high and TMB low tumor tissues regarding 16/18 genes investigated (Figure 2A-B). Only the 125 expression of β2 microglobulin (B2M) was found significantly lower in tumor tissues compared to 126 healthy colon samples in the analyzed data sets. When assessing differing genes between TMB high 127 and low CRC tissue samples, we observed, that the expression level of ERAP1, PDIA3, PSMB6, PSMB9, 128 PSMB10, CALR, CANX, TAP1, TAP2, TAPBP, B2M and HLA-C was found to be lower in tumors featuring 129 fewer mutations. In both tumor cell lines, t he mean expression of the selected genes resembled or 130 even exceeded the levels in CRC samples from TCGA, but due to the small size of the cell line cohort 131 (n=2), statistical analysis was inadmissible. Moreover, functional antigen processing was previous ly 132 assured through HLA ligandome analyses enabling the characterization of several thousand HLA 133 ligands for each CRC cell line, including tumor-specific antigens 20. 134 Flow-cytometry analyses showed that 98% of HROC113 and 96% of HROC285 T0 M2 cells stained 135 positive for HLA class I (Figure 3A). IFNγ pre-treatment increased the amount of HLA class I/II double 136 positive cells in HROC113 from <1% to 45% (p<0.001). In HROC285 T0 M2, the proportion of HLA class 137 I+/II+ cells even reached 72% following IFNγ treatment compared to 2% in untreated cells (p<0.001). 138 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 6 Accordingly, median fluorescence intensity of HLA class II increased by 20-fold after IFNγ stimulation 139 (p=0.002). Thus, IFNγ pre-treatment was performed for all the following experiments. 140 These results prove the integrity of antigen presentation in the selected tumor cell lines suggesting 141 functional immune interaction with autologous lymphocytes. Thus, the observed immunosuppression 142 by the tumor cells must be ascribed to other immune evasion mechanisms. 143 144 2.5 Tumor cells utilize several immunosuppressive mechanisms 145 Expression of the inhibitory checkpoint molecule programmed death-ligand 1 (PD-L1) was observed in 146 the majority of tumor cells (HROC113: 95%; HROC285 T0 M2: 79%; Figure 3B-C). Here, stimulation with 147 IFNγ increased median fluorescence intensity of PD-L1 expression about 4-fold in both tumor cell lines 148 (p=0.05 in both cases). When determining CD80/86 expression, this CTLA-4 ligand was detected on 149 30% and 17% of HROC113 and HROC285 T0 M2 cells, respectively (Figure 3D). Treatment with IFNγ did 150 not significantly change CD80/86 expression. 151 Further characterization of the tumor cells revealed granzyme B activity in HROC113 as well as 152 HROC285 T0 M2, which was found independent of IFNγ treatment (Figure S 1). As this apoptosis -153 propagating enzyme threatens cell survival, its presence is often accompanied by the expression of 154 proteinase inhibitor 9 (PI-9), which specifically inhibits the function of granzyme B. Indeed, at least 40% 155 of HROC113 as well as HROC285 T0 M2 tumor cells expressed this enzym e inhibitor (Figure 3E). This 156 finding seems to be common in CRC cells, since PI -9 expression was also observed in several other 157 patient-derived CRC cell lines in varying amounts and was independent of MSI (Figure S2). 158 The analysis of secreted cytokines showed that neither HROC113 nor HROC285 T0 M2 produced IL-10 159 in measurable amounts, which is known to impair T cell function. Additionally, the cytokine profile of 160 the tumor cell lines (Figure S 3) did not explain their immunosuppressive behavior in co -culture. 161 Fibrinogen-like protein 1 (Figure S4), an immunosuppressive ligand of LAG-3, was also not detectable 162 in tumor cell culture supernatants. 163 164 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 7 2.6 T cells characterization shows exhausted and regulatory subpopulations 165 Poor tumor cell recognition can be caused by an impaired ability for activation due to T cell exhaustion. 166 To assess the level of exhaustion in unspecifically expanded pTc and TiTc from patients HROC113 and 167 HROC285, expression levels of PD-1, CTLA-4 and LAG-3 were determined. LAG-3 (pTc HROC113: 82%; 168 pTc HROC285: 67%) and CTLA-4 (pTc HROC113: 68%; pTc HROC285: 51%) were found to be expressed 169 on the majority of pTc, while PD-1 was present on less than 20% of T cells (Figure 4A-B). Exhausted T 170 cells were classified as PD -1/CTLA-4/LAG-3 triple positive cells with 10% of pTc HROC113 and 9% of 171 pTc HROC285 identified as such, respectively. 172 The assessment of immune checkpoint molecules on TiTc (Figure 4C-D) revealed a larger quantity of 173 cells positive for PD -1 in TiTc of HROC113 and HROC285 compared to the respective matching pTc 174 (p=0.02). The large proportion of PD -1+ TiTc (60% in TiTc HROC113 as well as TiTc HROC285 ) was 175 accompanied by an overall enlarged population of exhausted cells in TiTc (p=0.05). 176 Even though, the amount of CD4+ cells was higher in TiTc than pTc (p<0.001), this did not coincide with 177 larger amount s of regulatory T cells (Figure 4E). In pTc HROC113, 5.2% of the cells were 178 CD4+/CD25+/FoxP3+, while this population represented less than 1% of cells among the matching TiTc 179 (p=0.006). A contrasting observation was made in pTc and TiTc HROC285, where 3.2 and 7.7% of the 180 cells were identified as regulatory T cells, respectively. 181 The cytokine detect ion assay in T cell and co -cultures revealed secretion of the pro -inflammatory 182 interleukins (IL) -5, -6 and -17a as well as GM-CSF (Figure S3). It was frequently seen, that the cytokine 183 concentration was highest in T cell culture and decreased in the co-culture setting, while secretion by 184 tumor cells alone was minimal. 185 186 2.7 Peptide-stimulated T cells recognize autologous tumor cells better than co-187 cultured T cells 188 In a previous study, we showed that T cell stimulation with mutated neoantigens and non -mutated 189 cryptic peptides improved tumor cell recognition in pTc and TiTc 20. In the present study, we compared 190 the strength of T cell activation mediated by these tumor-specific peptides with T cell stimulation by 191 autologous tumor cells. pTc HROC113 and pTc HROC285 pre-stimulated with cryptic peptides reached 192 significantly higher amounts of degranulating cells compared to all respective co-culture approaches 193 (p<0.001 in both cases) (Figure 1A and C). A similar effect concerning improved tumor cell recognition 194 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 8 by neoantigen stimulation was observed in pTc HROC113, but high variance prevented reaching 195 significance. Additionally, these peptide stimulation experim ents showed that tumor -specific T cell 196 responses can be induced similarly in both pTc and TiTc, while tumor cell recognition by p Tc partly 197 even proved superior (ppTc HROC113 (cryptic peptides) vs TiTc HROC113 (cryptic peptides) =0.01). Furthermore, these results 198 demonstrate, that the tumor cells are indeed immunogenic and the missing T cell response in co -199 culture tests m ust be ascribed to remarkable immunosuppressive tumor cell properties . However, 200 since the combined ICI treatment could not improve tumor cell recognition by peptide -stimulated T 201 cells, we assume that for these two MSI CRC cases, immunosuppression does not only depend on PD-202 1, PD-L1 and CTLA4. 203 204 2.8 Peptide-stimulated pTc HROC113 eliminate autologous tumor cells 205 Since granzyme B activity measurement was unsuitable for detecting T cell-mediated tumor cell killing, 206 we set up an in vitro cytotoxicity assay based on crystal violet staining of adherent tumor cells. These 207 were stimulated with IFNγ and treated with ICI and/or a PI-9i, which had been proven to successfully 208 control tumor growth in mice by Jiang et al. 18. The effect of the PI-9i on tumor and T cell viability was 209 tested beforehand with concentrations covering 25-1,000µM (Figure S5). The highest concentration 210 not inducing cytotoxic effects, i.e. 400µM, was chosen for application in the cytotoxicity assay. 211 Cytotoxicity was experimentally tested as a proof of concept with peptide-stimulated pTc HROC113, 212 as they showed significantly better tumor cell recognition than co -cultured T cells . After 48h of 213 incubation with autologous tumor cells, the cytotoxic capacity of untreated control pTc HROC113 was 214 low (3% tumor cell killing) , and was not significantly improved by the addition of ICI, PI-9i or the 215 combination of both (Figure 5) . On the contrary, pTc HROC113 pre-stimulated with tumor -specific 216 cryptic peptides or neoantigens eliminated on average 78% (p<0.001) or 77% (p=0.006) of the tumor 217 cells, respectively. However, similarly to the findings with untreated control pTc HROC113, the addition 218 of the above-mentioned immune modulators did not result in increased tumor cell killing. Instead, the 219 combination of ICI and PI-9i even seemed to worsen the outcomes with all three T cell populations. 220 221 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 9 3. Discussion 222 In this study, we investigated the interaction of carefully selected tumor and autologous T cells in vitro. 223 In initial co-culture experiments, no anti-tumor responses were observed, which was due to the clinical 224 manifestation of the original tumors rather expected than surprising. Disappointingly, subsequent ICI 225 treatment to re-establish T cell activity and tumor cell recognition did not work out as expected. 226 Resistance against ICI is either an innate tumor cell property or is observed as an acquired 227 phenomenon after therapy. ICI resistance can be categorized into three groups: resistance 228 mechanisms related to (I) antigen recognition; (II) T cell migration/infiltration; and (III) effector 229 functions of T cells 21. When comparing gene expression of proteins involved in antigen processing 14 230 between our two tumor models and normal colon tissue, the majority of genes showed a higher 231 expression in tumor than in normal tissue; hinting at a preservation of functional antigen processing. 232 Even though B2M expression was decreased, a HLA ligandome analysis revealed thousands of HLA -233 eluted peptides in both cell lines 20. Thus, the presentation of tumor -specific peptides with the 234 potential to activate T cells was maintained in these two CRC cases, contrasting the well-established 235 correlation of MSI and diminished HLA expression 22–24. However, despite preserved functional tumor 236 antigen presentation, the interaction of the T cell receptor and peptide-presenting tumor cells can be 237 impaired, for example, by an overexpression of cell surface glycosphingolipids sterically impeding the 238 interaction 25. 239 Since antigen presentation was likely not defective and the selected co-culture in vitro assay neglected 240 the issue of T cell infiltration, the observed ICI resistance was probably caused by resistance 241 mechanisms related to T cell effector functions. When ICI were used to interfere with PD-1 and PD-L1, 242 which was expressed on the majority of tumor cells, there was no improvement in anti-tumor response 243 observed, even when these ICI where combined with anti-CTLA-4. The detailed characterization of the 244 T cell populations revealed the expression of a further immune checkpoint, namely LAG -3, on the 245 majority of T cells, possibly impairing their activation after binding to tumor cell MHC II. Currently, 246 several anti-LAG-3 antibodies are tested in pre -clinical and clinical trials 26. Moreover, as TIM -3 and 247 TIGIT also seem to negatively regulate T cell activity, blocking antibodies are similarly under 248 investigation 27 and further immune checkpoints are identified continuously 28. By focusing the 249 investigations on clinically approved ICI the results of the study may be limited, but they also prove 250 that further research is urgently needed. 251 The effector function of T cells is not only suppressed directly by tumor cells, but is mainly influenced 252 by the tumor microenvironment. Here, i mmunosuppressive cell populations and cytokines are 253 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 10 responsible for decreased T cell activity. The determination of regulatory T cell subsets in our selected 254 cancer cases did not indicate strong cell-based immune inhibition as these did not exceed 10%, which 255 equals to regulatory T cell levels in healthy individuals 29. Studies claiming increased proportions of 256 regulatory T cells in peripheral blood of tumor patients compared to healthy donors 30–32 could thus 257 not be validated in the CRC cases analyzed. Of note, T cells underwent two weeks of unspecific 258 expansion prior to measurements, potentially changing the composition of cell subpopulations. Likely, 259 persisting immune suppression by tumor cells in the tumor microenvironment resulted in lower anti-260 tumor reactivity of TiTc compared to pTc, characterized by a higher proportion of exhausted T cells or 261 an increased state of T cell exhaustion. Moreover, secretion of known immunosuppressive cytokines 262 like IL-10 could be excluded as driver of T cell inhibition. 263 Additional examinations revealed granzyme B activity in both tumor cell lines HROC113 and 264 HROC285 T0 M2, accompanied by the expression of its inhibito r PI -9. Granzyme B, which is 265 physiologically expressed in cytotoxic T and natural killer cells 33, was already detected previously in 266 breast, lung and urothelial carcinoma cells 34–36. Moreover, the expression of PI-9, the protective shield 267 against intracellular granzyme B, was found in prostate, lung and rectal cancer 37–39 and we could 268 confirm this finding here for a small series of low-passaged, patient-derived CRC cell lines. Since several 269 studies showed a negative correlation between PI -9 expression and response to ICI 40,41, we 270 hypothesized, that the use of PI-9i might restore anti-tumor immune responses. 271 However, the proof-of-concept cytotoxicity assay of pTc HROC113 and the tumor cell line HROC113 272 revealed, that neither ICI nor PI-9i treatment resulted in significant changes and even the combination 273 failed to improve tumor cell elimination. It is thus safe to conclude, that t umor c ells utilize d 274 immunosuppressive mechanisms beyond the investigated cytokine secretion, immune checkpoint 275 expression and adaption to cytotoxic microenvironment. Clinically, failure of ICI would have been 276 interpreted probably as a n immunological cold tumor, but T cell stimulations disproved this 277 assumption. When T cells were stimulated with tumor -specific peptides, recognition and elimination 278 of tumor cells increased significantly. Both, cryptic peptides as well as neoantigens, sufficiently induced 279 T cell effector function, clearly exceeding the effects of ICI and/or PI -9i. By comparing stimulated pTc 280 and TiTc we further showed, that T cells from peripheral blood are a valuable source of lymphocytes, 281 which can be turned into tumor -reactive T cells by stimulation with tumor -specific peptides. In 282 connection with studies showing that the quality 42 and clonality 43 of neoantigens was superior in 283 predicting T cell response in comparison to mere neoantigen number, these results hint at a likely 284 broader applicability of immunotherapy beyond tumors with high TMB. 285 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 11 Nevertheless, the incomplete elimination of tumor cells by peptide -stimulated T cells might again be 286 an indicator of a complex network of immunosuppressive mechanisms in use by the tumor cells, which 287 must be examined further. Our data also imply , that the development of new immune modulators 288 interfering with immunosuppressive molecules (immune checkpoint ligands, enzymes, cytokines, etc.) 289 and tumor -individual selection of those will contribute equally to optimally support the effector 290 function of tumor -reactive T cells. By applying ex vivo expanded and selected T cells or genetically 291 modified CAR T cells or TCR T cells, the strength of the anti-tumor reactions increases dramatically and 292 can overcome immunosuppressive barriers. Additionally, the combinati on of strongly primed T cells 293 with further immune modulators could prevent acquired resistance to immunotherapy and tumor 294 relapse as several studies already showed durable responses in combination therapy 44,45. 295 Summarizing all results, our detailed investigation of two distinct sets of patient -derived cell 296 populations demonstrated that ICI-resistant tumor cells can be targeted by T cells primed with tumor-297 specific antigens. This peptide stimulation induced increased tumor cell recognition not only in TiTc, 298 but also in pTc, which partially even exceeded the former. These findings in pTc could hint towards an 299 equivalent or even superior performance in future immunotherapeutic approaches. In our 300 investigation, extensive T cell priming in a personalized approach was superior to standardized ICI 301 therapy, underlining the importance of precision medicine also in cancer immunotherapy. Additionally, 302 this patient-individual approach could be combined with drugs like ICI and enzyme blockers according 303 to the respective tumor characteristics. Such multimodal approaches could then tackle the complex 304 network of immunosuppressive strategies to successfully overcome immunological barriers erected by 305 any given tumor. 306 4. Material and Methods 307 4.1 Cell Culture 308 Patient-derived CRC cell lines HROC113 and HROC285 T0 M2 from the HROC collection 19 were cultured 309 under standard conditions in DMEM/Ham´s F12 medium supplemented with 10% FCS and 2mM L -310 glutamine. For several experiments, tumor cells were treated for 48h with 200IU/ml I FNγ (Imukin, 311 Boehringer Ingelheim, Ingelheim am Rhein, Germany). Cell culture reagents were obtained from PAN 312 Biotech, Aidenbach, Germany, unless stated otherwise. 313 314 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 12 4.2 T cell isolation and expansion 315 Isolation of p eripheral blood mononuclear cells and pTc from patients´ heparinized blood as well as 316 TiTc from vitally frozen tumor pieces was followed by a rapid Tc expansion protocol (REP) as described 317 recently 20. 318 319 4.3 Co-culture 320 Co-culture was conducted by adapting a previous protocol 46. Briefly, 20,000 IFNγ-treated tumor cells 321 were seeded with 200 ,000 T cells in a 24-well plate coated with anti-human CD28 (Immunotools, 322 Friesoythe, Germany) in DMEM/Ham´s F12 medium, 10% human AB serum, 2mM L -glutamine, 323 penicillin, streptomycin and amphotericin (T cell medium) supplemented with 300IU/ml IL-2. Every 2-324 3 days, half of the medium was exchanged. At day seven, cells were harvested and seeded with freshly-325 prepared IFNγ-treated tumor cells in a ratio of 10:1. The co -culture ended at day 14 and c ells were 326 further analyzed. The medium of several experiments was supplemented with ICI (20µg/ml 327 Pembrolizumab (Merck, Darmstadt, Germany); 10µg/ml Ipilimumab (Bristol-Myers Squibb, New York 328 City, NY, USA); or 20µg/ml Durvalumab (AstraZeneca, Cambridge, UK)). 329 330 4.4 T cell stimulation 331 Autologous B lymphoid cell lines (B -LCL) were used as antigen presenting cells. For peptide -loading, 332 3x106 B-LCL were incubated at 37°C with 10µg peptide pools in 1ml serum free medium for 1h. 333 Stimulations were performed in 24-well plates with irradiated (30Gy) peptide-loaded B-LCL added at a 334 ratio of 1:4 to 1x106 expanded Tc in 2ml of T cell medium supplemented with 1xITS solution IV and 335 300IU/ml IL-2 per well. After seven days of co-culture, Tc were harvested, counted and re-stimulated. 336 T cells stimulated with B-LCL without any peptide served as controls. 337 338 4.5 Flow cytometry 339 Tumor and T cells were collected and washed with phosphate -buffered saline (PBS). For extracellular 340 staining, cells were incubated in 100µl staining buffer (PBS, 200mM EDTA, 0.5% bovine serum albumin) 341 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 13 with the appropriate volume of antibody for 30min at 4°C protected from light. After centrifugation, 342 the staining solution was discarded and cells were washed again with staining buffer. Stained cells 343 were resuspended in staining buffer and measured promptly. Intracellular staining was performed 344 using the InsideStain Kit (Miltenyi Biotec) according to the manufacturer’s instructions and measured 345 promptly. The following anti -human antibodies were used from Im munotools: CD4-APC, CD8 -APC, 346 CD25-FITC, and FoxP3 -PE or Biol egend (San Diego, CA, USA): CD107a-FITC, CD152-PE, CD223-APC, 347 CD274-FITC, and CD279-FITC. Samples were measured using a BD FACSCalibur™ and data was analyzed 348 by using FCSalyzer 0.9.21 alpha (Sven Mostböck, Vienna, Austria; 349 https://sourceforge.net/projects/fcsalyzer/). 350 351 4.6 Degranulation assay 352 The ability of T cells to recognize tumor cells was tested in the degranulation assay 46. A 96-well plate 353 was coated with anti-human CD28 (Immunotools) and 100,000 IFNγ-treated tumor cells were seeded 354 together with 200 ,000 pTc or TiTc per well in Tc medium containing anti -human CD107a -FITC 355 (Biolegend). After 1h incubation, 1µg/ml Brefeldin A (MedChemExpress, Monmouth Junction, NJ, USA) 356 was added, followed by another 4h incubation. Intracellular flow cytometry staining was conducted as 357 described using CD8-APC (Immunotools) and IFNγ-PE (Biolegend). Samples were measured with gates 358 adjusted to detect CD8+/CD107a+/IFNγ+ cells. To receive values for relative degranulation, percentage 359 of triple-positive Tc was normalized to the triple -positive population in Tc not incubated with tumor 360 cells. 361 362 4.7 Cytokine detection and FGL1 ELISA 363 For cytokine detection, supernatants were collected from Tc, tumor cells and co-cultures, all cultured 364 for seven days without medium exchange. The concentrations of granulocyte-macrophage colony-365 stimulating factor (GM-CSF), IFNα, IFN γ, lL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and tumor 366 necrosis factor (TNF) were determined by MACSPlex Cytokine 12 Kit (Miltenyi Biotec). Supernatants of 367 tumor cell lines were also used for the measurement of FGL -1 with the Human FGL1/Fibrinogen Like 368 Protein 1 ELISA Kit (Assay Genie, Dublin, Ireland). 369 370 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 14 4.8 GranToxiLux 371 The GranToxiLux assay was performed according to manufacturer’s instructions (Oncoimmunin Inc, 372 Gaithersburg, MD, USA). 373 374 4.9 Cytotoxicity assay 375 Tumor cells were seeded into a 48 well plate and stimulated with 200IU/ml IFNγ for 48h. 376 Simultaneously, respective wells were treated with 400µM PI -9i (1,3-Benzoxazole-6-carboxylic acid, 377 Advanced ChemBlocks Inc, Hayward, CA, USA). Following pre-treatment, 5x105 peptide-stimulated or 378 control Tc were added to the approximately 1x105 tumor cells in each well. The number of tumor cells 379 was estimated using previously determined doubling times. The co-culture medium was supplemented 380 with ICI (20µg/ml Pembrolizumab; 10µg/ml Ipilimumab; 20µg/ml Durvalumab) and/or PI -9i (400µM). 381 After 48h of co-culture, medium was discarded, wells were washed once with PBS before 200µl of 0.2% 382 crystal violet solution was added. After 20min incubation at room temperature, staining solution was 383 discarded and wells were washed three times with PBS. When plates were dry, 500µl of sodium 384 dodecyl sulfate solution was added and optical density at 570nm was measu red at the microplate 385 reader Infinite 200 (Tecan, Männedorf, Switzerland). 386 4.10 RNA expression analysis 387 RNA sequencing of the patient-derived cell lines was performed by the Center for Quantitative Biology 388 (QBiC, University of Tübingen, Germany) and resulting data was provided. Moreover, publicly available 389 data was used for comparison. First, R package (R (4.2.1) version) was applied to The Cancer Genome 390 Atlas (TCGA, https://portal.gdc.cancer.gov/ (accessed on 20 .06.2022)) and the Genotype -Tissue 391 Expression (GTEx, gtexportal.org/home/ (accessed on 20.06.2022)) databases to analyze the difference 392 of the mRNA levels of 18 genes of interest including ERAP1 [ENSG00000164307.12], ERAP2 393 [ENSG00000164308.16], PDIA3 [ENSG00000167004.12], PSMB5 [ENSG00000100804.18] , PSMB6 394 [ENSG0000142507.9], PSMB 7 [ENSG00000136930.12], PSMB8 [ENSG00000204264.8], PSMB9 395 [ENSSG00000240065.7], PSMB10 [ENSG00000205220.11], B2M [ENSG00000166710.17] , CALR 396 [ENSG00000179218.13], CANX [ENSG00000127022.14], TAP1 [ENSG00000168394.10], TAP2 397 [ENSG00000204267.13], TAPBP [ENSSG00000231925.11], HLA -A [ENSG00000206503.11], HLA -B 398 [ENSG00000234745.9] and HLA-C [ENSG00000204525.14]. Gene expression was compared between 399 484 CRC and 359 normal colorectal mucosal tissue samples. Moreover, TMB was used to grou p the 400 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 15 cancer samples: Samples were sorted by TMB and top 20% were identified as TMB high. Remaining 401 samples were considered TMB low. 402 4.11 Statistical analyses 403 Statistical significance was determined by a n unpaired, two -sided t-test using GraphPad Prism 5 404 (Boston, MA, USA). Results were stated significant if the t-test resulted in p value <0. 05. Details on 405 compared groups are given in the figure legends. If not stated otherwise, graphs depicting the results 406 show mean values and the respective standard deviation. 407 408 Ethics approval and consent to participate 409 The study was conducted according to the guidelines of the Declaration of Helsinki. All patients gave 410 informed written consent to participate in the study and all procedures were approved by the Ethics 411 Committee of the University of Rostock University Medical Center (Reference numbers: A 2018-0054 412 and A 2019-0187) in accordance with generally accepted guidelines for the use of human material. 413 414

415 The authors express their gratitude to the patients for participating in the HROC collection. This 416 research was in part funded by grant number TBI-V-1-241-VBW-084 and TBI-V-240-VBW-084 from 417 the state Mecklenburg-Vorpommern. 418 419 Author Contributions 420 Design and conceptualization of the study was directed by ML. E xperimental design, data 421 interpretation and formal analysis was performed by SS. MK established the patient-derived cell lines 422 and SZ analyzed expression data. Identification and validation of cryptic peptides and neoantigens was 423 performed by MWL and AS. The initial draft and figures were prepared by SS and ML; SZ, MK, MWL 424 and AS reviewed and edited the manuscript. All authors have read and agreed to the final version of 425 the manuscript. 426 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 16 Competing Interests 427 MWL is an inventor of patents owned by Immatics Biotechnologies and has acted as a speaker and 428 paid consultant in cancer immunology for Boehringer Ingelheim. The other authors do not declare 429 conflicting interests. 430 431 Data Availability 432 The datasets analyzed during the current study available from the corresponding author on 433 reasonable request. 434 435

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 20 Figures 560 561 Figure 1: Effect of co-culture, ICI treatment and peptide stimulation on tumor cell recognition by Tc. 562 (A) pTc HROC113, (B) TiTc HROC113 and (C) pTc HROC285 underwent different (co-)culture conditions 563 or were stimulated with tumor -specific peptides to increase their anti tumor reactivity measured by 564 degranulation assay after 5h incubation with freshly prepared tumor cells. CD8 +/CD107a+/IFNγ+ cells 565 were considered tumor -reactive. Results were normalized to measurements of control Tc not 566 incubated with tumor cells. p<0.05 in t-test of sample vs. expanded T cell (*), control culture (#) or co-567 culture (&). Depicted are means of 2-6 biological replicates and the respective standard deviation. 568 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 21 569 Figure 2: Expression of genes involved in antigen processing. TCGA data of CRC (n=484) were 570 compared with normal colon tissue (TCGA: n=51, GTEX: n= 308) and tumor cell lines (n=2) , regarding 571 expression of 18 genes involved in antigen processing and presentation. Tumor cell line data contained 572 three technical replicates each, but due to the small group size, statistical calculations were omitted. 573 (A) Heatmap of analyzed data sets depicting high expression in blue and low expression in red, in regard 574 to TMB. (B) Expression of antigen processing genes ; t-test of tumor sample vs. normal tissue with 575 p<0.05 (*), p<0.01 (**) and p<0.001 (***); t-test of TMB high vs. TMB low with p<0.05 (#), p<0.01 (##) 576 and p<0.001 (###). 577 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 22 578 Figure 3: Tumor cell characterization. Untreated or IFNγ-treated (200IU/ml for 48h) tumor cells were 579 analyzed via flow cytometry. (A) shows PD-L1 positive cells. (B) Median fluorescence intensity was 580 determined and fold change in IFNγ-treated and untreated tumor cells was calculated. (C) Percentage 581 of HLA class I+ and II +, (D) CD80 +/86+ and (E) PI-9+ were determined. Depicted are means of 3-5 582 biological replicates and the respective standard deviation. 583 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 23 584 Figure 4: Characterization of T cells. Following 14 d ays of unspecific expansion, Tc were analyzed 585 regarding (A-D) exhaustion markers and (E) proportion of regulatory Tc defined as CD4+/CD25+/FoxP3+. 586 Depicted are means of 3-4 biological replicates and the respective standard deviation. 587 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint Interactions of T cells and autologous colon cancer cells 24 588 Figure 5: Cytotoxicity of pTc HROC113. Tumor cells were incubated for 48h with pTc HROC113, which 589 were pre-stimulated for 14 d ays with tumor-specific cryptic or neoantigen ic peptides. pTc HROC113 590 stimulated without peptide served as control. Medium was supplemented with three ICI (20µg/ml 591 pembrolizumab, 10µg/ml ipilimumab and 20µg/ml durvalumab) and/or 400µmol PI-9i. (*) p<0.05 in t-592 test of sample and unstimulated control without immune modulators. Depicted are two biologically 593 independent measurements, mean and standard deviation. 594 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 8, 2024. ; https://doi.org/10.1101/2024.02.06.579061doi: bioRxiv preprint