TCF4 Expression Research Articles

Reactivation of low avidity tumor-specific CD8+ T cells associates with immunotherapeutic efficacy of anti-PD-1

BackgroundCD8+ T cells are a highly diverse population of cells with distinct phenotypic functions that can influence immunotherapy outcomes. Further insights on the roles of CD8+ specificities and TCR avidity...

Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8+ T cells which enables an effective combination immunotherapy with anti-PD-1.

Despite the capability of extracellular vesicles (EVs) derived from Gram-negative and Gram-positive bacteria to induce potent anti-tumour responses, large-scale production of bacterial EVs remains as a hurdle for their development as novel cancer immunotherapeutic agents. Here, we developed manufacturing processes for mass production of Escherichia coli EVs, namely, outer membrane vesicles (OMVs). By combining metal precipitation and size-exclusion chromatography, we isolated 357mg in total protein amount of E. coli OMVs, which was equivalent to 3.93 × 1015 particles (1.10 × 1010 particles/μg in total protein amounts of OMVs) from 160 L of the conditioned medium. We show that these mass-produced E. coli OMVs led to complete remission of two mouse syngeneic tumour models. Further analysis of tumour microenvironment in neoantigen-expressing tumour models revealed that E. coli OMV treatment causes increased infiltration and activation of CD8+ T cells, especially those of cancer antigen-specific CD8+ T cells with high expression of TCF-1 and PD-1. Furthermore, E. coli OMVs showed synergistic anti-tumour activity with anti-PD-1 antibody immunotherapy, inducing substantial tumour growth inhibition and infiltration of activated cancer antigen-specific stem-like CD8+ T cells into the tumour microenvironment. These data highlight the potent anti-tumour activities of mass-produced E. coli OMVs as a novel candidate for developing next-generation cancer immunotherapeutic agents.

Doublecortin-likekinase3(DCLK3)isassociatedwithbadclinicaloutcomeofpatientswithgastriccancerandregulatestheferroptosisandmitochondriafunctioninvitroandinvivo.

Doublecortin-like kinase 3 (DCLK3), a member of tubulin superfamily, has been proved to be closely associated with the pathogenesis of numerous human tumors. However, the expression pattern and regulatory mechanisms of DCLK3 in gastric cancer (GC) remain unknown. DCLK3 expression in GC cells was assessed by RT-qPCR and western blotting. The correlation between DCLK3 levels and the overall survival of GC patients was assessed via TCGA, ACLBI, and Kaplan-Meier plotter databases. Additionally, key proteins (TCF4) involved in the regulation of DCLK3 on GC progression were screened by ACLBI database. Cell proliferation, ferroptotic cell death, and oxidative stress markers were measured by EdU staining, immunofluorescence, ELISA, and western blotting assays. DCLK3 was upregulated in GC, and high DCLK3 expression was significantly associated with poor survival of GC patients. Here, DCLK3 knockdown reduced GC cell proliferation, induced ferroptotic cell death, and exacerbated oxidative stress level. Logistic regression analysis showed that TCF4 was an independent prognostic indicator of GC. Mechanistically, DCLK3 promoted TCF4 expression and subsequently upregulated the expression of TCF4 downstream target genes (c-Myc and Cyclin D1). Furthermore, DCLK3 overexpression enhanced GC cell proliferation, but mitigating ferroptotic cell death and oxidative stress. The regulatory mechanism may involve the upregulation of TCF4, c-Myc, and cyclin D1. Our research suggests that DCLK3 modulates the levels of iron and reactive oxygen and may involve regulation of TCF4 pathway, thereby promoting the GC cell growth, indicating that DCLK3 may use as a prognostic marker and therapeutic target for GC patients.

Neoadjuvant pembrolizumab, GX-188E, and GX-I7 in patients with human papillomavirus-16- and/or 18-positive head and neck squamous cell carcinoma: Single-arm, phase 2 trial with single cell transcriptomic analysis and artificial intelligence-powered spatial analysis.

6075 Background: With the increasing prevalence of head and neck squamous cell carcinoma (HNSCC) associated with human papilloma virus (HPV) infection, effective treatment strategies for HPV-positive HNSCC are urgently needed. Here, we present the results of neoadjuvant checkpoint blockade of pembrolizumab, therapeutic HPV DNA vaccine of GX-188E, and long-acting interleukin-7 of GX-I7 for patients with resectable HPV-16 and/or 18-positive HNSCC. Methods: In this single-arm, phase 2 trial, patients with resectable HPV-16 and/or 18-positive HNSCC were enrolled. Patients were given pembrolizumab 200mg on day 1 and day 22; GX-188E 2mg on day 1, 8, and 22; and GX-I7 1200 ug/kg on day 8 before surgical resection. Major pathologic response (MPR; defined as residual viable tumor of less than or equal to 10%) was primary endpoint with null and alternative hypothesis of MPR rate ≤5% and ≥35%, respectively. Secondary objectives were safety, recurrence, and survival. Results: Of the 11 patients included, all underwent surgical resection after neoadjuvant treatment without delays in surgery or increased surgical complications. MPR was achieved in seven patients (63.6%) and pathologic complete response was achieved in four patients (36.3%), which met the primary endpoint. Single cell RNA sequencing of baseline and post-treatment paired specimens revealed that the proportion of follicular helper T cell cluster, featured with CXCR5 and BCL6 expression, was significantly increased among tumor-infiltrating immune cells, accompanied with the reinvigoration of CD8 T cell cluster toward less exhausted phenotypes, represented with the upregulation of TCF7 and CD28 expression. In addition, artificial intelligence-powered spatial analysis revealed that triple combination significantly increased the density of tumor-infiltrating lymphocytes, completely converting immune-desert and immune-excluded type of tumor to inflamed immune phenotype. Conclusions: Neoadjuvant pembrolizumab, GX-188E, and GX-I7 showed promising activity and manageable safety profile in patients with resectable HPV-16 and/or 18-positive HNSCC. Therapeutic induction of brisk immune responses significantly reshaped the tumor microenvironment and associated with pathologic regression, warranting further investigation of pembrolizumab, GX-188E, and GX-I7 for patients with HPV-positive HNSCC. Clinical trial information: NCT05286060 .

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.

Exogenous catalase treatment enhances CD8+ T cell functionality and hinders T cell exhaustion

Abstract ROS are small short-lived oxygen-containing molecules which are highly chemically reactive and involved in the regulation of biological processes like T cell activation and proliferation. The tumor microenvironment (TME) is a metabolically hostile environment with a deficiency in nutrients like glucose and glutamine, accumulation of tumor-driven metabolic waste, and extreme hypoxia. Hypoxia results in impaired mitochondrial function and generation of excess levels of ROS, which have been known to promote T cell exhaustion. Here, we investigated how exogenous treatment of antioxidant catalase altered CD8+ T cell effector responses and T cell exhaustion using OT-I TCR transgenic mice. During CD8+ T cell effector differentiation in vitro by stimulation with cognate antigen followed by culture with IL-2, addition of catalase significantly enhanced T cell activation and effector differentiation. Catalase-treated effector cells had higher expression of critical activation markers including CD69 and CD25, increased expression of the stemness marker TCF-1, and enhanced secretion of effector cytokines IFNγ and TNFα. During induction of T cell exhaustion in vitro by co-culture with B16 melanoma expressing ovalbumin (B16-Ova), catalase-treated OT-I T cells had significantly increased cytotoxic activity, enhanced expression of CD25 and lower co-expression of PD-1 and TIM-3 relative to non-treated OT-I T cells. Our findings uncover a new role for catalase in altering the fate of CD8+ effector T cells and potentially a new therapeutic tool for managing T cell exhaustion. NIH (R01 CA212605)

PI3Kδ: a key driver of effector differentiation under conditions of T cell exhaustion

Abstract In chronic infection and cancer, prolonged antigen exposure drives a state of T cell hyporesponsiveness, called exhaustion. A small subset of TCF1 +progenitor exhausted stem-like T cells (pTex) cells, is required to maintain exhausted T (Tex) cells and respond to checkpoint blockade. Understanding mechanisms regulating these populations may help identify approaches to counter exhaustion. Phosphatidylinositol 3-kinase-d (PI3Kd) is an important component of T cell signaling. We have found that TCF-1 is repressed in CD8 +T cells from patients with Activated PI3K Delta Syndrome and in a mouse model (Pik3cd E1020K/+mice) in acute viral infections. How activated-PI3Kδ affects T cell exhaustion is unknown. Using chronic LCMV clone 13 infection, we found that Pik3cd E1020K/+mice rapidly die, yet surviving mice recover faster than WT. RNA sequencing revealed decreased expression of Tcf7, encoding TCF-1, in antigen-specific CD8 +T cells, along with loss of TCF-1 +Tim3 −CD8 +pTex cells in Pik3cd E1020K/+mice upon cl13 infection. Tex cells that were maintained during chronic infection in Pik3cd E1020K/+mice displayed an effector-like phenotype, with expression of CX3CR1 +and KLRG1 +, and increased effector functions, suggesting that these cells might induce early immunopathology yet increase viral clearance. P14 cell transfers, in conjunction with CRISPR-mediated gene disruption, revealed that loss of FoxO1 leads to loss of TCF-1 +cells, whereas the increase of effector cells is at least partially cell-extrinsic. Our findings suggest that PI3Kδ is a critical rheostat that balances effector vs exhaustion differentiation, providing insight for therapeutic strategies for exhaustion.

Stem-like CD4 T differentiation controls CD8 T cell immunity or tolerance in cancer

Abstract In this work we wanted to understand how CD4 T cell differentiation shapes the CD8 T cell response in cancer. We identified a population of tumor specific CD4 T cells that maintain an activated, undifferentiated phenotype throughout the cancer response. These CD4 T cells were the dominant population within tumor draining lymph nodes (TDLNs), characterized by high expression of TCF1 and little to no expression of other lineage defining transcription factors. Tumor specific TCF1+ CD4s exhibited functional stem-like properties, as they self-renewed and differentiated almost exclusively into Tregs in tumor bearing mice. These cells, however, were not limited to Treg differentiation, as sorted TCF1+ CD4s from a 5-week tumor response expanded and gave rise to TH1 and TFH effectors in response to LCMV Armstrong infection. Interestingly, we also found a similar population of activated PD1+ TCF1+ CD4s with stem-like capabilities infiltrating various human tumors, indicating this phenotype of CD4s is comparable in human cancer and mouse models. Importantly, the choice of lineage differentiation of TCF1+ CD4s regulated the quality of the CD8 T cell anti-tumor response. When Tregs were selectively depleted, TCF1+ CD4s differentiated into TH1s, which enhanced tumor specific CD8 T cell effector differentiation in both tumor and TDLNs, resulting in reduced tumor burden. Using a bone marrow chimeric CRISPR-Cas9 approach, we found that IFNg from TH1s was intrinsically required for CD8 stem to effector differentiation within TDLNs, promoting an improved anti-tumor response. Overall, this work shows that CD4 differentiation dictates tolerance or immunity in cancer by regulating the phenotype and magnitude of the effector CD8 T cell response.

Reducing PI3Kδ signaling in a dose-dependent manner promotes human T cell stemness

Abstract Adoptive T Cell Therapy (ACT) is a promising type of highly personalized immunotherapy against cancer. However, during the clinical production of T cell products for ACT, the cells experience cues that lead to early senescence and poor quality. Thus, ways to improve T cell quality for ACT is warranted. Previous mouse data indicate that inhibiting the PI3Kδ pathway in T cells using Idelalisib, an FDA-approved selective inhibitor of the δ subunit of PI3K, produces less differentiated T cells with enhanced efficacy against cancer. We hypothesized that Idelalisib treatment in human T cells would recapitulate a dose-dependent acquisition of stemness features. Mouse T cells, bulk donor human peripheral blood mononuclear cells (PBMCs), and isolated human CD3+ T cells were cultured in vitrofor seven days with dose escalation of Idelalisib. We counted the number of T cells to track expansion and assessed the phenotype through flow cytometry. Reducing PI3Kδ signaling produced a less differentiated phenotype in both murine and human CD3+ T cells. Human CD3+ T cells had lower expression of TCF-1 and LEF-1, transcription factors regulating stemness, due to Idelalisib. However, other markers (CD62L and CD45RA) still suggested dose escalation up to 25 μM led to less differentiation. PBMCs and CD3+ T cells treated with dosages of Idelalisib above 10 μM had limited expansion and decreased viability compared to mouse T cells. Altogether, our data demonstrate that blocking PI3Kδ with Idelalisib in human T cells supports acquisition of stemness features.

SATB1 is a master gatekeeper that limits virus-specific CD8 +T cell differentiation.

Abstract Special AT-rich sequence binding protein-1 (SATB1) is a chromatin binding protein that has key roles in ensuring appropriate chromatin structure and remodelling during T cell development. While a role for SATB1 in both CD4 +T regulatory and CD4 +T H2 lineage specific function has been described, little is known about its role during virus-specific CD8 +T cell differentiation. Here we demonstrate that SATB1 down regulation is associated with naïve CD8 +T cell activation and subsequent differentiation. Genome wide SATB1 binding indicates that SATB1 is critical for maintaining the naïve CD8 +T cell transcriptional programs prior to activation. Overexpression of SATB1 upon CD8 +T cell activation results in major restraint of virus-specific CD8 +cell differentiation resulting in lower expression of inflammatory chemokine receptors, checkpoint molecules such as PD-1 and limits effector CD8 +T cell infiltration into infected tissues. Moreover, SATB1 over expression maintains high levels of TCF-1 expression and maintenance of the naïve T cell program. Our data suggest that SATB1 expression within naïve CD8 +T cells acts as gatekeeper for maintaining the naïve T cell program, and that down regulation upon activation is required for optimal effector CD8 +T cell differentiation. Supported by grants from the Australian Research Council (DP170102020).

CD38-cADPR axis mediated chronic Ca 2+signaling promotes the differentiation of TCF1 loterminal exhausted CD8 +T cells in tumor

Abstract Preponderance of terminally exhausted CD8 +T cells (Tex term) at the tumor site is the major impediment for successful immunotherapy of cancer. Despite the recent understanding in epigenetic, transcriptomic and metabolic features of Tex term, the cellular pathway underpinning the differentiation of Tex termremains elusive. Upon assessing intratumoral CD8 +T cells from both clinical samples and pre-clinical mouse models, we observed that CD8 +T cells co-expressing CD38 and PD1 displayed the characteristic features of Tex term. Single cell RNA sequencing analysis from both murine tumor infiltrating CD8 +T cells and in vitrochronically stimulated human CD8 +T cells revealed that clusters co-expressing Cd38and Pdcd1diplayed the transcriptomic signature of Tex term. Most importantly, clusters expressing Cd38and Tcf7were found to be mutually exclusive. Further studies to decipher the role of CD38 showed that both pharmacological and genetic knockdown of CD38 maintained TCF1 expression in chronically exhausted CD8 +T cells and significantly improved their ability to control tumor growth upon adoptive transfer in B16–F10 melanoma bearing mice. Mechanistic studies revealed that CD38 expression on Tex termled to chronic elevation of intracellular Ca 2+levels through cADPR-RyR2 axis, which activated Akt signalling. Activation of Akt restrained the nuclear localization of Ezh2, which was found to be pivotal in maintaining TCF1 expression in Tex termand inhibition of which in CD8 +T cells resulted in poor anti-tumor response. Thus, our data suggests that CD38 could be a drugable target to improve the immunotherapeutic efficacy of CD8 +T cells in cancer. Supported by grants from IA/I/19/1/504277/WTDBT_/DBT-Wellcome Trust India Alliance/India CRG/2019/001334 SERB Extramural Fund CSIR Intramural Fund

TCR signaling dynamically regulates TCF1 expression and effector differentiation

Abstract T Cell Factor 1, or TCF1, is a transcription factor required for thymic T cell development, memory T cell formation, and maintenance of exhausted/dysfunctional T cell stem/progenitors in tumors and chronic infection. However, TCF1 regulation and function during T cell activation is not well-understood. We analyzed the expression kinetics of Tcf7 mRNA, encoding TCF1 (RNA-SEQ) and TCF1 protein (flow cytometry and western immunoblotting) in CD8 T cells during the initial hours and cell divisions following activation in vivo. Naive CD8 T cells robustly express TCF1. Within 6 hours after activation in L. monocytogenes-infected B6 mice, antigen-specific CD8 T cells downregulated Tcf7 mRNA/TCF1 protein, only to re-express both prior to undergoing the first cell division. We observed a similar pattern of TCF1 kinetics in polyclonal CD8 and CD4 T cells activated with anti-CD3/CD28 antibody and PMA/ionomycin, which activates signaling pathways downstream of the T cell receptor (TCR). We further determined that the magnitude of pre-division TCF1 downregulation correlated with cognate peptide concentration and TCR avidity, but also with higher granzyme B expression during later cell divisions. Thus, our data suggests that the pre-division TCF1 drop is regulated by TCR signaling strength and may determine effector fate. By decoding TCF1 regulation during T cell activation, we could gain insights allowing us to target TCF1 to augment or inhibit effector T cell function in different disease contexts, including cancer, chronic infection, and autoimmunity. Supported by grants from NHI (T32 GM137793-02, R37 CA263614)

ASXL1 disruption preserves CD8 T cell exhaustion-progenitor epigenetic programming during chronic stimulation

Abstract Differentiation of CD8 T cells into a terminally exhausted state is a major hurdle limiting efficacy of both immune checkpoint blockade and adoptive cellular immunotherapy approaches. Prior studies describing disruption of the epigenetic regulators DNMT3A and TET2 in CD8 T cells have revealed a critical role of DNA methylation in repressing the proliferative capacity and effector potential of CD8 T cells during exhaustion. Disruption of these genes has also been noted to preserve the proliferative capacity of hematopoietic stem cells (HSCs), resulting in clonal hematopoiesis (CH) and establishing the broader concept that these epigenetic regulators control cellular developmental potential. In addition to DNMT3A and TET2, the histone modifier ASXL1 is a critical regulator controlling CH. Here we report that disruption of ASXL1 in CD8 T cells preserved a stem-like phenotype denoted by increased TCF1 expression, IFN-γ production during chronic infection. At the chronic stage of infection, ASXL1 KO CD8 T cells retained a heightened ability to undergo a proliferative burst during PD-L1 blockade. Genome-wide epigenetic profiling demonstrated that disruption of ASXL1 resulted in inhibition of DNA methylation accompanied by changes in chromatin accessibility. These data document crosstalk between histone modifications and DNA methylation in regulating the transition of stem-like progenitor T cells into terminally exhausted T cells. Collectively, these data demonstrate that ASXL1 is a novel epigenetic regulator controlling development of exhausted CD8 T cells and represents a novel immunotherapeutic target for improving responses to T cell-based cancer immunotherapy.

The Chromatin Regulator Mll1 Supports T Follicular Helper Cell Differentiation by Controlling Expression of Bcl6, LEF-1, and TCF-1.

T follicular helper (TFH) cells are essential for developing protective Ab responses following vaccination. Greater understanding of the genetic program leading to TFH differentiation is needed. Chromatin modifications are central in the control of gene expression. However, detailed knowledge of how chromatin regulators (CRs) regulate differentiation of TFH cells is limited. We screened a large short hairpin RNA library targeting all known CRs in mice and identified the histone methyltransferase mixed lineage leukemia 1 (Mll1) as a positive regulator of TFH differentiation. Loss of Mll1 expression reduced formation of TFH cells following acute viral infection or protein immunization. In addition, expression of the TFH lineage-defining transcription factor Bcl6 was reduced in the absence of Mll1. Transcriptomics analysis identified Lef1 and Tcf7 as genes dependent on Mll1 for their expression, which provides one mechanism for the regulation of TFH differentiation by Mll1. Taken together, CRs such as Mll1 substantially influence TFH differentiation.

CD4+ T cell memory is impaired by species-specific cytotoxic differentiation, but not by TCF-1 loss.

CD4+ T cells are typically considered as 'helper' or 'regulatory' populations that support and orchestrate the responses of other lymphocytes. However, they can also develop potent granzyme (Gzm)-mediated cytotoxic activity and CD4+ cytotoxic T cells (CTLs) have been amply documented both in humans and in mice, particularly in the context of human chronic infection and cancer. Despite the established description of CD4+ CTLs, as well as of the critical cytotoxic activity they exert against MHC class II-expressing targets, their developmental and memory maintenance requirements remain elusive. This is at least in part owing to the lack of a murine experimental system where CD4+ CTLs are stably induced. Here, we show that viral and bacterial vectors encoding the same epitope induce distinct CD4+ CTL responses in challenged mice, all of which are nevertheless transient in nature and lack recall properties. Consistent with prior reports, CD4+ CTL differentiation is accompanied by loss of TCF-1 expression, a transcription factor considered essential for memory T cell survival. Using genetic ablation of Tcf7, which encodes TCF-1, at the time of CD4+ T cell activation, we further show that, contrary to observations in CD8+ T cells, continued expression of TCF-1 is not required for CD4+ T cell memory survival. Whilst Tcf7-deficient CD4+ T cells persisted normally following retroviral infection, the CD4+ CTL subset still declined, precluding conclusive determination of the requirement for TCF-1 for murine CD4+ CTL survival. Using xenotransplantation of human CD4+ T cells into murine recipients, we demonstrate that human CD4+ CTLs develop and persist in the same experimental conditions where murine CD4+ CTLs fail to persist. These observations uncover a species-specific defect in murine CD4+ CTL persistence with implications for their use as a model system.

EXCESSIVE WNT SIGNALLING ALTERS THE CARTILAGE RESPONSE TO PHYSIOLOGIC LOADING

Regulation of articular cartilage homeostasis is a complex process in which biologic and mechanical factors are involved. Hyperactivation of Wnt signaling, associated with osteoarthritis (OA), could jeopardize the protective anabolic effect of physiological loading. Here, we investigated the role of excessive Wnt signalling in cartilage molecular responses to loading.Human cartilage explants were harvested from hips of donors without OA. The Wnt agonist CHIR99021 was used to activate Wnt signalling 24 hours before cartilage explants were subjected to a loading protocol consisting of 2 cycles of 1 hour of 10% compression at 1 Hz, followed by 1-hour free swelling. Mechano-responsiveness was evaluated using the expression of type II collagen, aggrecan and MMP-13. Expression of known target genes TCF-1 and c-JUN was evaluated as positive control for Wnt and mechanical stimulation, respectively.In the absence of loading, CHIR99021 decreased the expression of the cartilage anabolic genes type II collagen and aggrecan, and increased the levels of MMP-13, corroborating that Wnt hyperactivation disrupts cartilage homeostasis. In the absence of Wnt hyperactivation, the applied loading protocol, representative for a physiologic stimulation by mechanical loading, led to an increase in type II collagen and aggrecan levels. However, when cartilage explants were subjected to mechanical stimulation in the presence of CHIR99021, the expression of cartilage anabolic genes was decreased, indicating changes to the cells’ mechano-responsiveness. Interestingly, mechanical stimulation was able to reduce the expression levels of MMP-13 compared to the condition of CHIR stimulation without loading.Hyperactivation of Wnt signaling switches the anabolic effect of physiologic compressive loading towards a potential catabolic effect and could contribute to the development and progression of OA.

Abstract 4157: Tumor context dictates reliance on TCF1 for response to immunotherapy

Abstract Stem-like CD8 T cells are regulated by the transcription factor TCF1 and are key players in the response to immune checkpoint blockade (ICB). However, recent findings indicate that the dependance on TCF1+ stem-like T cells for ICB efficacy may not be equal across patients or in different tumor contexts. Here we leveraged TCF1 conditional knock-out (TCF1 cKO) mice to investigate how TCF1 instructs the early fate and functions of CD8 T cells upon ICB therapy in tumors that differ for immunogenicity and levels of tumor antigen expression. Strikingly, we discovered that TCF1 expression in CD8 T cells is required for ICB efficacy in poorly immunogenic B16OVA melanomas but is dispensable in highly immunogenic MC38OVA colorectal tumors. Single-cell RNA sequencing and immunophenotyping in the tumor draining lymph node (TDLN) revealed defective priming and expansion of tumor-specific TCF1 cKO T cells in B16OVA- but not MC38OVA-bearing mice treated with ICB. Conversely, ICB therapy efficiently expanded tumor-specific TCF1 WT T cells in the TDLN of both tumor models. In vitro, we found defective proliferation, reduced PD1 and CD28 up-regulation and reduced phosphorylation of key molecules downstream the T cell receptor pathway in TCF1 cKO T cells stimulated with low but not high TCR signals. These data indicate that TCF1 poises T cells for optimal responsiveness in suboptimal priming conditions such as those found in low antigen expressing tumors. Furthermore, in the absence of TCF1 we found the accumulation in the TDLN of a subset of tumor-specific naïve T cells poised to give rise to short-lived effectors, which are less suited to sustain anti-tumor responses in poorly immunogenic tumors where expansion of T cells retaining memory potential is required for durable responses. Single-cell RNA sequencing and immunophenotyping within the tumor microenvironment showed that in MC38OVA tumors both WT and TCF1 cKO mice expanded a CD8 subset sharing a signature with transitory effectors, which were shown to mediate ICB efficacy in chronic viral infection models. Expansion of cytotoxic CD8 T cells likely accounted for the strong anti-tumor response observed in both WT and TCF1 cKO mice. Conversely, B16OVA tumors failed to expand transitory effectors and accumulated Tox+ dysfunctional CD8 T cells. Importantly, in the absence of TCF1 dysfunctional T cells became destabilized, failed to persist and shared features with CD8 T cells found in patients that fail ICB. Altogether, the reduced priming of stem-like T cells in the TDLN combined with a destabilized dysfunctional T cell state in the tumor contributed to the failure of TCF1 cKO mice to sustain effective ICB responses in poorly immunogenic tumors. Our study highlights a role for TCF1 during the priming and early stages of the anti-tumor CD8 T cell response with important implications for guiding optimal therapeutic interventions in cancers with low frequency of TCF1+ CD8 T cells and low neoantigen expression. Citation Format: Giulia Escobar, Katherine Tooley, Joan Pages Oliveras, Linglin Huang, Hanning Cheng, Chang Xue, Davide Mangani, Natanael Hazel, Carola Rutigliani, Luca Biasco, Ana C. Anderson. Tumor context dictates reliance on TCF1 for response to immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4157.

Abstract 4167: Translational control of T cell exhaustion in melanoma, perspectives in therapeutics

Abstract Background: Eukaryotic initiation factor 4F (eIF4F) complex plays a pivotal role in the selective translational control of protein expression in cancer. We previously showed that the inhibition of eIF4F had both a direct antitumor effect that could overcome resistance to BRAF inhibition in BRAFV600 melanoma and an indirect immune-mediated antitumor effect via PD-L1 down regulation resulting from STAT1 decreased translation. We now explore further the translational control of immune checkpoints (ICs) on T cell and its link with lymphocyte exhaustion. Methods: We performed polysome profiling (PP) on peripheral blood-derived activated and resting T lymphocytes as well as tumor-infiltrating lymphocytes (TILs) isolated from melanoma specimens in the presence or absence of silvestrol as an eIF4A (the helicase component of eIF4F) inhibitor. We studied the expression of ICs and exhaustion-associated transcription factors on lymphocytes at the various stages of exhaustion. The tumor cell lysing capacity of PBMC was evaluated with and without silvestrol. We also assessed the effect of silvestrol on tumor microenvironment in the BP syngeneic melanoma tumor model. Results: Silvestrol decreased the translation of multiple ICs in activated CD4+ and CD8+ T cells, including TOX, NFAT1, NFAT2, LAG3, and TIM3 (the two latter being also transcriptionally regulated). FACS analysis confirmed the dose-dependent decrease of protein expression of exhaustion-associated transcription factors TOX, NFATc1, TCF1 and T-bet in activated lymphocytes. This effect was more prominent in more exhausted T cells. Silvestrol decreased T cell proliferation but, in co-culture experiments, it increased immune cell killing of melanoma cells by T-lymphocytes. In the BP murine model, the antitumor effect of silvestrol was associated with denser T cell infiltration and less exhausted TILs than control tumors. Conclusion: Our findings demonstrate the role of eIF4F in the orchestrated regulation of functionally related proteins involved in T cell exhaustion. They highlight the drugability of eIF4A for the cancer treatment and suggest that eIF4A inhibitors could be useful with IC inhibitors in combination or sequentially. Citation Format: Samad Muhammadnejad, Biswendu Biswas, Ramdane Guemiri, Naima Benannoune, Jérémy Lavigne, Virginie Quidville, Jean-Yves Scoazec, Stephan Vagner, Caroline Robert. Translational control of T cell exhaustion in melanoma, perspectives in therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4167.

Abstract 4615: Differential immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes according to ProMisE classifier in endometrial cancer

Abstract Endometrial cancer (EC), one of the most common gynecological malignancies, has been gradually increasing worldwide. The Cancer Genome Atlas endometrial collaborative project discovered 4 distinct prognostic genomic subtypes, and the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) was developed: POLE-mutated, mismatch repair deficiency (MMR-D), non-specific molecular profile (NSMP), and p53-abnormal (p53abn). In the era of immunotherapy, immune checkpoint inhibitors (ICIs) become a definite management of EC, both as single agent or in combination with other targeted agents. However, the characteristics of the tumor immune microenvironment according to molecular classification in EC are not well known. This study was conducted to identify distinct immune properties of tumor infiltrating lymphocytes (TILs) according to molecular classification in EC and find optimal therapeutic targets in each molecular subtypes. We isolated peripheral blood mononuclear cells (PBMCs) and TILs from 81 patients with EC. The expression of immune checkpoint receptors and T-cell transcription factors of TILs were examined using flow cytometry. Sixty-six patients were evaluated according to the ProMisE algorithm based on sequencing for the presence of POLE mutations, immunohistochemistry for MMR proteins and p53 (POLE-mut, n=6; MMR-D, n=17; p53abn, n=20; NSMP, n=23). Tumors were categorized into one of the four molecular subtypes. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1, and their proliferation was assessed to predict ICI-induced reinvigoration capacity of CD8 TILs. Comparing T-cell transcription factors (TCF-1 and TOX), CD8 TILs showed higher expression of TOX, and lower expression of TCF-1. When comparing the immune characteristics of TILs according to molecular subtypes, Tregs of POLE-mut showed significantly lower expression of CTLA-4 and Ki-67 than that of MMR-D. In addition, CD8 TILs of MMR-D showed higher expression of immune checkpoint receptors, including PD-1, CTLA-4, and TIM-3, and lower expression of TCF-1 than those of p53abn. CD8+TILs of p53abn showed significantly higher expression of CD226 than those of MMR-D. In particular, the frequency of antigen reactive CD39+CD103+CD8 T cells significantly lower in p53abn than in MMR-D. CD8 TILs of NSMP showed higher expression of TOX than those of MMR-D. In ex vivo T cell proliferation assay (n=49), 80.0% POLE-mut (4 of 5), 46.2% MMR-D (6 of 13), 35.7% p53abn (5 of 14) and 35.36% NSMP (6 of 17) showed enhanced proliferation of CD8 TILs upon anti-PD-1 treatment. In endometrial cancer, TILs showed distinct immune properties according to ProMisE classification. Further study is needed to develop optimal treatment targets for the p53abn and NSMP subtype, which is non-immunogenic and hardly reinvigorated by ICIs. Citation Format: Jung Chul Kim, Junsik Park, Yong Jae Lee, Sunghoon Kim, Sang Wun Kim, Jung-Yun Lee. Differential immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes according to ProMisE classifier in endometrial cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4615.

Single-Cell RNA sequencing highlights the regulatory role of T cell marker genes Ctla4, Ccl5 and Tcf7 in corneal allograft rejection of mouse model.

A mouse corneal allograft model was induced and single-cell RNA sequencing (scRNA-seq) data of corneal tissues and T cells were analyzed to reveal a T cell-mediated mechanism for corneal allograft rejection in mice. Corneal tissue samples from a mouse model of corneal allograft were collected for scRNA-seq analysis, followed by quality control, dimensionality reduction, cluster analysis and enrichment analysis. A large number of highly variable genes were identified in mice with corneal allograft. Significant difference existed in immune T cells, especially in CD4+T cells. It was found that T cell marker genes Ctla4, Ccl5, Tcf7, Lgals1, and Itgb1 may play key roles in the corneal allograft rejection. Mice with allograft rejection showed a significant increase in the proportion of CD4+T cells in the corneal tissues. Besides, Ccl5 and Tcf7 expression was increased in mice with allograft rejection and positively linked to the proportion of CD4+T cells. Whereas, Ctla4 expression was downregulated and negatively associated with the proportion of CD4+T cells. Collectively, Ctla4, Ccl5 and Tcf7 may participate in the rejection of corneal allograft in mice by affecting CD4+T cell activation.