Terminal Exhaustion Research Articles

TET2 regulates early and late transitions in exhausted CD8+ T cell differentiation and limits CAR T cell function.

CD8+ T cell exhaustion hampers control of cancer and chronic infections and limits chimeric antigen receptor (CAR) T cell efficacy. Targeting TET2 in CAR T cells provides therapeutic benefit; however, TET2's role in exhausted T cell (TEX) development is unclear. In chronic lymphocytic choriomeningitis virus (LCMV) infection, TET2 drove conversion from stem cell-like TEX progenitors toward terminally differentiated and effector (TEFF)-like TEX. TET2 also enforced a terminally differentiated state in the early bifurcation between TEFF and TEX, indicating broad roles for TET2 in acquisition of effector biology. To exploit the therapeutic potential of TET2, we developed clinically actionable TET2-targeted CAR T cells by disrupting TET2 via knock-in of a safety switch alongside CAR knock-in at the TRAC locus. TET2-targeted CAR T cells exhibited restrained terminal exhaustion in vitro and enhanced antitumor responses in vivo. Thus, TET2 regulates fate transitions in TEX differentiation and can be targeted with a safety mechanism in CAR T cells for improved tumor control.

IMMU-53. ANTIGEN PRESENTATION BY TUMOR-ASSOCIATED MACROPHAGES MEDIATES PROGENITOR TO TERMINAL EXHAUSTION TRANSITION IN GBM AND OTHER SOLID TUMORS

Abstract While terminally exhausted T cells (Tex_term) retain important anti-tumor cytotoxic function, it is the relative preservation of renewable, stem-like progenitor exhaustion (Tex_prog) that better indicates immunotherapeutic responsivity. Although restraining the progression from Tex_prog to Tex_term thus takes on clinical significance, the cellular interactions in a tumor microenvironment (TME) governing such progression remain less established. Employing glioblastoma (GBM) and other solid tumors as models of severe exhaustion, we provide a detailed characterization of the progression from Tex_prog to Tex_term within the TME, where we observe a striking and disproportionate loss of Tex_prog over time, leading to a low progenitor exhaustion to terminal exhaustion ratio (PETER). We find exhaustion concentrated within tumor-specific T cell subsets, with cognate antigenic exposure requisite for acquisition of the Tex_term phenotype. However, we implicate tumor-associated macrophages (TAM), and not tumor cells, as the source of antigenic exposure governing the Tex_prog to Tex_term transition. Using cell – cell interaction analysis, we additionally highlight candidate receptor–ligand communications that may be specifically mediating the progression to Tex_term and resultant decline in PETER within the TME.

Abstract A047: Activation of conventional type 2 dendritic cells (cDC2) by anti-CD47 may enhance antitumor T cell function in mouse liver cancer models

Abstract Anti-CD47 has been extensively studied for its effects on activating phagocytosis by blocking the ‘don’t eat me’ signal presented by CD47 expressed on cancer cells. This study explored the effects of anti-CD47 on enhancing antigen presentation by dendritic cells and improving antigen-specific antitumor immunity. Mouse Hepa1-6 liver cancer cells with or without over-expression of LCMV-GP protein and splenocytes from P14 transgenic mice were used to test antigen-specific antitumor immunity. Bone marrow-derived conventional type 1 and type 2 dendritic cells (cDC1 and cDC2) or MutuDC1/ DC2 cell lines were co-cultured with CD8 T cells with or without exhaustion. Effects of anti-CD47 on the cross-priming ability of DC were analyzed by T cell proliferation (CSFE labeling), activation (expression of IFNγ, granzyme B, TNFa, and IL-2), cell killing (in vitro cytotoxicity assay) and T cell exhaustion (LAG3, TOX, PD1 expression). In vivo studies indicated that anti-CD47 treatment may suppress tumor growth, activate intra-tumoral cDC2 but not type 1 dendritic cells (cDC1), and delay CD8 T cells from entering terminal exhaustion in the tumor microenvironments. In vitro studies indicated that anti-CD47 may enhance the priming ability of cDC2, but not cDC1, and increase ISG15 expression in cDC2. When co-cultured with exhausted T cells, anti-CD47-treated cDC2 may delay the exhaustion process and restore antigen-specific effector function and cytotoxic capacity. Conclusion: Anti-CD47 may enhance antitumor T cell function in mouse liver cancer models by activation of cDC2, which may serve as a new strategy of developing immunotherapeutic agents for liver cancer (supported by grants NSTC 111- 2314-B-002-039 and 113-2314-B-002-113-MY3). Citation Format: Cheng-Zhe Jian, Da-Liang Ou, Chia-Lang Hsu, Chien-Kuo Lee, Chiun Hsu. Activation of conventional type 2 dendritic cells (cDC2) by anti-CD47 may enhance antitumor T cell function in mouse liver cancer models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A047.

Calcineurin inhibitor blocks tolerance by suppressing donor T cell terminal exhaustion after allogeneic hematopoietic cell transplantation.

Calcineurin inhibitor blocks tolerance by suppressing donor T cell terminal exhaustion after allogeneic hematopoietic cell transplantation.

Inhibitory Fcγ receptor deletion enhances CD8 T cell stemness increasing anti-PD-1 therapy responsiveness against glioblastoma

BackgroundCertain cancers present challenges for treatment because they are resistant to immune checkpoint blockade (ICB), attributed to low tumor mutational burden and the absence of T cell-inflamed features. Among these,...

TCellSI: A novel method for T cell state assessment and its applications in immune environment prediction.

T cell is an indispensable component of the immune system and its multifaceted functions are shaped by the distinct T cell types and their various states. Although multiple computational models exist for predicting the abundance of diverse T cell types, tools for assessing their states to characterize their degree of resting, activation, and suppression are lacking. To address this gap, a robust and nuanced scoring tool called T cell state identifier (TCellSI) leveraging Mann-Whitney U statistics is established. The TCellSI methodology enables the evaluation of eight distinct T cell states-Quiescence, Regulating, Proliferation, Helper, Cytotoxicity, Progenitor exhaustion, Terminal exhaustion, and Senescence-from transcriptome data, providing T cell state scores (TCSS) for samples through specific marker gene sets and a compiled reference spectrum. Validated against sizeable pseudo-bulk and actual bulk RNA-seq data across a range of T cell types, TCellSI not only accurately characterizes T cell states but also surpasses existing well-discovered signatures in reflecting the nature of T cells. Significantly, the tool demonstrates predictive value in the immune environment, correlating T cell states with patient prognosis and responses to immunotherapy. For better utilization, the TCellSI is readily accessible through user-friendly R package and web server (https://guolab.wchscu.cn/TCellSI/). By offering insights into personalized cancer therapies, TCellSI has the potential to improve treatment outcomes and efficacy.

Soluble Tim-3 serves as a tumor prognostic marker and therapeutic target for CD8+ T cell exhaustion and anti-PD-1 resistance

Resistance to PD-1 blockade in onco-immunotherapy greatly limits its clinical application. Tcell immunoglobulin and mucin domain containing-3 (Tim-3), a promising immune checkpoint target, is cleaved by ADAM10/17 to produce its soluble form (sTim-3) in humans, potentially becoming involved in anti-PD-1 resistance. Herein, serum sTim-3 upregulation was observed in non-small cell lung cancer (NSCLC) and various digestive tumors. Notably, serum sTim-3 is further upregulated in non-responding patients undergoing anti-PD-1 therapy for NSCLC and anti-PD-1-resistant cholangiocarcinoma patients. Furthermore, sTim-3 overexpression facilitates tumor progression and confers anti-PD-1 resistance in multiple tumor mouse models. Mechanistically, sTim-3 induces terminal Tcell exhaustion and attenuates CD8+ Tcell response to PD-1 blockade through carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1). Moreover, the ADAM10 inhibitor GI254023X, which blocks sTim-3 production, reduces tumor progression in Tim-3 humanized mice and reverses anti-PD-1 resistance in human tumor-infiltrating lymphocytes (TILs). Overall, human sTim-3 holds great predictive and therapeutic potential in onco-immunotherapy.

Tuning CAR T-cell therapies for efficacy and reduced toxicity

Chimeric antigen receptor (CAR) T-cell therapies are a standard of care for certain relapsed or refractory B-cell cancers. However, many patients do not respond to CAR T-cell therapy or relapse later, short- and long-term toxicities are common, and current CAR T-cell therapies have limited efficacy for solid cancers. The gene engineering inherent in CAR T-cell manufacture offers an unprecedented opportunity to control cellular characteristics and design products that may overcome these limitations. This review summarises available methods to “tune” CAR T-cells for optimal efficacy and safety. The components of a typical CAR, and the modifications that can influence CAR T-cell function are discussed. Methods of engineering passive, inducible or autonomous control mechanisms into CAR T-cells, allowing selective limitation or enhancement of CAR T-cell activity are reviewed. The impact of manufacturing processes on CAR T-cell function are considered, including methods of limiting CAR T-cell terminal differentiation and exhaustion, and the use of specific T-cell subsets as the CAR T starting material. We discuss the use of multicistronic transgenes and multiplexed gene editing. Finally, we highlight the need for innovative clinical trial designs if we are to make the most of the opportunities offered by CAR T-cell therapies.

Lrp10 suppresses IL7R limiting CD8 T cell homeostatic expansion and anti-tumor immunity

Signals emanating from the T-cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T-cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T-cell function. Through forward genetic screening in mice, we discover that loss-of-function mutations in LDL receptor-related protein 10 (Lrp10) cause naive and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. T-cell activation induces Lrp10 expression, which post-translationally suppresses IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhances T-cell homeostatic expansion through IL7R signaling. Lrp10-deficient mice are also intrinsically resistant to syngeneic tumors. This phenotype depends on dense tumor infiltration of CD8 T cells, which display increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T-cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy.

NRF2-dependent regulation of the prostacyclin receptor PTGIR drives CD8 T cell exhaustion.

The progressive decline of CD8 T cell effector function-also known as terminal exhaustion-is a major contributor to immune evasion in cancer. Yet, the molecular mechanisms that drive CD8 T cell dysfunction remain poorly understood. Here, we report that the Kelch-like ECH-associated protein 1 (KEAP1)-Nuclear factor erythroid 2-related factor 2 (NRF2) signaling axis, which mediates cellular adaptations to oxidative stress, directly regulates CD8 T cell exhaustion. Transcriptional profiling of dysfunctional CD8 T cells from chronic infection and cancer reveals enrichment of NRF2 activity in terminally exhausted (Texterm) CD8 T cells. Increasing NRF2 activity in CD8 T cells (via conditional deletion of KEAP1) promotes increased glutathione production and antioxidant defense yet accelerates the development of terminally exhausted (PD-1+TIM-3+) CD8 T cells in response to chronic infection or tumor challenge. Mechanistically, we identify PTGIR, a receptor for the circulating eicosanoid prostacyclin, as an NRF2-regulated protein that promotes CD8 T cell dysfunction. Silencing PTGIR expression restores the anti-tumor function of KEAP1-deficient T cells. Moreover, lowering PTGIR expression in CD8 T cells both reduces terminal exhaustion and enhances T cell effector responses (i.e. IFN-γ and granzyme production) to chronic infection and cancer. Together, these results establish the KEAP1-NRF2 axis as a metabolic sensor linking oxidative stress to CD8 T cell dysfunction and identify the prostacyclin receptor PTGIR as an NRF2-regulated immune checkpoint that regulates CD8 T cell fate decisions between effector and exhausted states.

Hallmarks of tumor-experienced T cells are absent in multiple myeloma patients from diagnosis through maintenance therapy.

Dysregulation of the bone marrow (BM) niche in multiple myeloma (MM) alters the composition and state of resident immune cells, potentially impeding anti-tumor immunity. One common mechanism of immune inhibition in solid tumors is the induction of exhaustion in tumor-specific T cells. However, the extent of T cell tumor recognition and exhaustion is not well-characterized in MM. As the specific mechanisms of immune evasion are critical for devising effective therapeutic strategies, we deeply profiled the CD8+ T cell compartment of newly-diagnosed MM (NDMM) patients for evidence of tumor reactivity and T cell exhaustion. We applied single-cell multi-omic sequencing and antigen-specific mass cytometry to longitudinal BM and peripheral blood (PB) samples taken from timepoints spanning from diagnosis through induction therapy, autologous stem cell transplant (ASCT), and maintenance therapy. We identified an exhausted-like population that lacked several canonical exhaustion markers, was not significantly enriched in NDMM patients, and consisted of small, nonpersistent clones. We also observed an activated population with increased frequency in the PB of NDMM patients exhibiting phenotypic and clonal features consistent with homeostatic, antigen-nonspecific activation. However, there was no evidence of "tumor-experienced" T cells displaying hallmarks of terminal exhaustion and/or tumor-specific activation/expansion in NDMM patients at any timepoint.

The cell cycle regulator p16 promotes tumor infiltrated CD8+ T cell exhaustion and apoptosis

The therapeutic efficacy of adoptive T cell therapy is largely restricted by reduced viability and dysfunction of CD8+ T cells. Continuous antigen stimulation disrupts the expansion, effector function, and metabolic fitness of CD8+ T cells, leading to their differentiation into an exhausted state within the tumor microenvironment (TME). While the function of the cell cycle negative regulator p16 in senescent cells is well understood, its role in T cell exhaustion remains unclear. In this study, we demonstrated that TCR stimulation of CD8+ T cells rapidly upregulates p16 expression, with its levels positively correlating with TCR affinity. Chronic TCR stimulation further increased p16 expression, leading to CD8+ T cell apoptosis and exhaustion differentiation, without inducing DNA damage or cell senescence. Mechanistic investigations revealed that p16 downregulates mTOR, glycolysis, and oxidative phosphorylation (OXPHOS) associated gene expression, resulting in impaired mitochondrial fitness, reduced T cell viability, and diminished effector function. Furthermore, the deletion of p16 significantly enhances the persistence of CD8+ T cells within tumors and suppresses the terminal exhaustion of tumor-infiltrating T cells. Overall, our findings elucidate how increased p16 expression reshapes T cell intracellular metabolism, drives T cell apoptosis and exhaustion differentiation, and ultimately impairs T cell anti-tumor function.

Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ Tcells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ Tcells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ Tcell subset by coupled Tcell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ Tcells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ Tcell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ Tcell exhaustion and could be targeted to potentiate cancer immunotherapy.

Modulating the epigenetic interplay in exhausted CD8 T cells for effective immunotherapy

Abstract Epigenetic imprinting of terminal exhaustion in chronically stimulated CD8 T cells is a major impediment to T cell immunotherapies during chronic viral infections or cancer. Foundational work establishes that exhausted CD8 T cells (TEX) acquire heritable DNA methylation programs that silence genes critical for T cell stemness/function, restraining responses to immune checkpoint blockade (ICB). However, the molecular interplay between histone post-translational modifications and de novo DNA methylation during the progression to terminal exhaustion remains poorly understood. To address this, we employ our novel in vitro model of human T cell dysfunction and the chronic LCMV infection model to reveal a temporal linkage in which progressive histone changes prime the acquisition of de novo DNA methylation in TEX. Via a selective inhibitor or CRISPR editing in human CD8 T cells, we identify specific histone-modifying enzymes as key epigenetic drivers that orchestrate DNA methylation programming during terminal exhaustion. Notably, targeting the histone-modifying enzymes reinvigorated effector function and cytotoxicity in dysfunctional human T cells. Moreover, combined epigenetic therapy and anti-PD-L1 enhanced the ICB responses of both progenitor and cytolytic TEX during chronic viral infection. Collectively, our findings reveal novel epigenetic drivers of CD8 T cell dysfunction, supporting therapeutic potential of targeting the epigenetic crosstalk underpinning T cell exhaustion.

Deciphering the role of Klf2 in CD8 T cell differentiation and exhaustion during chronic infection

Abstract T cell exhaustion, a state of dysfunction occurring during chronic infections and cancer, is marked by a progressive loss of effector functions and increased expression of inhibitory receptors. In chronic infection, CD8 T cells differentiate into TCF1+ progenitor (TPRO), CX3CR1+ effector (TEFF), and terminally exhausted (TEXH) subsets. The differentiation pathways are governed by complex transcription factor (TF) networks. Employing regulon analysis enabled by single-cell RNA+ATAC multiome sequencing, we dissected the TF networks orchestrating the fate of exhausted CD8 T cell populations. Our integrative analysis, complemented by single-cell CRISPR screening, highlighted Krüppel-like factor 2 (KLF2) as a pivotal TF necessary for maintaining TPRO cells and generating TEFF cells. Our research also revealed that KLF2 regulates the distinct distribution patterns of CD8 T cell subsets across organs. More intriguingly, multiomic analysis uncovered a novel function of Klf2 in limiting T cell exhaustion. Klf2-deficient CD8 T cells exhibited a marked increase in exhaustion signature genes. ATAC-seq and CUT&TAG assays further revealed different chromatin accessibility and histone modifications within Klf2 and its enhancers across CD8 T cell subsets. These insights underscore the differential transcriptional and epigenetic regulation of Klf2, presenting a novel mechanism that influences the cell fate decision of CD8 T cells during chronic infections.

Understanding the role of TNFR2 signaling in CD8 T cell exhaustion.

Abstract Glioblastoma (GBM) is the most common primary brain cancer in adults with a median survival less than 15 months. Approaches harnessing the immune response are hindered by multiple factors, including T cell exhaustion. Exhausted T cells are less capable of limiting tumor progression. As cells progress along the exhaustion pathway, SLAM Family Member 6 (SLAMF6) gets downregulated and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and TOX are upregulated. TOX is both necessary and sufficient to initiate the terminal exhaustion program. However, our understanding of the pathways that lead to the upregulation of TOX remains incomplete. We hypothesized that tumor necrosis factor (TNF) could be involved in the upregulation of TOX, as TNF has been shown to induce exhaustion. We observed upregulation of the TNF receptor type II (TNFR2) in tumor-infiltrating T cells, but not in lymphoid organs. Furthermore, TNFR2 expression is correlated with canonical exhaustion markers. Interestingly, TNFR2 knock out (KO) CD8 T cells express TIM3 but exhibit significantly less TOX. These data suggest that TIM3 and TOX are regulated independently. Finally, TNFR2 KO mice have significantly better CD8-mediated tumor control against subcutaneous GBM tumors. Further studies will need to be performed to determine whether TNFR2 expression on CD8 T cells is sufficient to mediate this anti-tumor effect and whether TNFR2 blockade could improve the efficacy of current treatments.

Investigating the potential of CD8α:MyD88 to restrain terminal exhaustion in tumor-specific T cells

Investigating the potential of CD8α:MyD88 to restrain terminal exhaustion in tumor-specific T cells

Tumor immune dysfunction and exclusion subtypes in bladder cancer and pan-cancer: a novel molecular subtyping strategy and immunotherapeutic prediction model

BackgroundMolecular subtyping is expected to enable precise treatment. However, reliable subtyping strategies for clinical application remains defective and controversial. Given the significance of tumor immune dysfunction and exclusion (TIDE), we aimed to develop a novel TIDE-based subtyping strategy to guide personalized immunotherapy in the bladder cancer (BC).MethodsTranscriptome data of BC was used to evaluate the heterogeneity and the status of TIDE patterns. Subsequently, consensus clustering was applied to classify BC patients based on TIDE marker-genes. Patients’ clinicopathological, molecular features and signaling pathways of the different TIDE subtypes were well characterized. We also utilize the deconvolution algorithms to analyze the tumor microenvironment, and further explore the sensitivity and mechanisms of each subtype to immunotherapy. Furthermore, BC patient clinical information, real-world BC samples and urine samples were collected for the validation of our findings, which were used for RNA-seq analysis, H&E staining, immunohistochemistry and immunofluorescence staining, and enzyme-linked immunosorbent assay. Finally, we also explored the conservation of our novel TIDE subtypes in pan-cancers.ResultsWe identified 69 TIDE biomarker genes and classified BC samples into three subtypes using consensus clustering. Subtype I showed the lowest TIDE status and malignancy with the best prognosis and highest sensitivity to immune checkpoint blockade (ICB) treatment, which was enriched of metabolic related signaling pathways. Subtype III represented the highest TIDE status and malignancy with the poorest prognosis and resistance to ICB treatment, resulting from its inhibitory immune microenvironment and T cell terminal exhaustion. Subtype II was in a transitional state with intermediate TIDE level, malignancy, and prognosis. We further confirmed the existence and characteristics of our novel TIDE subtypes using real-world BC samples and collected patient clinical data. This subtyping method was proved to be more efficient than previous known methods in identifying non-responders to immunotherapy. We also propose that combining our TIDE subtypes with known biomarkers can potentially improve the sensitivity and specificity of these biomarkers. Moreover, besides guiding ICB treatment, this classification approach can assist in selecting the frontline or recommended drugs. Finally, we confirmed that the TIDE subtypes are conserved across the pan-tumors.ConclusionsOur novel TIDE-based subtyping method can serve as a powerful clinical tool for BC and pan-cancer patients, and potentially guiding personalized therapy decisions for selecting potential beneficiaries and excluding resistant patients of ICB therapy.

Abstract LT02: Regulation of Exhausted CD8+ T cell Differentiation by IKZF Transcription Factors

Abstract CD8+ T cells are indispensable for pathogen and tumour clearance, although can induce immunopathology if left unrestricted. T cell exhaustion, progressively programmed in the context of persistent antigen exposure, ensures partial control while mitigating immunopathological risk. However, exhaustion is characterised by curtailed proliferative capacity, cytokine production and cytotoxic functions, which tumours and chronic pathogens exploit to persist. As such, transiently disrupting exhaustion has emerged as a therapeutic strategy for treating cancer. Exhausted cells transition through multiple differentiation states, from stem-like progenitors that mediate response to checkpoint blockade, to terminal effector or exhausted cells. Unfortunately, only a fraction of patients respond to checkpoint blockade long-term, in part due to loss of the therapy responsive progenitor population. Therefore, understanding the molecular pathways moderating the exhausted states, hindering the effector populations, and promoting terminal exhaustion is essential for augmenting cancer immunotherapy. We identify IKZF transcription factors, Ikaros (IKZF1), Helios (IKZF2) and Aiolos (IKZF3), as key mediators of T cell exhaustion using gene knock-out mice and a CRISPR-mediated genetic knock-out system in both chronic infection and tumour models. We elucidate the molecular pathways involved in regulation of T cell exhaustion by IKZF transcription factor using an array of RNA sequencing, ATAC sequencing and flow cytometric validation technologies. IKZF1 and IKZF3 are critical regulators of exhausted subset expansion, retention, phenotype and cytokine function. Strikingly, dual IKZF1 and IKZF3 ablation boosts the response of anti-tumour and anti-viral exhausted T cells, potentially via the formation of atypical, exhausted T cell populations and the enrichment of functional exhausted subsets. Excitingly, dual ablation vastly improves survival and tumour control in a solid tumour, HER2-specific CAR T cell model. Cumulatively, our data highlight a novel role for IKZF transcription factors in biasing exhausted T cell differentiation and provides potentially important clinical implications, directing future cancer immunotherapies targeting T cell exhaustion. Citation Format: Sinead M. Reading, Isabelle Munoz, Maria N. de Menezes, Nicole Y. L. Saw, Krutika Ambani, Antonio Ahn, Simone Nussing, Sara Roth, Shienny Sampurno, Kelly M. Ramsbottom, Joseph A. Trapani, Kim L. Good-Jacobson, Ricky W. Johnstone, Shom Goel, Paul A. Beavis, Ian A. Parish. Regulation of Exhausted CD8+ T cell Differentiation by IKZF Transcription Factors [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT02.

Abstract LB070: In vitro modeling of antigen-driven exhaustion in human CAR T cells

Abstract A major hurdle for chimeric antigen receptor (CAR) T cells to function in solid tumors is chronic antigen exposure leading to terminal lymphocyte exhaustion and impaired anti-tumor activity. Therefore, it is necessary to establish improved in vitro models of antigen-driven CAR T cell exhaustion to evaluate new CAR design candidates prior to validation with costly in vivo studies. Using the human tumor antigen mesothelin (MSLN) and MSLN-targeting CAR T cells as a model, we developed two in vitro assays that drive an exhausted phenotype in human CAR T cells and can be used to predict anti-tumor responses in vivo: 1) a chronic stimulation assay with plate-bound recombinant antigen (MSLN), ICAM-1, and CD58 and 2) a high antigen-expressing 3D tumor spheroid co-culture assay. In the chronic antigen stimulation assay, repetitive exposure to plate-bound MSLN (alongside co-stimulation signals provided by recombinant ICAM-1 and CD58) reprogrammed the transcriptomic profile of MSLN CAR T cells leading to increased surface expression of inhibitory checkpoint molecules and reduced cytokine production. Moreover, chronically stimulated MSLN CAR T cells demonstrated a drastically impaired ability to kill MSLN+ tumor cells compared to their unstimulated or acutely stimulated counterparts. A similar exhausted phenotype was observed in the high-antigen tumor spheroid model: when MSLN CAR T cells were co-cultured with MSLN-high human NCI-H226 spheroids for 7 days in a 96-well plate, expression of inhibitory checkpoint molecules markedly increased on the surface of CAR T cells. This increased expression of exhaustion markers coincided with impairment of NCI-H226 spheroid clearance. By contrast, other tumor spheroid models expressing low to intermediate levels of MSLN (NCI-H1650, NCI-H2052, NCI-H292, and SKOV-3 tumor cells) did not substantially induce expression of inhibitory checkpoint molecules and the spheroids were successfully cleared by MSLN CAR T cells. Importantly, the ability (or inability) of a CAR construct to clear NCI-H226 high-antigen tumor spheroids in vitro accurately predicted CAR T cell performance in in vivo solid tumor studies: the CAR construct that showed effective killing in the in vitro spheroid model was the only condition to provide durable regression of NCI-H1650 tumors in a NSG xenograft mouse model. Additionally, the rank order of CAR efficacy for a series of MSLN CAR binders obtained using the tumor spheroid model mirrored in vivo efficacy. As the CAR T cell field develops novel strategies to combat antigen-driven exhaustion for use in solid tumors, these new antigen-dependent in vitro models of exhaustion can enable more cost-efficient and effective evaluation of those strategies than using in vivo models alone. Citation Format: Maria Steele, Tina Tan, Jason Yokoyama, Allan Wang, Ty Crowl, Jared Hammer, Jerry Chen, Kevin Haworth, Howell Moffett, Aaron Foster, Marc Lajoie, Scott Boyken, Rupesh Amin. In vitro modeling of antigen-driven exhaustion in human CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB070.