Heterogeneity of Tumor-Infiltrating CD8+ T Cells: Implications for Systemic Immunity and Therapy

Abstract

CD8+ T cells are essential mediators of adaptive immunity, orchestrating immunosurveillance and providing long-lived protection against recurrent infections. Upon pathogen clearance, these cells generate durable memory capable of rapidly eliminating secondary threats. However, the formation of functional memory T cells is severely compromised in cancer. Notably, metastatic disease, rather than primary tumors, accounts for the majority of cancer-related mortality. Persistent antigen exposure within the tumor microenvironment promotes T cell exhaustion and tissue residency, impairing their ability to mount systemic anti-tumor responses. In this dissertation, we investigate the mechanisms by which agonistic antibodies enhance systemic anti-tumor immunity in the neoadjuvant setting. Furthermore, we identify and characterize key regulators of TCR signaling and transcriptional programs that govern the differentiation and function of intratumoral T cells targeted by these therapeutic agents.Neoadjuvant immunotherapy seeks to harness the primary tumor as a source of relevant tumor antigens to enhance systemic anti-tumor immunity through improved immunological surveillance. Despite having revolutionized the treatment of patients with high-risk early-stage triple-negative breast cancer (TNBC), a significant portion of patients remain unresponsive and succumb to metastatic recurrence post-treatment. We found that optimally scheduled neoadjuvant administration of anti-4-1BB monotherapy was able to counteract metastases and prolong survival following surgical resection. Phenotypic and transcriptional profiling revealed enhanced 4-1BB expression on tumor-infiltrating intermediate (Tint), relative to progenitor (Tprog) and terminally exhausted (Tterm) T cells. Furthermore, Tint was enriched in low-affinity T cells. Treatment with anti-4-1BB drove clonal expansion of Tint, with reduced expression of tissue-retention marker CD103 in Tprog. This was accompanied by increased TCR clonotype sharing between paired tumors and pre-metastatic lungs. Further interrogation of sorted intratumoral T cells confirmed enhanced T cell egress into circulation following anti-4-1BB treatment. In addition, the gene signature extracted from anti-4-1BB treated Tint was consistently associated with improved clinical outcomes in BRCA patients. Combinatorial neoadjuvant anti-4-1BB and ablation of tumor-derived CXCL16 resulted in enhanced therapeutic effect. These findings illustrate the intratumoral changes underpinning the efficacy of neoadjuvant anti-4-1BB, highlighting the reciprocity between local tissue-retention and distant immunologic fortification, suggesting treatment can reverse the siphoning of intratumoral T cells to primary tumor, enabling redistribution to distant tissues and subsequent protection against metastases. Having shown that neoadjuvant anti-4-1BB therapy promotes the expansion and egress of a specific subset of TILs, and synergizes with CXCL16 depletion in the TME, we next investigated the regulators potentially driving the development of these CXCR6+ T cells within the primary tumor. Gene ontology analysis indicated a significant enrichment of processes related to the negative regulation of cell activation, and we identified the adaptor molecule LAT2 as being highly expressed in CXCR6+ T cells. Given that LAT2 can competitively inhibit LAT and disrupt TCR signaling – a pathway frequently dysregulated in the TME due to chronic TCR stimulation – we sought to determine whether LAT2 plays a critical role in the development of CXCR6+ T cells. To this end, we utilized Lat2KO mice in both whole-body knockout and competitive adoptive transfer models to assess the intrinsic requirement of LAT2. Our in vivo studies demonstrated that LAT2 is essential for the engraftment and tumor-infiltrating capacity of T cells, which correlates with effective tumor control. Furthermore, our in vitro studies revealed that LAT2 is preferentially expressed in low-affinity T cells, suggesting its potential role in fine-tuning TCR signaling thresholds and modulating T cell responses in the context of suboptimal antigen recognition. Finally, we investigated potential transcriptional regulators governing the expression of CXCR6 and 4-1BB in TILs. While it is understood that the process of T cell exhaustion is modulated by a diverse array of transcriptional regulators, transcriptional control of these specific markers remains less understood. Recent studies have suggested that the transcription factor Runx2 may play a significant role in mitigating T cell exhaustion in the context of adoptive T cell therapy for leukemia. However, its involvement in driving T cell differentiation and exhaustion within solid tumor malignancies is still not fully elucidated. We found that Runx2 was highly expressed only in exhausted T cells within the TME of solid tumors. Overexpression of Runx2 in CD8+ T cells in vitro promoted effector and exhaustion profile. However, in vivo knockout of Runx2 in T cells demonstrated an increase in exhaustion marker TIM-3, accompanied by loss stem-like marker CD62L in murine lung cancer model. This was accompanied by loss of tumor control. Furthermore, competitive adoptive transfer of polyclonal wild-type and Runx2KO T cells showed Runx2-dependent 4-1BB expression in exhausted T cells in TNBC which was not evident in lung cancer. Notably, Runx2 repressed CXCR6 expression in a TCF-1 independent manner in exhausted T cells from TNBC. The studies described in this dissertation collectively demonstrate that the heterogeneity of tumor-infiltrating CD8+ T cells provides an opportunity to identify promising targets where immunotherapeutic research efforts should be focused. Understanding the molecular processes governing the development of specific exhausted T cell subsets may provide further insights into potential avenues for enhancing anti-metastatic immunotherapy treatment.