Infectious diseases are a worldwide problem that have been brought into focus by the 2020 SARS-CoV-22020 outbreak. The respiratory coronavirus is responsible for a global pandemic that has claimed over 6 million lives and affected hundreds of millions more. To slow the spread of the infection and ease the strain on healthcare systems, new vaccines and therapeutics were developed that protected against severe diseases. However, multiple waves of new variants emerged as the virus evolved to escape immunity afforded by vaccination attempts. To generate more effective vaccines that would provide life-long, pan-variant protection against infectious pathogens, we must first understand the immune response and the memory cells that confer immunity.Following infection, memory CD8 T cells can be found surveilling the body in circulation or persisting in the tissues. In the circulating compartment, different subsets of memory CD8 T cells have been identified based on localization pattern, phenotype, and function. Effector memory T cells (TEM) can be found traversing the non-lymphoid organs and are of high effector function, low proliferative, and low differentiation potentials. Central memory T cells (TCM), however, home to the secondary lymphoid tissues and are of low effector function but high differentiation potential. The distinction between memory CD8 T cell subsets is important not only for understanding the immune response, but also for predicting T cell protection post infection or vaccination. While TCM cells are homogeneous based of expression of cell surface markers, several studies have reported heterogeneity within the TEM population. We found that the conventional definition of TEM cells is conflated with a terminally-differentiated (t-TEM) subset, which consists of memory T cells with effector characteristics. The t-TEM population is of distinct ontogeny and fate from TEM and TCM. All three subsets differentially rely on key lineage-defining transcription factors and are transcriptionally and functionally distinct. In human blood, we identified analogous subsets with corresponding cell surface molecules and gene expression patterns. The delineation of t-TEM further clarified our understanding of circulating memory CD8 T cell biology. Through the characterization of memory CD8 T cell subsets, we observed a subset- and tissue-specific expression of granzyme A (GzmA), a serine protease that can induce apoptosis as well as mediate cytokine production. We showed that expression of GzmA by t-TEM in the circulation and tissue-resident (TRM) T cells in the intestines conferred heightened protection against viral reinfections. However, CD8 T cell-specific GzmA in the colon can contribute to pathology in the DSS-induced model of colitis. In addition to enhancing cell-death, GzmA can also mediate in inflammatory cytokine response in vitro and in vivo. Together, these studies clarified the definition of memory T cell subsets and provided insights on the role of CD8 T cell-dependent GzmA in conferring immunity against reinfection as well as contributing to inflammation.

Immunological memory is a hallmark property of the adaptive immune system. Following antigen clearance, T cells form specialized subsets capable of rapidly responding to antigen re-encounter. A recently described subset of memory T cells is tissue resident memory (TRM), which provide frontline protection against pathogens and emerging malignancies. Tumor infiltrating lymphocytes with TRM features are associated with improved clinical outcomes. While tremendous progress has been made deciphering the transcriptional regulation of T cell subsets, the cellular interactions which govern T cell differentiation and function in non-lymphoid tissues are not well understood. Here, we leveraged genetic models and state of the art spatial transcriptomics to identify a key role for the chemokine XCL1 and its functional partner, XCR1+ dendritic cells in CD8+ T cell tissue residency and anti-tumor activity.

CD8 + T cells are a central component of the adaptive immune system. Upon infection, a naive CD8 + T cell will differentiate into a heterogeneous population of effector T cells composed of terminal-effector and memory-precursor CD8 + T cells. Terminal-effector T cells rapidly decay after pathogens are eradicated while memory-precursor T cells survive during the contraction phase to become memory T cells, providing long-term protection from reinfection. Similar to the heterogeneity of effector T cells, memory T cells can also be divided into central-memory, effector-memory and tissue-resident memory subsets based on trafficking, location, proliferation potential and cytotoxic function. These memory subsets collaborate together to enhance the pathogen clearance and vaccine efficacy. The differentiation of a naive CD8 + T cell into a specific effector or memory subset is influenced by cell-extrinsic environmental signals and cell-intrinsic factors including transcription factors (TFs), epigenetic modification and chromatin organization. Considerable advances have been made to identify key TFs that regulate these T cell fate decisions. However, the TF-mediated transcriptional network responsible for specific subset differentiation and how epigenetic modifications and chromatin configuration modulate CD8 + T cell fate determination is still largely unknown. To address these questions, we deciphered epigenetic landscapes in effector and memory CD8 + T cells in response to bacterial infection by characterizing the genome-wide histone modification, chromatin accessibility and transcriptional program. Integrative analysis of epigenomics data showed that subset-specific enhancers established by key TFs foreshadow the specific lineage differentiation. To better identify cruicial TFs from multilayered epigenetic landscapes, we developed a webpage ranking-based algorithm (PageRank) to rank the importance of TFs from transcriptional network and identified a novel function of two TFs: YY1 and Nr3c1 to regulate terminal-effector and memory-precursor subset differentiation, respectively. By leveraging the PageRank analysis and chromatin accessibility data, we developed a computational screen to predict key TFs for tissue-resident memory T cell differentiation. Combining this approach with shRNA functional screen, we identified the role of Runx3 in programming tissue-residency signatures in non-lymphoid tissues and tumors. Finally, we discovered a novel role for the genome organizer CTCF in CD8 + T cell fate decisions illustrating the impact of chromatin organization on effector and memory T cell differentiation. Taken together, we uncovered a multi-layered regulation of chromatin state, accessibility and organization, that influences T cell fate decisions by fine-tuning transcriptional circuits. We further constructed a computational framework that integrates these high-dimensional data, facilitating identification of key transcriptional regulators and providing valuable biological insights.

CD8+ T cells play an indispensable role in the host protection from infections and malignancies. As such, many current immunotherapies target molecules that alter CD8+ T cell function and differentiation. Although genome organization is known to be important for regulating cell development and function, the changes in spatial chromatin organization accompanying effector and memory CD8+ T cell differentiation remain unknown. Here, we studied how genome organization is integrated with other molecular mechanisms regulating CD8+ T cell differentiation and targeted CTCF, a key factor that regulates genome organization through blocking or facilitating chromatin interactions, to determine how altering interactions affect the CD8+ T cell response. We observed T cell subset-specific changes in intra-TAD interactions at sites related to transcriptional rewiring, such as genes encoding for transcription factors that regulate CD8+ T cell differentiation. Further, terminally-differentiated effector cell differentiation was accompanied by enrichment of interactions among subset-specific enhancers and promoters, reflecting their terminally differentiated state. We next characterized the binding profile of CTCF, a known regulator of chromatin interactions. CTCF binding changed with CD8+ T cell differentiation, and weak-affinity CTCF binding is needed to promote terminal differentiation in both an infection and tumor setting. Strikingly, disruption of a single CTCF binding site upregulated expression of corresponding memory-associated molecules, providing clear evidence that CD8+ T cell differentiation is regulated through chromatin interactions. Thus, this study not only provides key insights into the remodeling of chromatin architecture during the CD8+ T cell response to infection, but also provides high quality sequencing data to act as a resource to further identify novel regulators of the CD8+ T cell response.

CD4+ T lymphocytes are a key element of adaptive immunity, acting to coordinate and enhance functions of innate cells, B cells, and CD8+ T cells in response to diverse pathogens. Following clearance of the pathogen, a small proportion of effector CD4+ T cells persists and differentiates into long-lived memory cells, which enable a robust secondary response against reinfection and are pivotal in conferring lasting cellular immunity. While the majority of memory cells circulate between tissues and the secondary lymphoid organs (SLOs), tissue-resident memory T cells (TRM) remain lodged in non-lymphoid barrier tissues, particularly at mucosal surfaces like the intestine and serve as sentinels at sites of potential re-exposure to pathogens.

In this dissertation, I aimed to address two overarching questions regarding the biology of virus-specific CD4+ TRM in the small intestine (SI) following acute lymphocytic choriomeningitis virus (LCMV) infection. First, I addressed the developmental origins of CD4+ TRM cells by examining how the resident population is related to circulating CD4+ T helper subsets in SLOs. Second, I investigated potential transcriptional regulators in CD4+ TRM cells, specifically factors with known roles in driving effector versus memory T cell diffentiation. My work revealed that LCMV-specific CD4+ TRM at day 7 of infection shared a gene-expression program and chromatin profile with TH1 cells and progressively acquired a mature TRM program by a memory time point, supporting a developmental relationship between TRM and TH1 subsets. Furthermore, I demonstrated that TRM cells expressed genes associated with both effector and memory T cell fates, including the transcriptional regulators Blimp1, Id2, and Bcl6 which were necessary for CD4+ TRM differentiation. TH1-associated Blimp1 and Id2 were both required for early TRM formation, while TFH-associated Bcl6 initially inhibited TRM differentiation but was critical for development of long-lived TRM cells. These results identified new significance for transcription factors previously associated with circulating CD4+ T cell populations and their roles in driving SI CD4+ TRM differentiation. This work may provide the basis to exploit the protective capacity of this essential memory T cell population and modulate their activity in the immune response.

T cell responses are initiated by detection of cognate peptide presented by antigen presenting cells in secondary lymphoid tissues and result in migration of T cells to various tissues with disparate local conditions. Hypoxia Inducible Factors (HIFs) are uniquely poised to integrate physiological and disease-derived signals to modulate T cell responses making them an attractive target for further study. Understanding how T cells have taken advantage of the HIF pathway to interpret local conditions and integrate these with the array of mitogenic signals produced during infection is essential for the advancement of our understanding of T cell biology and critical for the development of novel therapeutic approaches. This dissertation takes advantage of genetic models to modulate the oxygen sensing HIF pathway to define how HIFs regulate CD8+ T cell differentiation and function.

By employing genetic deletion of the von Hippel Lindau tumor suppressor protein (VHL), a crucial negative regulator of HIF signaling, we identfied a novel role for HIF activity in modulating effector CD8+ T cell function following chronic viral infection. We demonstrated that sustained HIF signaling not only enhances effector function in the face of persistent antigen, but drives resistance to immune exhaustion in antigen-specific CD8+ T cells. Constitutive HIF signaling also drove enhanced glycolytic metabolism and pharmacological inhibition of glycolysis resulted in partial rescue of HIF-dependent alterations in expression of effector molecules, activation-associated receptors, and critical transcription factors suggesting that modulating cellular metabolism can directly impact T cell function and possibly fate decisions.

Extensive metabolic changes accompany T cell activation including a switch to glycolytic energy production and increased biosynthesis. Recent studies suggest that a subsequent return to reliance on fatty acid oxidation to fuel oxidative phosphorylation and increasing spare respiratory capacity are essential for the differentiation of memory CD8+ T cells. Constitutive HIF activity in CD8+ sustains glycolytic metabolism and suppresses oxidative phosphorylation providing a powerful model by which to define the role of metabolic pathway choice in CD8+ T cell differentiation. Here we demonstrate that surprisingly, constitutive glycolytic metabolism does not inhibit the formation of long-lived memory CD8+ T cells, but predominantly supports the formation of effector memory (TEM) CD8+ cells. Moreover, the departure from a reliance on oxidative phosphorylation also drives accelerated emergence of memory precursor cells following the peak of the response to acute viral infection. Importantly, these long-lived memory CD8+ T cells upregulated IL-7 receptor, expressed critical transcription factors at similar levels to wildtype cells, and demonstrated a heightened response upon secondary challenge demonstrating that restricting CD8+ T cells to glycolytic metabolism does not inhibit the differentiation of a protective memory pool.

While constitutive HIF signaling has provided a powerful model to define targets of HIF activity in CD8+ T cells, we also sought to understand how loss of HIF signaling and HIF-mediated glycolytic metabolism may impact CD8+ T cell differentiation and function. In this dissertation we begin to explore how deletion of HIF-1a and HIF-2a impact CD8+ T cell metabolism and function following acute viral infection and place these findings in the context of CD8+ T cell immunity as well as propose areas of future study.

Patients with metastatic and locally advanced cutaneous squamous cell carcinoma (CuSCC) undergoing anti-programmed death-1(PD-1) immune checkpoint blockade therapy have shown durable anti-tumor activity. Response to anti-PD-1 checkpoint blockade therapy partially depends on an existing anti-tumor T cell population within the tumor microenvironment (TME) directed against neoantigens specific for the tumor. The development and ultimately escape from this immune response is not well understood in cutaneous squamous cell cancer development. Using gene expression profiles including those that predict response to anti-PD-1 therapy, I will investigate normal skin to precancerous skin to invasive CuSCC from both immunocompetent and immunocompromised patients with quantitative molecular profiling of metabolic pathway and immune related gene expression. We expect to show differential expression of genes involved in shaping the TME and immune cell infiltration from normal skin to invasive cancer. Therefore, these findings can help identify steps in cancer development and perhaps insight into prevention strategies. In addition, we will compare the development of tumors in the context of a normal immune system and an impaired immune response (ie organ transplant patients). We hope these profiles may help identify additional therapeutic strategies for treating both immune competent and immune suppressed patients.