Influenza A virus (IAV) is one of the most ubiquitous respiratory viruses in the world, causing significant disease burden in the United States and abroad. Current vaccination strategies that target the generation of humoral immunity offer limited heterosubtypic protection; T cells offer cross-strain protection and the promise of universal immunity against IAV. Local tissue immunity plays a key role in pathogen clearance and tissue protection, particularly in the form of tissue resident memory T cells (TRM), which are a non-circulating memory T cell subset that have been shown in a variety of tissue sites to be superior mediators of protection compared to circulating memory T cells. At the same time, T cell immunity has been associated with inflammatory processes that may also lead to lung immunopathology. How lung tissue localized T cell immunity mediates its protection during a recall response to IAV challenge is not well understood. Using the lymphocyte sequestering drug FTY720, we show that primary infection with H3N2 IAV strain X31 provides tissue localized heterosubtypic immunity independently of humoral immunity against an H1N1 PR8 IAV strain. Within the lung resident niche, the recall response drives faster CD4+ and CD8+ T cell expansion compared to a primary infection. This rapid T cell expansion resulted from in situ TRM proliferation that was augmented by the migration of peripheral T cells. By tracking a naïve T cell population specific for the IAV strain used in secondary challenge, we demonstrate that influenza-specific T cells, including those specific for newly introduced antigens, migrate to the lung niche from the local mediastinal lymph node (medLN) where both CD4+ and CD8+ T cells experience enhanced priming and proliferation. We further show that primary infection fortifies the medLN with persistently increased numbers of T cells as well as both CD103+ and CD103- conventional dendritic cells (cDCs) that are transcriptionally similar to cDCs in an infection naïve mouse. By depleting Zbtb46+ cDCs, we determine that cDC fortification is a crucial mechanism for enhanced T cell priming and expansion in the medLN during a recall response. We also found that lung localized CD4+ T cell responses exhibit significant immunomodulatory function. Polyclonal lung CD4+ TRM generated by influenza infection as well as lung OT-II TRM exhibit increased production of antiviral inflammatory cytokines in addition to enhanced IL-10 family cytokine production compared to splenic CD4+ effector memory T cells (TEM). During a heterosubtypic challenge, we further observed that lung niche non-TRM CD4+ T cells produce significantly more in situ IL-10 compared to a primary infection, which modulated airway IFN-ɣ and TNF-α production without any depreciation in viral clearance. Immunomodulatory characteristics of a recall response was reflected in lung tissue-wide transcriptional downregulation of innate responses such as type I IFN responses compared to a primary infection. This work demonstrates the dual antiviral and immunomodulatory protective role of enhanced tissue-localized T cell responses during the recall response to IAV challenge.

The hematopoietic system has long been charactered for its essential function in protecting against pathogens, but it is increasingly established that immune cells play integral roles in resolving inflammation and driving tissue repair. While many cell types are recruited to the site of injury and participate in coordinated immune responses, regulatory T (Treg) cells have emerged as key players of tissue protection by limiting damage and promoting regeneration in multiple organ systems. A conserved feature of “pro-repair” Treg cells is their expression of amphiregulin (Areg), an epidermal growth factor (EGFR) ligand associated with many formative processes in organismal development, tissue regeneration, and cancer. Many hematopoietic and non-hematopoietic cells produce Areg, yet Treg–specific expression has been found to be uniquely important and non-redundant in a number of damage models such as ischemic stroke, muscle injury, and influenza infection. In the lung, re-establishing epithelial barrier integrity is essential for recovery after acute viral injury. Rapid activation of renewal pathways preserves respiratory function during active inflammation and prevents against secondary infections and sequela. It has been previously reported that during influenza virus infection, Treg cell-production of Areg supports host resilience and thwarts severe alveolar damage. Animals that genetically lack Areg from Treg sources suffer a sharp loss of blood oxygenation and worse pathology. Although this growth factor signaling heavily influences disease outcome, the mechanisms by which Areg signals and how Treg cells engage with parenchymal and stromal cells within the alveolar niche are poorly understood. Given that Treg cells constitute only a small fraction of Areg-producing cells in the lung, we hypothesized that spatially restricted signaling and local tissue interactions enable this minority population to exert a major impact on organ function. Here, I used a multidisciplinary approach to interrogate the ability of lung Treg cells to promote alveolar lung repair during H1N1 influenza infection in a murine system. Through high-resolution immunofluorescence imaging, I characterize the unique distribution of Treg cells within lung tissues and their rapid recruitment to sites of active viral replication. Treg cells co-localize with a distinct population of Collagen-14+ EGFR+ mesenchymal cells (Col14+) that are Areg–responsive and robustly promote alveolar epithelial cell development. In the absence of Treg–derived Areg, Col14+ cells exhibit aberrant transcriptional programming, reduced expression of important alveolar growth factors, Fgf7 and Fgf10, and a dramatic increase in apoptotic cell death that together results in impaired alveolar epithelial progenitor cell differentiation. Following genetic ablation of stromal Egfr expression, mice experience a stark decline in blood oxygen saturation and dysplastic alveolar repair similar to loss of Areg from Treg cells, providing evidence that Areg from Treg cells instead signals through Col14+ cell intermediates. These findings underscore that localized delivery of distinct growth factors within tissue stem niches profound impacts whole organ physiology and regeneration. Lastly, I developed a novel Areg reporter mouse strain to better understand Areg producing cells in vivo. Through multiplexed, gene expression and TCR single-cell RNA sequencing, I identified the distinct factors and TCR repertoire that distinguishes “pro-repair” Treg cells in both influenza and bleomycin-induced lung injury. This system can be used as a platform for investigating the unique mechanisms by which reparative Treg cells and other Areg-producing immune cells migrate within tissues and deliver context-specific signals that orchestrate regenerative programming.

T-cells are central to our success as a species. They confer specific and long-term immunity in a process known as adaptive immunity. During adaptive immune response, pathogen ingested by peripheral sentinel cells are brought to the local lymph nodes and presented to T-cells. T-cell recognizes the antigen via its receptor complex (TCR-CD3). The high affinity binding primes the cell for activation. With a positive costimulationary signal from CD28, the T-cell is fully activated, resulting in IL-2 secretions and cellular proliferation. Clinicians are increasingly harnessing the adaptive immune system to combat diseases such as cancer. Specifically, T-cells are activated and expanded ex vivo for adoptive immunotherapies. The ability to modulate T-cell activation is crucial in engineering appropriate effector cell populations for therapeutics. The focus of this thesis is to address the functional impact of CD28 spatial organization on T-cell activation. It has been observed that the spatial segregation of CD3 and CD28 by a few microns has resulted in poor activation of human T-cells. Lck, a Src family kinase (SFK) emerges as the instigator of the phenomenon. The kinase is associated with both CD3 and CD28 signal cascades. We propose a reaction diffusion model to describe the delicate balance between protein mobility and Lck de-activation. The work in this dissertation describes two probes to investigate Lck kinase activity, which permit real-time imaging of both the initiation of pLck activity and its duration. A FRET reporter is constructed to study the spatial and temporal initiation of the kinase activity. Embedded with the Lck membrane domain and contained a substrate for pLck to phosphorylate, the FRET biosensor reports the Lck kinase activity in real-time. Using microprinting to control CD3 and CD28 spatial organizations, the FRET reporter reveals that while T-cells require CD28 for significant IL-2 secretion, CD3 engagement is essential to initiate cellular activation through a spike in pLck kinase activity. Spatially, the reporter shows heightened kinase activity concentrated at the center of the cells upon CD3 engagement. To study the duration of pLck activity, a recruitment reporter is made. CD3 is found ubiquitously throughout the cellular membrane. And its activation by pLck induces the recruitment of a pair of tandem SH2-domain. The recruitment probe (also containing a pair of tandem SH2-domain) revealed curtailed pLck kinase activity due to CD3-CD28 segregation. Ultimately, understanding CD28 modulation of T-cell activation is clinically relevant as it provides new opportunities and targets for the development of therapeutics.

Cell therapies are an emerging form of therapy, with the potential to treat and cure a variety of diseases. As more cell therapies become approved and commercialized, challenges remain in the manufacturing of these often single-batch products due to their complexity and patient-to-patient variability, which limit their cost-effectiveness and reproducibility. In this dissertation, we aim to improve the manufacturing of two different cell therapies, namely, organoid-based cell therapies using hydrogel scaffolds, and adoptive cell therapies using deep learning and microfluidics, to facilitate their widespread clinical use. First, we develop new tools to manufacture organoids, which are widespread in drug-screening technologies, but have been sparingly used for cell therapy as current approaches for producing self-organized cell clusters lack scalability or reproducibility. Here, we use alginate microwell scaffolds to form pre-vascularized organoids composed of endothelial cells and mesenchymal stem cells, where the size and structure can be readily tuned by varying the cell source, ratio of cells, or size of the microwells. Furthermore, by uncrosslinking the alginate scaffold, the organoids can be harvested in a gentle manner without damaging their structure or impairing their functionality. Finally, we assess the ability of the pre-vascularized organoids to restore vascular perfusion in a mouse model of hindlimb ischemia. By making use of the dynamic nature of hydrogels, this method can offer high yields of reproducible, self-organized multicellular aggregates for use in cell therapies. Next, we shift our focus to the identification of antigen-specific T cells, which is a critical step in the manufacturing of adoptive cell therapy. Conventional techniques for selecting antigen-specific T cells are time-consuming, making them difficult to adapt for large-scale manufacturing, and are limited to pre-defined antigenic peptide sequences. Here we train a deep learning model to rapidly classify videos of antigen-specific CD8+ T cells by distinguishing the distinct interaction dynamics (in motility and morphology) between cognate and non-cognate T cells and dendritic cells (DCs). The model is able to classify high affinity antigen-specific CD8+ T cells from OT-I mice with an area under the curve (AUC) of 0.91, and generalizes well to other types of high and low affinity CD8+ T cells. We also show that the experimental addition of anti-CD40 antibodies amplifies the differences between cognate and non-cognate T cells and DCs, thereby improving the model’s ability to discriminate between them. This workflow can be used to better understand the role of cognate T cell – DC interactions in the pathogenesis of cancer and autoimmune diseases, and can be integrated into a device to simplify and accelerate the selection of antigen-specific T cells for use in adoptive cell therapy. Finally, we sought to develop a device to address two other issues associated with the selection of antigen-specific T cells: low-throughput screening, and the inability to assess a mixed population of T cells against a library of antigens, both of which are necessary to identify rare T cells, and improve clinical outcomes of the corresponding cell therapy. A few specialized assays exist that can assess T cells against multiple antigens, but they are often limited by an increased manufacturing burden. Here, we develop a microfluidic artificial lymph node, which is inspired by the efficient selection of antigen-specific T cells in vivo. In particular, our flow-through design consists of multiple compartments, each containing microcarrier beads coated with DCs presenting a distinct antigen, such that T cells that are flowed sequentially through each compartment can stably arrest to cognate DCs, becoming captured in the appropriate compartment. We test a single-compartment device computationally using agent-based simulations, and experimentally using a mixed population of antigen-specific and wild-type (WT) (non-specific) T cells, and in both cases we observe a preferential accumulation of cognate, antigen-specific T cells. This proof-of-concept single-compartment device can be readily scaled up to systematically test many T cells against multiple antigens. Underlying this work is the development of technologies to enable the large-scale manufacturing of cell therapies. Cell therapies are undergoing a transformation to a new class of therapeutic modality, and there are many emerging questions, especially related to the scale-up and scale-out of production processes. Together, this work aims to engineer technologies to improve cell therapy manufacturing processes, facilitate their clinical translation, and ensure their availability to all patients who would benefit from them.

The first reports of bacteria as a cancer therapy date back to the pioneering work of Dr. William Coley–now widely regarded as the father of immunotherapy. As far back as 1891, Coley demonstrated that the intratumoral injection of live and later heat-killed isolates of Streptococcus pyogenes and Serratia marcescens could induce durable remission in patients with bone and soft tissue sarcoma. While this therapy was deemed to unsafe at the time, Coley’s findings have formed the basis for our modern understanding of immunology and cancer immunotherapy. Over the past two decades, the advent of synthetic biology is driving a new era of medicine through the genetic programming of living cells. This transformative approach enables the creation of engineered systems that sense and respond to diverse environments, permitting safe and effective targeted delivery of therapeutic payloads within disease sites. In this thesis, I seek to utilize principles from synthetic biology and immunology to engineer bacteria for immunotherapeutic delivery. I have developed multiple strains of non-pathogenic E. coli capable of colonizing solid tumors and delivering diverse immunotherapeutic payloads specifically within the tumor. This local delivery approach enables the utilization of therapeutic agents that may be otherwise systemically toxic. In one instance, we engineered an encoded nanobody antagonist of CD47 (CD47nb), an anti-phagocytic receptor commonly overexpressed in several human cancers. We show that delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Thus, engineered bacteria may be used for safe and local delivery of diverseimmunotherapeutic payloads leading to systemic antitumor immunity.

The human body is exposed to a multitude of prevalent viruses, requiring ongoing surveillance and protection by the immune system. Maintenance of human anti-viral adaptive immunity in diverse tissue sites is determined by a multitude of factors and critical for long-term protection against repeat exposure to viral infection. Yet, studies of anti-viral immunity have primarily been limited to animal studies and studies of peripheral blood in humans. Studies in mice have demonstrated that memory T cells in tissues provide superior protection against viral infection compared to circulating T cells, particularly tissue-resident memory T cells (TRM), which remain in tissues long-term without re-entering circulation. However, much remains to be understood about how anti-viral immune responses are maintained in human tissues and how adaptive immune cells in various tissues sites function upon re-exposure to viral antigens. We have established a human tissue resource through a collaboration with LiveOnNY, a local organ procurement organization, to obtain blood and multiple lymphoid and mucosal sites from donors of all ages. Using this tissue resource, we employed comprehensive cellular and molecular analysis to investigate tissue immunity to three prevalent but distinct viruses—influenza A, CMV, and SARS-CoV-2. We compared CD8+ T cells recognizing ubiquitous and longstanding viruses influenza A and CMV across multiple tissue sites of 58 organ donors ages 1-78 years in order to elucidate how covariates of virus, tissue, age, and sex impact the anti-viral immune response. Using flow cytometry, T cell receptor repertoire sequencing, functional assays, and single-cell transcriptional profiling, we showed that virus specificity and tissue localization are the primary drivers of anti-viral T cell immune responses in the human body, with age and sex further influencing T cell subset differentiation. Specifically, virus specificity correlated with virus-specific T cell distribution, memory subset differentiation, and clonal repertoire, while tissue localization determined overall subset distribution and functional responses. We further investigated the tissue-localized immune response to emergent SARS-CoV-2. By examining multiple tissues of organ donors who had recovered from natural infection by SARS-CoV-2, we showed that adaptive memory immune responses persisted months after infection, with memory T and B cells preferentially localized in the lung and lung-associated lymph node. Persisting memory cell populations included tissue-resident T and B cells, particularly in the lung, as well as germinal center B cells in the lung-associated lymph node along with follicular helper T cells, indicating ongoing generation of humoral immunity. Together, these findings highlight the importance of tissue-localized anti-viral immunity and help to define characteristics of site-specific protective immunity that may be leveraged for the development of more effective treatment and prevention strategies.

Treatment of acute myeloid leukemia, an aggressive hematopoietic malignancy of myeloid progenitors, has remained rather stagnant over the course of several decades. Infusions of cytarabine and anthracycline antibiotics have dominated the landscape of AML therapy, with minor changes to dosing schedule occasionally making slight adjustments to efficacy or tolerability. Improvements in prognosis have been bittersweet, with most progress seen in younger populations less likely to get the disease, and already more likely to achieve remission and to meet survival milestones. Much of this progress is attributed to other factors, such as improved supportive care and availability of hematopoietic stem cell and platelet transfusion. In most patients, occupying the 60-and-above demographic, improvements in survival have not been significant. In turn, the population impact of AML has changed little over time. While accounting for about one-third of total leukemia cases and one percent of total cancer cases, AML accounts for about one half of total leukemia deaths and two percent of total cancer deaths. Most advances straying away from standard treatment have been in important pathways that could be impactful in subsets of the overall AML patient population. Tyrosine kinases are implicated in numerous cancers including AML, with activity-enhancing mutations conferring growth advantages to malignant cells. About one-third of AML patients have mutations in one such kinase, FLT3, and may benefit from inhibitors to tyrosine kinases overall and from FLT3- specific agents. Mutations in isocitrate dehydrogenases highlight another subpopulation, about one-fifth of AML patients, who might benefit from emerging agents that inhibit these pathways from creating a leukemia-favoring environment in the bone marrow. Other pathways similarly implicated in numerous cancers including AML are being targeted with new agents that can benefit some AML patients, such as Hedgehog signaling and apoptotic regulation. Still, breakthroughs are needed that can help most AML patients, particularly in the cases of relapsed leukemia that occurs in most patients within a year or two after remission is achieved. CD33 is among a few molecular targets for AML, though it is just as ubiquitously expressed on healthy myeloid cells. Antibody-drug conjugates like Mylotarg have made progress in this approach, though hematopoietic toxicities have made treatment difficult in older populations. Clever techniques such as ablation of CD33 from healthy myeloid progenitors may be supportive in CD33-based approaches, and immunotherapy involving CD33-targeting is a rapidly growing research focus. This dissertation describes a new type of bispecific antibody that binds CD33 on AML and CD3 on cytotoxic T cells in a proof-of-concept study. Various formats for bifunctional molecules have been created and used clinically, including antibody-drug conjugates and bispecific antibodies that simultaneously engage antigens on two different types of cells. Those like the one described here, bispecific T-cell engagers, have typically taken the form of single-chain fusion proteins containing the variable regions binding to both antigens of interest. Other bispecific antibodies have imitated naturally-occurring immunoglobulin structures, boasting superior pharmacokinetics while facing steep obstacles in large-scale production. The single-chain fusions, easier to produce, can face difficulties in full engagement, with loss of function sometimes seen in fusion partners at the C-terminus. We propose a new format, believed to present two antigen-binding domains in N-terminal positions on a two-chain heterodimeric structure. Capitalizing on an elegantly designed system of hydrophobic cores and hydrogen-bonding networks generating an orthogonal heterodimer, we added an immunoglobulin hinge region to secure a permanently-bound heterodimer, and attached domains binding to CD3 and CD33. We hypothesized that this design, ensured to present its antibody components at N-termini, could bind two antigens at a distance appropriate for facilitating T cell cytotoxicity to AML. After expressing and purifying these proteins in mammalian cells, we demonstrated their ability to persist as a bispecific heterodimer. We showed in vitro that our bispecific heterodimers could bind both CD3+ and CD33+ cells, and that they bolstered T cell cytotoxicity to AML cell lines in a dose-dependent manner. Monomeric components bound only CD3+ or CD33+ cells depending on antibody variable domain present, and had no effect on T cell cytotoxicity. In a mouse model of minimal residual disease, T cells alone did not have a significant effect on the growth of AML, nor did they have an effect on overall survival. T cells with bispecific heterodimer greatly extended survival, and mice of this treatment group were free of leukemia. These findings suggest that this format for bispecific proteins allows for robust simultaneous engagement with both antigens of interest in a manner conducive to T cell cytotoxicity against AML. We believe this presents a compelling modular system for bispecific antibodies, where CD3- and CD33-binding domains can be readily swapped with domains binding to other cancer- or immune cell-specific antigens, and can be further developed into a trispecific system engaging other immune cells or extending half-life with anti-albumin or Fc domains.

The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the U.S. population over the age of 50. In a similar reach but different clinical realm, nearly 40% of all men and women will be diagnosed with cancer at some point during their lifetimes. The impact of both of these diseases is compounded by the limited knowledge of cellular mechanisms and the insufficiency of effective treatment options. At the microscopic level of the cell cytoskeleton, increasing evidence has led researchers to further explore microtubules (MTs) and MT-based structures, such as primary cilia, as potential keys to unlocking improved treatment options. However, the way in which microtubules regulate the processes giving rise to these diseases remains a critical gap in knowledge. The works outlined here aimed to elucidate mechanisms that may be used to combat diseases attacking the skeletal and immune systems. In order to characterize the influence of primary cilia with respect to osteoclast differentiation, we implemented a series of treatments to an immortalized macrophage cell line: cilia lengthening (using Fenoldopam) and mechanical stimulation (using oscillatory fluid flow). The results were analyzed by a combination of immunocytochemistry and quantitative PCR. Our first result showed definitively that while osteoclasts do not possess primary cilia, their macrophage precursors do. We also discovered that these macrophage primary cilia are dynamic and can be modulated; cells whose cilia had been lengthened showed a significant decrease in osteoclast formation, indicating that macrophage cilia resorption may be a necessary step for osteoclast differentiation to occur. Combined with findings from previous studies, there is increasing evidence that the primary cilium, as a therapeutic target for bone diseases, may offer a dual beneficial approach to both promote bone formation and downregulate osteoclast activity. We then explored the possibility of directional MT translocation during T-cell activation being linked to Rho GTPases, which regulate actin polymerization. WASp and WAVE2, known to have functional roles in T-cell activation, were identified as primary candidates. In order to investigate this relationship, we implemented a stepwise micropatterning procedure by which PDMS was used to transfer local areas of activation (presenting fluorescently-tagged antibodies against CD3 and CD28) which, upon T-cell receptor (TCR) triggering, could mimic immune synapse (IS) formation. We showed that, although there was no correlation between the spatial organization of MTs and WASp, MTs and WAVE2 location were highly correlated, providing strong evidence for a link between these two systems. In addition, MT disruption via nocodazole resulted in a significant decrease in T-cell activation and mechanosensing capabilities. Given the role of WAVE2 in promoting cell spreading and adhesion during IS formation, this result provides additional evidence that this cytoskeletal filament is in fact connected to proteins involved in actin nucleation and elongation. We anticipate the work in Aim 1 to help reveal a previously unexplored therapeutic target for osteoporosis, a disease that currently has no clinical manifestations prior to a fracture event. Further investigation has the potential to contribute to diagnosis and prevention techniques, as well as new treatments. Similarly, given the emergence of adoptive T-cell immunotherapy for immune-related disorders, the findings of Aim 2 will advance our understanding of both the biological and mechanical influence of the cytoskeleton and motivate microtubules as one component of a more comprehensive armamentarium of treatment approaches.

T cells can fight cancer, but an immunosuppressive tumor microenvironment (TME) disallows them from carrying out their function over time. Upregulation of inhibitory checkpoint molecules such as programmed cell death protein 1 (PD1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) can lead to such an immunosuppressive TME. Despite their widespread use, immune checkpoint blockade (ICB) antibodies targeting checkpoint molecules remain ineffective in most cancer patients. We do not understand why some patients respond to ICB better than others. To understand the heterogeneity of ICB response, we must understand the heterogeneity of the T cell subsets acted upon by such therapies. Here, we ask how T cell subsets change in the presence and absence of ICB. We track T cell clones through their T cell receptor sequences and link phenotypes with T cell receptor specificities. Through multiplexed single cell TCR sequencing, single cell RNA sequencing, and the use of cell- surface CITE-seq antibodies, coupled with surgical biopsy, we longitudinally tracked the fate of individual T cell clones within tumors at baseline and in response to ICB in an immunogenic mouse tumor model. Furthermore, computational clustering of T cells solely based on their gene expression profiles may ignore upstream regulatory mechanisms that control T cell gene expression. Hence, we employed Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to cluster CD8+ and CD4+ T cell phenotypes. VIPER leverages inference of gene regulatory networks to allow full quantitative characterization of protein activity for transcription factors, co-factors, and signaling molecules by assessing the enrichment of their transcriptional targets cell-by-cell among expressed genes. This gave us a window into the transcriptional states and their inferred protein activity. We next developed a computational analysis toolkit to study TCR clonality incorporating sub-sampling of TCR clonotypes, forward and back tracing of shared clones between timepoints, and in turn, inferred shared clonal evolution. We employed the above workflow to MC38 tumor-infiltrating and tumor-draining lymph node-derived CD8+ and CD4+ T cells. We found that T cell phenotypes are highly dynamic within tumors at baseline, in the absence of ICB, particularly within the window that they are responsive to therapy. In the absence of ICB, effector phenotype of CD8+ T cells diminished, while the exhaustion phenotype was enhanced as tumors progressed. Within the CD4+ population, a heterogenous subset of regulatory CD4+ T cells (Tregs) changed phenotype over time, and CD4+ Th1 like effectors, along with stem like progenitor CD4+ showed distinct dynamism. Next, by analyzing responses to therapy within his context, we found that both anti-PD1 and anti-CTLA4 act through distinct mechanisms on CD8+ and CD4+ T cells. Anti-PD1 acted upon intra-tumoral effector CD8+ T cells to slow their progression to terminally differentiated exhausted cells, i.e., increased their persistence within tumors. Anti-CTLA4 therapy increased recruitment of novel effector CD8+ T cell clones to tumors from lymph nodes while diminishing tumor-infiltrating Tregs. ICB also potentiated CD4+ Th1 like phenotype. These results uncovered a behavior pattern of CD8+ and CD4+ T cells within tumors at baseline tumor progression, and then in the presence of ICB. We believe these findings have added to our understanding of the subtleties of T cell phenotypes in tumors, specifically in response to ICB. This will provide a practical framework for designing and validating novel checkpoint blockade therapies in the future.

Immune checkpoint blockade (ICB) therapies have transformed the treatment landscape for advanced melanoma, extending patient survival and improving quality of life for numerous patients with a disease that was once considered to be universally fatal. However, despite the success of ICB for many patients, over half are either resistant to initial therapy, or develop resistance over time after an initial response. The mechanisms underlying this therapy resistance remain unclear for the majority of patients. We have recently identified loss of the co-stimulatory and adhesion molecule CD58 on melanoma cells as a driver for cancer immune evasion and ICB resistance. In this thesis, we use in vitro co-culture models of patient-derived melanoma cells and tumor infiltrating lymphocytes as well as in vivo patient-derived xenograft models to demonstrate the necessity of CD58 interactions with its ligand CD2 on T cells for T cell activation, tumor infiltration, and effector cytotoxicity. Furthermore, using genome-wide genetic and protein screening approaches, we identify CMTM6 as a positive regulator of CD58, and uncover its role in mediating CD58’s regulation of inhibitory PD-L1 signaling by binding to both proteins and preventing their lysosomal degradation. Thus, CMTM6 co-regulates these co- inhibitory and co-stimulatory signals such that, in the absence of CD58, CMTM6 becomes available to bind and stabilize additional PD-L1, enhancing its inhibitory signals to T cells. Finally, we identify a potential role for CD58 on T cells as a marker of effector memory T cells with enhanced effector and progenitor function. The CD58-CD2 axis therefore serves a multi-faceted, underappreciated role in melanoma cancer immunity, and may serve as a therapeutic target for enhancing anti-tumor T cell responses.