Summary How the TCR repertoire, in concert with risk-associated MHC, imposes susceptibility for autoimmune diseases is incompletely resolved. Due largely to recombinatorial biases, a small fraction of TCR α or β chains are shared by most individuals, or public. If public TCR chains modulate a TCRαβ heterodimer’s likelihood of productively engaging autoantigen, because they are pervasive and often high frequency, they could also broadly influence disease risk and progression. Prior data, using low resolution techniques, have identified the heavy use of select public TCR in some autoimmune models. Here we assess public repertoire representation in mice with experimental autoimmune encephalomyelitis (EAE) at high resolution. Saturation sequencing was used to identify >18×106 TCRβ sequences from the central nervous systems, periphery, and thymi of mice at different stages of autoimmune encephalomyelitis and healthy controls. Analyses indicated the prominent representation of a highly diverse public TCRβ repertoire in the disease response. Preferential formation of public TCR implicated in autoimmunity was identified in pre-selection thymocytes and, consistently, public, disease-associated TCRβ were observed to be commonly oligoclonal. Increased TCR sharing and a focusing of the public TCR response was seen with disease progression. Critically, comparisons of peripheral and CNS repertoires and repertoires from pre-immune and diseased mice demonstrated that public TCR were preferentially deployed relative to non-shared, or private, sequences. Our findings implicate public TCR in skewing repertoire response during autoimmunity, and suggest that subsets of public TCR sequences may serve as disease-specific biomarkers or influence disease susceptibility or progression.

Although the TCR repertoire is highly diverse, a small fraction of TCR chains, referred to as public, preferentially form and are shared by most individuals. Prior studies indicated that public TCRβ may be preferentially deployed in autoimmunity. We hypothesized that if these TCRβ modulate the likelihood of a TCRαβ heterodimer productively engaging autoantigen, because they are widely present in the population and often high frequency within individual repertoires, they could also broadly influence repertoire responsiveness to specific autoantigens. We assess this here using a series of public and private TCRβ derived from autoimmune encephalomyelitis-associated TCR. Transgenic expression of public, but not private, disease-associated TCRβ paired with endogenously rearranged TCRα endowed unprimed T cells with autoantigen reactivity. Further, two of six public, but none of five private TCRβ provoked spontaneous early-onset autoimmunity in mice. Our findings indicate that single TCRβ are sufficient to confer on TCRαβ chains reactivity toward disease-associated autoantigens in the context of diverse TCRα. They further suggest that public TCR can skew autoimmune susceptibility, and that subsets of public TCR sequences may serve as disease- specific biomarkers or therapeutic targets.

Profiling tumors at single-cell resolution provides an opportunity to understand complexities underpinning lymph-node metastases in head and neck squamous-cell carcinoma. Single-cell RNAseq (scRNAseq) analysis of cancer-cell trajectories identifies a subpopulation of pre-metastatic cells, driven by actionable pathways including AXL and AURK. Blocking these two proteins blunts tumor invasion in patient-derived cultures. Furthermore, scRNAseq analyses of tumor-infiltrating CD8 + T-lymphocytes show two distinct trajectories to T-cell dysfunction, corroborated by their clonal architecture based on single-cell T-cell receptor sequencing. By determining key modulators of these trajectories, followed by validation using external datasets and functional experiments, we uncover a role for SOX4 in mediating T-cell exhaustion. Finally, interactome analyses between pre-metastatic tumor cells and CD8 + T-lymphocytes uncover a putative role for the Midkine pathway in immune-modulation and this is confirmed by scRNAseq of tumors from humanized mice. Aside from specific findings, this study demonstrates the importance of tumor heterogeneity analyses in identifying key vulnerabilities during early metastasis.

The major histocompatibility complex (MHC) is the strongest genetic risk factor for autoimmunity. It acts together with a corresponding TCR repertoire, yet, considering the extent of the repertoire's diversity, how this imposes disease susceptibility on a population is not well understood. We address the hypothesis that shared or public TCR, those present in most individuals, modulate autoimmune risk. High resolution analyses of autoimmune encephalomyelitis-associated T-cell receptor chain (TCR showed preferential utilization of public TCR sequences, implicating them in pathogenesis. Disease-associated public TCR, when transgenically expressed in association with endogenously rearranged T-cell receptor chain (TCR, could further endow unprimed T cells with autoantigen reactivity. Enforced expression of two of six public but no private TCR further provoked spontaneous, early-onset autoimmunity in mice. These findings implicate public TCR in skewing repertoire response characteristics and autoimmune susceptibility, demonstrate how single TCR chains can bias autoantigen specificity, and suggest that subsets of public TCR sequences may serve as diseasespecific biomarkers or therapeutic targets. vi TABLE OF CONTENTS CHAPTER 1.