Browsing by Subject "Dendritic Cell"

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    Evaluating thymocyte negative selection within the polyclonal population
    (2019-09) Breed, Elise
    The development of a self-tolerant and effective T cell receptor repertoire is dependent on interactions coordinated by various antigen presenting cells (APC) within the thymus. T cell receptor–self-peptide–MHC interactions are essential for determining T cell fate, where high affinity interactions can result in clonal deletion or regulatory T (Treg) cell differentiation of potentially autoreactive T cells. The APCs that provide these signals have distinct localization, different antigen processing features, and can provide different co-stimulatory signals that are also critical to these selection processes and may distinguish the ultimate fate of a T cell. Clonal deletion and Treg differentation of T cells specific for self-antigens in the thymus have been widely studied, primarily by approaches that focus on a single receptor (using TCR transgenes) or a single specificity (using pMHC tetramers). However, little is known about how distinct APCs coordinate clonal deletion and Treg cell development at the population level. Here, we report an assay that measures cleaved caspase 3 to define clonal deletion at the population level. This assay distinguishes clonal deletion from apoptotic events caused by neglect and approximates the anatomic site of deletion using CCR7. This approach showed that 78% of clonal deletion events occur in the cortex in mice. Medullary deletion events were detected at both the semi-mature and mature developmental stages, although mature events were associated with failed Treg cell induction. Using this assay, we showed that bone marrow derived APC drive approximately half of deletion events at both stages. We also found that both cortical and medullary deletion rely heavily on CD28 co-stimulation. We further assessed the contribution of distinct APC subsets to clonal deletion and Treg cell selection using cell type ablation or deficiency. We found that total deletion and nascent Treg cell events were not altered in the absence of B cells, pDC, or XCR1+ cDC1. In an effort to eliminate SIRPa+ cDC2, we discovered that a fraction of thymic SIRPa+ cDC2 express the lectin CD301b. These cells resemble the type 2 immune response-promoting CD301b+ DC that are present in skin draining LN. CD301b expression was localized primarily within the thymus medulla and depended on IL-4R. Deficiency of these IL-4 and IL-13 signaled cDC2 caused a measurable reduction in clonal deletion events, suggesting a non-redundant role for tolerance induction. These findings demonstrate useful strategies for studying clonal deletion and nascent Treg cell development within the polyclonal population. Additionally, they provide valuable insight into how and when thymocytes undergo clonal deletion as they traverse through the thymus and interact with distinct APC during development.
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    The role of T cell receptor affinity in CD4+ T cell differentiation
    (2019-02) Kotov, Dmitri
    Naïve helper T cells become activated when their T cell receptor (TCR) recognizes a microbial peptide presented on MHCII (p:MHCII) by dendritic cells (DCs). During this interaction, DCs provide polarizing cues to guide T cell differentiation towards specific fates, like T follicular helper cells which help B cells. The affinity of TCR for its cognate p:MHCII is known to influence the fate adopted by CD4+ T cells, but the mechanisms responsible for this effect are not completely understood. The mechanism could be T cell extrinsic, by affecting the DC subset a T cell contacts thereby influencing the polarizing factors received, or T cell intrinsic, by modulating TCR signal strength thus regulating genes involved in T cell differentiation. The T cell extrinsic hypothesis was tested with a quantitative, imaging approach called histo-cytometry to analyze the T cell-DC interactions. This technique involves time-consuming manual image analysis. Therefore, we wrote software to automate histo-cytometry analysis, reducing hands-on analysis time by up to 90%. With histo-cytometry, we determined that TCR affinity did not affect the DC subset contacted by CD4+ T cells, rendering the cell extrinsic hypothesis unlikely. We therefore focused on the T cell intrinsic hypothesis by performing a CRISPR/Cas9 screen to identify TCR regulated genes that influence T cell differentiation. This screen revealed two novel regulators of helper T cell differentiation, Eef1e1 and Gbp2, which are expressed at a higher level in high affinity than low affinity T cells. These results suggest that TCR affinity influences helper T cell differentiation intrinsically by regulating genes that control the differentiation process. We next explored the relationship between CD4+ T cell differentiation and cytotoxic activity. This study determined that cytotoxic function of helper T cells was dependent on the Fas pathway and Fas was upregulated on target cells proportional to the TCR affinity of the responding T cells. Additionally, we identified that many different helper T cell subsets express FasL and have cytotoxic potential. Thus, my work could benefit future vaccines by providing greater understanding of how helper T cell fate decisions occur and how these decisions influence cytotoxic function.