Evaluating thymocyte negative selection within the polyclonal population

Abstract

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.