We investigated how TCR intrinsic determinants and extrinsic cues co-operatively generate ‘holes’ in the repertoire (clonal deletion) or allocate autoreactive T cell-specificities to the Treg repertoire (Treg cell diversion). To asses TCR intrinsic determinants, we developed methods for high-resolution antigen-specific TCR repertoire analyses in combination with in vitro and in vivo assays of functional TCR characteristics to comprehensively assess how tolerance to myelin proteoplipid protein (PLP), a paradigmatic example of a disease relevant autoantigen of the central nervous system, shapes the TCR repertoire (Aim 1). These global repertoire inventories allowed us to delineate TCRs that were ‘lost’ from the CD4 T cell pool whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance, and the contribution of different ‘diverter’ TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. Together, this reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal ‘permissive’ antigen reactivity (Hassler et al., 2019).
In a related line of investigation, we assessed the quantitative and qualitiative impact of T cell extrinsic determinants on shaping of the TCR repertoire (Aim 2). Here, we focused on thymic antigen presenting cells and their unique functional attributes. Commonly, dendritic cells and medullary thymic epithelial cells are believed to represent the key antigen presenting cells (APCs) for central T cell tolerance induction. We previously found that the thymus also harbours a distinct population of B cells that display unique properties: unlike their peripheral counterparts, thymic B cells express high levels of co-stimulatory molecules and MHC II. Moreover, they also express AIRE, a transcriptional co-activator that serves a crucial tolerogenic function in medullary thymic epithelial cells by promoting the expression of peripheral tissue-antigens (Yamano et al., 2015). Together, these potent APC features of thymic B cells suggested that they might serve a non-redundant role in central T cell tolerance induction. We established high throughput methodology for the generation and comparison of global inventories of TCR repertoires that are generated in the absence or presence of thymic B cells or under various circumstances in which thymic B cells were manipulated to lack the capacity to directly present antigens, to express autoantigens driven by AIRE, or to undergo B cell receptor class-switching, respectively. Together, these studies reveal that the tolerogenic ‘footprint’ of thymic B cells on the composition of the nascent TCR repertoire in the thymus is much larger than previously anticipated (or considered at all). These findings have not been published yet, but were presented and well received at a recent international conference on autoimmunity (Crete, 2022).
