CORDIS - EU research results

Clonal Deletion versus Clonal Diversion: Footprints of Self-Tolerance in the T Cell Repertoire

Periodic Reporting for period 4 - TOLERANCE FOOTPRINT (Clonal Deletion versus Clonal Diversion: Footprints of Self-Tolerance in the T CellRepertoire)

Reporting period: 2022-04-01 to 2022-09-30

The immune system efficiently protects the body from 'foreign' invaders such as bacteria and viruses. The major effector cells of the ‘adaptive’ arm of the immune system are T and B cells. Millions of these constantly patrol the body. Importantly, every single one of these cells carries a unique antigen receptor. Hence, at the population level, a hugely diverse ‘receptor repertoire’ is established. Once activated by a pathogen, individual immune cells generate thousands of copies of themselves through rapid cell division, and this process of ‘clonal expansion’ allows minute numbers of pathogen specific precursor cells to mount protective responses against virtually any pathogen that an individual may encounter.
The antigen receptor repertoire is generated during immune cell development. In the case of T cells this happens in the thymus, where each developing cell is equipped with an individual antigen receptor (the T cell receptor; TCR) through random rearrangement of TCR gene segments. Importantly, owing to its random nature, this assembly of TCRs not only produces ‘useful’ T cells that potentially recognize invading microbes and viruses, but inevitably also leads to the emergence of potentially ‘dangerous’ T cells that carry autoreactive TCRs specific for the body’s own structures (so-called self-antigens).
A variety of mechanisms have been described that prevent autoreactive T cells from causing damage to tissues, collectively leading to a state of ‘self-tolerance’. A failure of self-tolerance can result in a variety of diseases such as type-1-diabetes or multiple sclerosis; collectively, these pathological conditions are referred to as autoimmune diseases. The precise aetiology of many autoimmune diseases is only insufficiently understood, and treatment regimens are mostly limited to general and unspecific immune suppression. Hence, there is a clear need for a deeper understanding of the physiological processes that maintain self-tolerance.
A classical concept of self-tolerance envisioned that autoreactive T cells would be eliminated early in their development, and this mechanism of clonal deletion has indeed been shown to purge a substantial fraction of autoreactive T cells from the T cell repertoire during thymic selection. More recently, it has become clear that there is a second, fundamentally different mechanism that operates through the ‘re-education’ of potentially dangerous autoreactive cells into harmless and beneficial regulatory T cells (Treg cells). Like clonal deletion, this process of cell-fate diversion into the Treg cells lineage can also be instructed during early T cell differentiation in the thymus, and the key parameters that distinguish between the two modalities of central T cell tolerance remain incompletely understood (Klein et al., 2019). The project ‘TOLERANCE FOOTPRINT’ has provided important insights into the determinants that specify whether an autoreactive CD4+ T cell is deleted or diverted into the Treg cell lineage and how both mechanisms together shape the composition of the T cell repertoire.
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).
So far, experimental evidence in favor of an avidity model of thymic selection was largely limited to observations related to variations of antigen dose in cell receptor (TCR) transgenic systems that harbor unphysiological frequencies of cells of a given antigen specificity. However, it has become clear that monoclonal models only poorly recapitulate physiological Treg cell ‘instruction’, and close to nothing was known with regards to how clonal deletion and Treg cell diversion together shape the quality and quantity of polyclonal cohorts of antigen-specific CD4 T cells. Our work on the fate of autoreactive cells within naturally diverse T cell repertoires has shed light on TCR affinity as a crucial determinant of the decision between clonal deletion and clonal diversion (Treg cell differentiation). In the context of the role of thymic antigen presenting cells as TCR extrinsic determinants of deletion verus Treg cell differentiation, our data reveal that, in qualitative terms, thymic B cells shape the TCR repertoire in a non-redundant manner and, in quantitative terms, to an extent that is very similar to the respective impact of dendritic cells.
Clonal Deletion versus regulatory T cells differentiation