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In the thymus, strongly self-reactive T cells may undergo apoptotic deletion or differentiate into Foxp3+ T-regulatory (T-reg) cells. Mechanisms that partition T cells into these two fates are unclear. Here, we show that IL-2 signalling is required to prevent deletion of CD4+ CD8– CCR7+ Helios+ thymocytes poised to upregulate Foxp3. The deletion prevented by IL-2 signalling is Foxp3 independent and occurs later in thymocyte development than the deletion that is prevented by Card11 signalling. Our results distinguish two bottlenecks at which strongly self-reactive thymocytes undergo deletion or progress to the next stage of T-reg differentiation; Card11 regulates the first bottleneck and IL-2 regulates the second.

The differentiation of hematopoietic precursors into the many functionally distinct T‐cell types produced by the thymus is a complex process. It proceeds through a series of stages orchestrated by a variety of thymic microenvironments that shape the T‐cell developmental processes. Numerous cytokine and cell surface receptors direct thymocyte differentiation but the primary determinant of cell fate is the engagement of the T‐cell antigen receptor (TCR ). The strength of the TCR signal and the maturation stage of the thymocyte receiving it can direct the various differentiation programs or, alternatively, end the process by inducing cell death. The regulation of thymocyte death is critical for the efficiency of thymic T‐cell differentiation and the preservation of immune tolerance. A detailed knowledge of mechanisms that eliminate thymocytes from the T‐cell repertoire is essential to understand the “logic” of T‐cell selection in the thymus. This review focuses on the central role of the BCL ‐2 family of proteins in the apoptotic checkpoints that punctuate thymocyte differentiation and the consequences of defects in these processes.

Thymocytes that bind strongly to self‐antigens are prevented from becoming naive T cells by several mechanisms. They undergo clonal deletion at two stages of development; wave 1 in immature thymocytes lacking the medulla‐homing chemokine receptor, CCR7, or wave 2 in more mature CCR7+ thymocytes. Alternatively, self‐reactive thymocytes upregulate Foxp3 to become T‐regulatory cells. Here, we describe the differential timing of the two waves of deletion and Foxp3 upregulation relative to the immature proliferating stage. Proliferating thymocytes were pulse‐labeled in normal C57BL/6 mice with 5‐ethynyl‐2'‐deoxyuridine (EdU). Thymocytes progressed into wave 1 (CCR7−) and wave 2 (CCR7+) of clonal deletion ~2 and 5 days after proliferation, respectively. Foxp3 upregulation occurred between 4 and 8 days after proliferation, predominantly in thymocytes with a Helios+ CCR7+ phenotype. These findings establish a timeline that suggests that wave 1 of clonal deletion occurs in the thymic cortex, whereas wave 2 and Foxp3 upregulation both occur in the thymic medulla.

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation.

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1Anaef, with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1Anaef mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44hi Helios+ PD-1+ CD4+ T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1Anaef is mostly normal in vivo, although CD44 is overexpressed on naïve thymocytes and T cells in a T-cell-autonomous manner. We identify CD44 expression as a sensitive reporter of tonic mTOR-S6 kinase signaling through a novel mouse strain, chino, with a reduction-of-function mutation in Mtor. Elevated tonic mTOR-S6 signaling occurs in Rasgrp1Anaef naïve CD4+ T cells. CD44 expression, CD4+ T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1AnaefMtorchino double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1Anaef T cell dysregulation.