Cell death and thymic tolerance.

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.

A timeline demarcating two waves of clonal deletion and Foxp3 upregulation during thymocyte development.

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.

Deletion of self-reactive CCR7- thymocytes in the absence of MHC expression on thymic epithelial cells.

The selection of αß T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αß T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRß + CD5 + Helios + CCR7- cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRß chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.

IL-2 prevents deletion of developing T-regulatory cells in the thymus.

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.

Helios marks strongly autoreactive CD4+ T cells in two major waves of thymic deletion distinguished by induction of PD-1 or NF-κB.

Acquisition of self-tolerance in the thymus requires T cells to discriminate strong versus weak T cell receptor binding by self-peptide-MHC complexes. We find this discrimination is reported by expression of the transcription factor Helios, which is induced during negative selection but decreases during positive selection. Helios and the proapoptotic protein Bim were coinduced in 55% of nascent CCR7(-) CD4(+) CD69(+) thymocytes. These were short-lived cells that up-regulated PD-1 and down-regulated CD4 and CD8 during Bim-dependent apoptosis. Helios and Bim were also coinduced at the subsequent CCR7(+) CD4(+) CD69(+) CD8(-) stage, and this second wave of Bim-dependent negative selection involved 20% of nascent cells. Unlike CCR7(-) counterparts, Helios(+) CCR7(+) CD4(+) cells mount a concurrent Card11- and c-Rel-dependent activation response that opposes Bim-mediated apoptosis. This "hollow" activation response consists of many NF-κB target genes but lacks key growth mediators like IL-2 and Myc, and the thymocytes were not induced to proliferate. These findings identify Helios as the first marker known to diverge during positive and negative selection of thymocytes and reveal the extent, stage, and molecular nature of two distinct waves of clonal deletion in the normal thymus.

Rasgrp1 mutation increases naive T-cell CD44 expression and drives mTOR-dependent accumulation of Helios⁺ T cells and autoantibodies.

Missense variants are a major source of human genetic variation. Here we analyze a new mouse missense variant, Rasgrp1(Anaef), with an ENU-mutated EF hand in the Rasgrp1 Ras guanine nucleotide exchange factor. Rasgrp1(Anaef) mice exhibit anti-nuclear autoantibodies and gradually accumulate a CD44(hi) Helios(+) PD-1(+) CD4(+) T cell population that is dependent on B cells. Despite reduced Rasgrp1-Ras-ERK activation in vitro, thymocyte selection in Rasgrp1(Anaef) 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 Rasgrp1(Anaef) naïve CD4(+) T cells. CD44 expression, CD4(+) T cell subset ratios and serum autoantibodies all returned to normal in Rasgrp1(Anaef)Mtor(chino) double-mutant mice, demonstrating that increased mTOR activity is essential for the Rasgrp1(Anaef) T cell dysregulation. DOI: http://dx.doi.org/10.7554/eLife.01020.001.

Anti-islet autoantibodies trigger autoimmune diabetes in the presence of an increased frequency of islet-reactive CD4 T cells.

OBJECTIVE: To define cellular mechanisms by which B cells promote type 1 diabetes. RESEARCH DESIGN AND METHODS: The study measured islet-specific CD4 T cell regulation in T-cell receptor transgenic mice with elevated frequencies of CD4 T cells recognizing hen egg lysozyme (HEL) autoantigen expressed in islet ß-cells and thymic epithelium under control of the insulin-gene promoter. The effects of a mutation in Roquin that dysregulates T follicular helper (Tfh) cells to promote B-cell activation and anti-islet autoantibodies were studied, as were the effects of HEL antigen-presenting B cells and passively transferred or maternally transmitted anti-islet HEL antibodies. RESULTS: Mouse anti-islet IgG antibodies-either formed as a consequence of excessive Tfh activity, maternally transmitted, or passively transferred-caused a breakdown of tolerance in islet-reactive CD4(+) cells and fast progression to diabetes. Progression to diabetes was ameliorated in the absence of B cells or when the B cells could not secrete islet-specific IgG. Anti-islet antibodies increased the survival of proliferating islet-reactive CD4(+) T cells. FcγR blockade delayed and reduced the incidence of autoimmune diabetes. CONCLUSIONS: B cells can promote type 1 diabetes by secreting anti-islet autoantibodies that act in an FcγR-mediated manner to enhance the expansion of islet-reactive CD4 T cells and cooperate with inherited defects in thymic and peripheral CD4 T-cell tolerance. Cooperation between inherited variants affecting CD4 T-cell tolerance and anti-islet autoantibodies should be examined in epidemiological studies and in studies examining the efficacy of B-cell depletion.