The type 1 diabetes susceptibility locus Idd5 favours robust neonatal development of highly autoreactive regulatory T cells in the NOD mouse.

Regulatory T lymphocytes expressing the transcription factor Foxp3 (Tregs) play an important role in the prevention of autoimmune diseases and other immunopathologies. Aberrations in Treg-mediated immunosuppression are therefore thought to be involved in the development of autoimmune pathologies, but few have been documented. Recent reports indicated a central role for Tregs developing during the neonatal period in the prevention of autoimmune pathology. We therefore investigated the development of Tregs in neonatal NOD mice, an important animal model for autoimmune type 1 diabetes. Surprisingly, we found that, as compared with seven other commonly studied inbred mouse strains, in neonatal NOD mice, exceptionally large proportions of developing Tregs express high levels of GITR and PD-1. The latter phenotype was previously associated with high Treg autoreactivity in C57BL/6 mice, which we here confirm for NOD animals. The proportions of newly developing GITRhighPD-1+ Tregs rapidly drop during the first week of age. A genome-wide genetic screen indicated the involvement of several diabetes susceptibility loci in this trait. Analysis of a congenic mouse strain confirmed that Idd5 contributes to the genetic control of GITRhighPD-1+ Treg development in neonates. Our data thus demonstrate an intriguing and paradoxical correlation between an idiosyncrasy in Treg development in NOD mice and their susceptibility to type 1 diabetes.

IL-2 and IL-15 drive intrathymic development of distinct periphery-seeding CD4+Foxp3+ regulatory T lymphocytes.

Development of Foxp3-expressing regulatory T-lymphocytes (Treg) in the thymus is controlled by signals delivered in T-cell precursors via the TCR, co-stimulatory receptors, and cytokine receptors. In absence of IL-2, IL-15 or their receptors, fewer Treg apparently develop in the thymus. However, it was recently shown that a substantial part of thymic Treg are cells that had recirculated from the periphery back to the thymus, troubling interpretation of these results. We therefore reassessed the involvement of IL-2 and IL-15 in the development of Treg, taking into account Treg-recirculation. At the age of three weeks, when in wt and IL-15-deficient (but not in IL-2-deficient) mice substantial amounts of recirculating Treg are present in the thymus, we found similarly reduced proportions of newly developed Treg in absence of IL-2 or IL-15, and in absence of both cytokines even less Treg developed. In neonates, when practically no recirculating Treg were found in the thymus, the absence of IL-2 led to substantially more reduced Treg-development than deficiency in IL-15. IL-2 but not IL-15 modulated the CD25, GITR, OX40, and CD73-phenotypes of the thymus-egress-competent and periphery-seeding Treg-population. Interestingly, IL-2 and IL-15 also modulated the TCR-repertoire expressed by developing Treg. Upon transfer into Treg-less Foxp3sf mice, newly developed Treg from IL-2- (and to a much lesser extent IL-15-) deficient mice suppressed immunopathology less efficiently than wt Treg. Taken together, our results firmly establish important non-redundant quantitative and qualitative roles for IL-2 and, to a lesser extent, IL-15 in intrathymic Treg-development.

Recirculating Foxp3+ regulatory T cells are restimulated in the thymus under Aire control.

Thymically-derived Foxp3+ regulatory T cells (Treg) critically control immunological tolerance. These cells are generated in the medulla through high affinity interactions with medullary thymic epithelial cells (mTEC) expressing the Autoimmune regulator (Aire). Recent advances have revealed that thymic Treg contain not only developing but also recirculating cells from the periphery. Although Aire is implicated in the generation of Foxp3+ Treg, its role in the biology of recirculating Treg remains elusive. Here, we show that Aire regulates the suppressive signature of recirculating Treg independently of the remodeling of the medullary 3D organization throughout life where Treg reside. Accordingly, the adoptive transfer of peripheral Foxp3+ Treg in AireKO recipients led to an impaired suppressive signature upon their entry into the thymus. Furthermore, recirculating Treg from AireKO mice failed to attenuate the severity of multiorgan autoimmunity, demonstrating that their suppressive function is altered. Using bone marrow chimeras, we reveal that mTEC-specific expression of Aire controls the suppressive signature of recirculating Treg. Finally, mature mTEC lacking Aire were inefficient in stimulating peripheral Treg both in polyclonal and antigen-specific co-culture assays. Overall, this study demonstrates that Aire confers to mTEC the ability to restimulate recirculating Treg, unravelling a novel function for this master regulator in Treg biology.

Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages.

Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control the precursors of Aire+ mTEChi that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce key transcriptional regulators in mTEClo and control the composition of mTEClo subsets, including Aire+ mTEChi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEClo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4+ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.

The Repertoire of Newly Developing Regulatory T Cells in the Type 1 Diabetes-Prone NOD Mouse Is Very Diverse.

Regulatory T lymphocytes expressing the forkhead/winged helix transcription factor Foxp3 (Treg) play a vital role in the protection of the organism from autoimmune disease and other immunopathologies. The antigen specificity of Treg plays an important role in their in vivo activity. We therefore assessed the diversity of the T-cell receptors (TCRs) for antigen expressed by Treg newly developed in the thymus of autoimmune type 1 diabetes-prone NOD mice and compared it to the control mouse strain C57BL/6. Our results demonstrate that use of the TCRα and TCRß variable (V) and joining (J) segments, length of the complementarity determining region (CDR) 3, and the diversity of the TCRα and TCRß chains are comparable between NOD and C57BL/6 mice. Genetic defects affecting the diversity of the TCR expressed by newly developed Treg therefore do not appear to be involved in the etiology of type 1 diabetes in the NOD mouse.

Regulatory T Cell Heterogeneity in the Thymus: Impact on Their Functional Activities.

Foxp3+ regulatory T cells (Treg) maintain the integrity of the organism by preventing excessive immune responses. These cells protect against autoimmune diseases but are also important regulators of other immune responses including inflammation, allergy, infection, and tumors. Furthermore, they exert non-immune functions such as tissue repair and regeneration. In the periphery, Foxp3+ Treg have emerged as a highly heterogeneous cell population with distinct molecular and functional properties. Foxp3+ Treg mainly develop within the thymus where they receive instructive signals for their differentiation. Recent studies have revealed that thymic Treg are also heterogeneous with two distinct precursors that give rise to mature Foxp3+ Treg exhibiting non-overlapping regulatory activities characterized by a differential ability to control different types of autoimmune reactions. Furthermore, the thymic Treg cell pool is not only composed of newly developing Treg, but also contain a large fraction of recirculating peripheral cells. Here, we review the two pathways of thymic Treg cell differentiation and their potential impact on Treg activity in the periphery. We also summarize our current knowledge on recirculating peripheral Treg in the thymus.

Robust intrathymic development of regulatory T cells in young NOD mice is rapidly restrained by recirculating cells.

Regulatory T lymphocytes (Treg) play a vital role in the protection of the organism against autoimmune pathology. It is therefore paradoxical that comparatively large numbers of Treg were found in the thymus of type I diabetes-prone NOD mice. The Treg population in the thymus is composed of newly developing cells and cells that had recirculated from the periphery back to the thymus. We here demonstrate that exceptionally large numbers of Treg develop in the thymus of young, but not adult, NOD mice. Once emigrated from the thymus, an unusually large proportion of these Treg is activated in the periphery, which causes a particularly abundant accumulation of recirculating Treg in the thymus. These cells then rapidly inhibit de novo development of Treg. The proportions of developing Treg thus reach levels similar to or lower than those found in most other, type 1 diabetes-resistant, inbred mouse strains. Thus, in adult NOD mice the particularly large Treg-niche is actually composed of mostly recirculating cells and only few newly developing Treg.