Medullary Thymic Epithelial Cell Antigen-presentation Assays.

Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context. Key features • This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022). • This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA323-339, to assess mTEC antigen presentation to CD4+ T cells. • This requires specific equipment such as a Miltenyi Biotec AutoMACS® Pro Separator, a BD FACSAriaTM III cell sorter, and a BD® LSR II flow cytometer.

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.

Lymphotoxin: from the physiology to the regeneration of the thymic function.

The members of the Tumor Necrosis Factor (TNF) superfamily, the ligand lymphotoxin α1ß2 (LTα1ß2) and its unique receptor lymphotoxin ß receptor (LTßR), play a pivotal role in the establishment and regulation of the immune system by allowing a tight communication between lymphocytes and stromal cells. Recent advances using transgenic mice harboring a specific deletion of the Ltbr gene in distinct stromal cells have revealed important roles for LTßR signaling in the thymic function that ensures the generation of a diverse and self-tolerant T-cell repertoire. In this review, we summarize our current knowledge on this signaling axis in the thymic homing of lymphoid progenitors and peripheral antigen-presenting cells, the trafficking and egress of thymocytes, the differentiation of medullary thymic epithelial cells, and the establishment of central tolerance. We also highlight the importance of LTα1ß2/LTßR axis in controlling the recovery of the thymic function after myeloablative conditioning regimen, opening novel perspectives in regenerative medicine.

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.