ABSTRACT
Regulatory T cells (Treg) are critical players of immune tolerance that develop in the thymus via two distinct developmental pathways involving CD25+Foxp3- and CD25-Foxp3lo precursors. However, the mechanisms regulating the recently identified Foxp3lo precursor pathway remain unclear. Here, we find that the membrane-bound lymphotoxin α1ß2 (LTα1ß2) heterocomplex is upregulated during Treg development upon TCR/CD28 and IL-2 stimulation. We show that Lta expression limits the maturational development of Treg from Foxp3lo precursors by regulating their proliferation, survival, and metabolic profile. Transgenic reporter mice and transcriptomic analyses further reveal that medullary thymic epithelial cells (mTEC) constitute an unexpected source of IL-4. We demonstrate that LTα1ß2-lymphotoxin ß receptor-mediated interactions with mTEC limit Treg development by down-regulating IL-4 expression in mTEC. Collectively, our findings identify the lymphotoxin axis as the first inhibitory checkpoint of thymic Treg development that fine-tunes the Foxp3lo Treg precursor pathway by limiting IL-4 availability.
Subject(s)
Forkhead Transcription Factors , Interleukin-4 , Lymphotoxin beta Receptor , Lymphotoxin-alpha , Signal Transduction , T-Lymphocytes, Regulatory , Animals , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Interleukin-4/metabolism , Mice , Lymphotoxin-alpha/metabolism , Lymphotoxin-alpha/genetics , Lymphotoxin beta Receptor/metabolism , Lymphotoxin beta Receptor/genetics , Thymus Gland/immunology , Thymus Gland/cytology , Thymus Gland/metabolism , Epithelial Cells/metabolism , Mice, Inbred C57BL , Cell Differentiation , Mice, Transgenic , Interleukin-2/metabolism , Cell Proliferation , Lymphotoxin alpha1, beta2 Heterotrimer/metabolism , Lymphotoxin alpha1, beta2 Heterotrimer/geneticsABSTRACT
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.
ABSTRACT
Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1ß and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD ß-cells underscores the role of deamidation in amplifying T-cell reactivity.