To test for anergy, we adoptively transferred CD25neg CTV-labelled Bg2 cells into recipient mice and re-challenged them 28 days later with -gal721-739-pulsed bone marrow-derived DCs (BMDCs)


To test for anergy, we adoptively transferred CD25neg CTV-labelled Bg2 cells into recipient mice and re-challenged them 28 days later with -gal721-739-pulsed bone marrow-derived DCs (BMDCs). chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer -gal Mouse monoclonal to KLHL22 to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3. Immune tolerance is imposed through multiple processes that begin during thymic T-cell development and continue in the periphery. During negative selection in the thymus, medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) present self-antigens to tolerize auto-reactive T cells. Intrinsic tolerance mechanisms induce deletion or anergy of high-affinity self-reactive T cells, whereas lower affinity CD4 cells are converted into regulatory T cells (Treg) that mediate extrinsic tolerance1,2. DCs can acquire antigen in the periphery and migrate into the thymus3, or thymic resident DCs can capture circulating antigen4. In addition to presenting ubiquitous antigens, mTECs also transcribe and present a variety of peripheral tissue antigens (PTAs) under the control of the autoimmune regulatory element (Aire)5,6, increasing the diversity of self-antigens presented in the thymus. Thymic tolerance does not eliminate all self-reactive T cells, necessitating mechanisms of peripheral tolerance. Immature DCs continually survey peripheral tissues to acquire self-antigens, which are presented in the draining lymph nodes (LNs) to induce T-cell deletion, anergy or Treg formation7. In contrast to DCs, which are specialized for acquiring antigens from other tissues, several subsets of LN cells transcribe PTAs, analogous to mTECs. Extrathymic Aire-expressing cells transcribe and present PTAs in an Aire-dependent manner, leading to CD8 T-cell deletional tolerance and CD4 T-cell anergy8,9. Extrathymic Aire-expressing cells are developmentally related to DCs9. PTAs are transcriptionally expressed independently of Aire by several subsets of radioresistant LN stromal cells (LNSCs), including lymphatic endothelial cells (LECs), fibroblastic reticular cells (FRCs) and blood endothelial cells (BECs)10,11. Although the effects of PTAs expressed in BECs have not been tested, LECs and CHIR-090 FRCs both induce deletional tolerance of CD8 T cells10,11,12,13. We previously showed that LECs transcribe and present an CHIR-090 epitope from the melanocyte differentiation protein tyrosinase, leading to proliferation and deletion of tyrosinase-specific CD8 T cells10,14. Proliferating tyrosinase-specific CD8 T cells activated by LECs in the absence of 4-1BB co-stimulation upregulate PD-1, which binds to PD-L1 on CHIR-090 a radioresistant stromal cell, inhibits the upregulation of the IL-2 receptor and leads to death12. LECs express the highest level of PD-L1 among the LNSC. LECs also express herpesvirus entry mediator (HVEM) and major histocompatibility complex (MHC)-II12, which are ligands for the BTLA/CD160 and LAG-3 inhibitory pathways, respectively15. Tyrosinase and PD-L1 are more highly expressed CHIR-090 by LECs in the LN (LN-LECs) compared with LECs from tissue lymphatics in the diaphragm or colon16, suggesting the LN microenvironment endows LN-LECs with tolerogenic properties not found in tissue CHIR-090 LECs. In this study, we investigated whether MHC-II expression on LN-LECs is related to their tolerogenic role, and whether MHC-II is used to induce CD4 T-cell tolerance. The MHC-II antigen presentation pathway has been extensively studied in professional antigen-presenting cells (APC) and in cell lines. MHC-II molecules are synthesized in the ER and associated with invariant chain (Ii), which targets the complex into late endosomal MHC-II-loading compartments (MIICs). Next, Ii is cleaved by cathepsins, leaving the class II Ii-associated peptide (CLIP) in the peptide-binding groove. CLIP is exchanged for antigenic peptides by the non-classical MHC-II molecule H2-M. H2-M can be inhibited by H2-O, altering the representation of peptides presented17. LECs express MHC-II12, but the ability of LECs to load and present self-peptides on MHC-II molecules has not been investigated. In addition, it is unknown whether PTA expression in LECs leads to CD4 T-cell tolerance. To investigate whether LECs present PTAs on MHC-II molecules and induce CD4 T-cell tolerance, we created transgenic systems where the model antigens -galactosidase (-gal) or haemagglutinin (HA) are expressed in LECs under the control of LEC-specific Lyve-1 or Prox1 promoters. Using these complementary models, we demonstrate that LECs do not directly present these PTAs on MHC-II molecules, but instead provide antigen to DCs.