Capture and Molecular Control of CD1a-Autoreactive T Cells in Human Skin

Abstract

Functional studies of T cell response have revealed the existence of CD1a autoreactive T cells in human skin, and CD1a tetramers might directly enumerate T cells (TCRs) and dissect the molecular basis for activation. We employed CD1a tetramers without a priori selection of bound lipids, finding that untreated CD1a tetramers carrying unspecified cellular lipids stained a large skin T cell pool (0.1-13 %) in every subject tested (n=9). Tetramer-selected cells were CD4-biased and produced IL-22 and other cytokines in a CD1a-dependent manner. We performed mass spectrometry of eluted CD1a ligands, and found that tetramer staining occurred in the presence of ~100 distinct lipids. This pattern differs from CD1-lipid and MHC-peptide recognition models where defined antigen is an absolute requirement for binding. Tetramers treated with representative CD1a ligands - diacylglycerol, ceramide, hexosylceramide, phosphatidylcholine, or lysophosphatidylcholine – similarly stained skin T cell lines. Staining inversely correlated with lipid head-group size: sulfatide, sphingomyelin, and phosphatidylinositol inhibited CD1a binding to T cells. Further, mutational CD1a tetramer studies pointed to a candidate TCR binding surface on the outside of CD1a required for recognition. Thus, CD1a-autoreactive T cells are unexpectedly abundant in skin and mainly recognize the surface of CD1a, raising the question of what mechanisms regulate this T cell pool with autoimmune potential. By comparing the profiles of total cellular lipids against those captured by CD1a, we found that CD1a ligands were strongly biased toward sphingomyelins (SM), with >40-fold enrichment of a long chain, unsaturated (C42:2, combined lengths) SM over the C34:1 form dominant in cells. 42:2 SM strongly inhibited CD1a tetramer binding to skin derived CD1a autoreactive T cells from all donors tested, identifying 42:2 SM as an endogenous antagonist of CD1a recognition. Crystallography of CD1a-SM complexes revealed that longer lipid chains in 42:2 SM force the phosphocholine group through the roof of CD1a, disrupting the putative TCR contact surface. However, an SM just 6 carbons shorter was mostly contained within CD1a and stabilized CD1a surface residues. Our studies demonstrate molecular features of lipids preferentially captured by CD1a match those interfering with autoreactive CD1a-TCR binding, informing design of therapeutic inhibitors for CD1a-mediated skin inflammation.