The development of responsive and self-tolerant T cells is vital to immunity and autoimmunity and requires a narrow range of TCR signal strength in response to selfpeptide-MHC (pMHC) in the thymus. No or weak signals induce death of thymocyte (death by neglect), while strong signals also promote cell death (negative selection). Only intermediate signals encourage survival and maturation of thymocytes (positive selection). The upper and lower affinity boundaries that trigger positively selecting TCR signals are poorly understood and thought to be largely set by the affinity of newly rearranged TCR for pMHC with downstream modulation via signaling mechanisms. All eukaryotic cells are covered with a dense coating of sugars or glycans, namely glycocalyx, which includes cell surface glycoproteins. N-glycans of surface glycoproteins can interact with soluble galectins forming a molecular lattice that regulate cellular functions. Deletion of the Golgi branching enzyme N-acetylglucosaminyl tranferase V (Mgat5) markedly increases TCR clustering and signaling at the immune synapse in peripheral T cells, but surprisingly does not appear to alter thymocyte development. Therefore, we investigated this apparent paradox by deleting the upstream Golgi branching enzymes Mgat1 and Mgat2 in thymocytes. We find that Nglycan branching of TCR and the CD4/CD8 co-receptors act upstream of signaling to separately control the upper and lower affinity boundaries from which TCR-pMHC interactions positively select T cells.

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How environmental factors combine with genetic risk at the molecular level to promote complex trait diseases such as multiple sclerosis (MS) is largely unknown. In mice, N-glycan branching by the Golgi enzymes Mgat1 and/or Mgat5 prevents T cell hyperactivity, cytotoxic T-lymphocyte antigen 4 (CTLA-4) endocytosis, spontaneous inflammatory demyelination and neurodegeneration, the latter pathologies characteristic of MS. Here we show that MS risk modulators converge to alter N-glycosylation and/or CTLA-4 surface retention conditional on metabolism and vitamin D(3), including genetic variants in interleukin-7 receptor-α (IL7RA*C), interleukin-2 receptor-α (IL2RA*T), MGAT1 (IV(A)V(T-T)) and CTLA-4 (Thr17Ala). Downregulation of Mgat1 by IL7RA*C and IL2RA*T is opposed by MGAT1 (IV(A)V(T-T)) and vitamin D(3), optimizing branching and mitigating MS risk when combined with enhanced CTLA-4 N-glycosylation by CTLA-4 Thr17. Our data suggest a molecular mechanism in MS whereby multiple environmental and genetic inputs lead to dysregulation of a final common pathway, namely N-glycosylation.