Seaburg, Lauren

Abstract Invariant Natural Killer T (iNKT) cells are an innate lineage of lymphocytes that can produce copious amount of cytokines within hours of stimulation. In the thymus, positive selection into the iNKT cell lineage occurs at the double positive (DP) stage, before maturation through various stages (stage 0-3) to complete development. NKAP, a transcriptional repressor, is required for conventional T lymphocyte development and maturation. NKAP interacts with Hdac3 for its repressor function. Previously, using CD4-cre NKAP cKO mice, we have shown that NKAP is required for positive selection into the iNKT cell lineage. To study the role of NKAP in later developmental stages, we generated PLZF-cre NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0. In the PLZF-cre NKAP cKO mice we observed a significant decrease in the absolute number of iNKTs with a 20-fold reduction at stage 2 and 200-fold reduction at stage 3. This defect is cell intrinsic and cannot be rescued by over-expression of Bcl-XL. Our preliminary results indicate that there may be a block in iNKT cell maturation, as expression of transcription factors Tbet, Gata3, and ROR-γt that define NKT1, NKT2 and NKT17 lineages respectively, are decreased in PLZF-cre NKAP cKO. PLZF-cre Hdac3 cKO mice have a similar defect in iNKT development. Thus, NKAP together with Hdac3 may regulate iNKT cell development and maturation.

Abstract Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naive T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. In this study, we demonstrate that NKAP-deficient immature RTEs do not undergo apoptosis, but are eliminated by complement. C3, C4, and C1q are bound to NKAP-deficient peripheral T cells, demonstrating activation of the classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they increase sialic acid incorporation into cell surface glycans. This is essential to peripheral lymphocyte survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to IgM recruitment. We demonstrate that the defect in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1, ST8sia4, and ST8sia6) that mediate α2,8 sialylation are downregulated in NKAP-defcient RTEs. The maturation of peripheral NKAP-deficient T cells is partially rescued in a C3-deficient environment. Thus, sialylation during T cell maturation is critical to protect immature RTEs from complement in the periphery.