Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to
Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature na?ve T cells. 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. Introduction T cell maturation occurs continuously after thymocytes pass positive selection. The maturation process for single-positive (SP) thymocytes starts with down-regulation of CD24 and CD69, and up-regulation of Qa2 and CD62L. Mature SP thymocytes (defined as CD24?Qa2+) express sphingosine-1-phosphate receptor 1 (S1P1) and gain the ability to egress from thymic cortico-medullary junctions (1, 2). In the periphery, newly generated 1320288-17-2 manufacture T cells, designated as recent thymic emigrants(RTEs), continue post-thymic maturation in secondary lymphoid organs to acquire functional competency and enter the long-lived na?ve T cell pool(3, 4). The mechanism of T cell maturation is poorly understood, in part due to the current inability to distinguish between RTEs and long-lived mature na?ve T cells with surface markers. In addition, RTEs and long-lived mature na?ve T cells share overlapping survival niches in the periphery (5). T cell maturation is not dependent on TCR engagement (3) or IL-7R signaling (6, 7), but requires interactions of RTEs within secondary lymphoid organs (4). NKAP was identified in a genetic complementation screen for novel regulators of T cell activation. NKAP is a transcriptional repressor that binds to histone deacetylase 3 (HDAC3) and also corepressor interacting with RBP-J (CIR), a component of the Notch corepressor complex(8). NKAP is required at several points in hematopoiesis. NKAP is essential for the double-negative 3 (DN3) to double-positive (DP) transition in early T cell development(8), for hematopoietic stem cell (HSC) maintenance and survival(9), and for invariant NKT (iNKT) cell development(10). NKAP was also the first molecule shown to be required for T cell maturation(11). When NKAP is conditionally deleted at the DP stage using CD4-cre, positive selection and egress of SP thymocytes occur normally, but NKAP-deficient RTEs fail to mature (11). The failure of T cell maturation in the absence of NKAP is cell intrinsic. CD4-cre NKAP cKO mice have a 20-fold decrease in the absolute number of splenic na?ve T cells, and the cells in 1320288-17-2 manufacture this population are almost entirely comprised of functionally and phenotypically immature RTEs(11). The complement system is a component of the innate immune system that is activated in a cascade-like manner in the presence of pathogen-associated molecular patterns (PAMPs) such as interaction of C1 with antibodies (the classical pathway), activation of mannose-binding lectin (MBL) with pathogen-specific carbohydrate S1PR4 patterns (the lectin pathway), and the spontaneous hydrolysis of complement C3 (the alternative pathway). Deposition of C3b and C4b leads to opsonization for phagocytosis, and formation of a C3 convertase at the cell surface leads to direct lysis by the pore-forming membrane-attack complex (MAC)(12). Here, we show that NKAP-deficient RTEs do not die by apoptosis, but 1320288-17-2 manufacture are eliminated by complement. C3 is deposited on the cell surface of NKAP-deficient but not wild-type (WT) RTEs. As WT RTEs mature, they upregulate the complement inhibitor CD55/DAF as well as increase the incorporation of sialic 1320288-17-2 manufacture acids into cell surface glycoproteins which leads to the recruitment of the complement inhibitor Factor H(13). NKAP-deficient RTEs express less CD55, and have decreased incorporation of sialic acid in their cell surface glycoproteins as demonstrated by increased binding of the lectin peanut agglutinin (PNA). RTEs that lack NKAP have C4 and C1q deposited on their cell surface, in addition to C3, indicating activation of the classical arm of the complement pathway. Activation of the classical complement pathway is strongest when cells are bound by IgM. Interestingly, it had been previously shown that treatment of cells with neuraminidase, which cleaves sialic acid from the cell surface, leads to the binding.