Recent thymic emigrants (RTEs) are newly generated T cells that need to undergo post-thymic maturation to gain functional competency and enter the long-lived naïve T cell pool. The mechanism of T cell maturation remains incompletely understood. Previously, we demonstrated that the transcriptional repressor NKAP is required for T cell maturation. As NKAP associates with histone deacetylase 3 (HDAC3), we examined whether HDAC3 is also required for T cell maturation. While thymic populations are similar in CD4-cre HDAC3 conditional knockout (cKO) mice compared to wild-type (WT) mice, however, the peripheral numbers of CD4+ and CD8+ T cells are dramatically decreased. In the periphery, the majority of HDAC3-deficient naïve T cells are RTEs, indicating a block in T cell maturation. CD55 upregulation during T cell maturation is substantially decreased in HDAC3-deficient T cells. Consistent with a block in functional maturation, HDAC3-deficient peripheral T cells have a defect in TNF licensing after TCR/CD28 stimulation. CD4-cre HDAC3 cKO mice do not have a defect in intrathymic migration, thymic egress, T cell survival or homeostasis. In the periphery, similar to immature NKAP-deficient peripheral T cells, HDAC3-deficient peripheral T cells were bound by IgM and complement proteins, leading to the elimination of these cells. In addition, HDAC3-deficient T cells display decreases in the sialic acid modifications on the cell surface that recruit natural IgM to initiate the classical complement pathway. Therefore, HDAC3 is required for T cell maturation.

Recent thymic emigrants (RTEs) must undergo phenotypic and functional maturation to become long-lived mature naïve T cells. In CD4-cre NKAP conditional knockout mice, NKAP-deficient RTEs fail to complete T cell maturation. Here, 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 down regulated 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.

To generate functional peripheral T cells, proper gene regulation during T cell development is critical. Here, we found that histone deacetylase 3 (HDAC3) is required for T cell development. T cell development in CD2-icre HDAC3 conditional knockout mice (HDAC3-cKO) was blocked at positive selection, resulting in few CD4 and CD8 T cells, and could not be rescued by a TCR-transgene. These SP thymocytes failed to upregulate Bcl-2, leading to increased apoptosis. HDAC3-cKO mice failed to downregulate RORγt during positive selection, and phenocopied the block in positive selection in RORγt-transgenic mice. In the absence of HDAC3, the RORC promoter was hyperacetylated. In the periphery, the few CD4 T cells present were skewed towards RORγt+ IL-17-producing Th17 cells, leading to inflammatory bowel disease. Positive selection of CD8SP thymocytes was restored in RORγt-KO Bcl-xl-transgenic HDAC3-cKO mice, demonstrating that HDAC3 is required at positive selection to down-regulate RORγt.

Invariant Natural Killer T (iNKT) cells are a unique lineage with characteristics of both adaptive and innate lymphocytes, and recognize glycolipid presented by an MHC Class I-like CD1d molecule. During thymic development, iNKT cells also differentiate into NKT1, NKT2 and NKT17 functional subsets that preferentially produce cytokines IFN-γ, IL-4 and IL-17, respectively, upon activation. Newly selected iNKT cells undergo a burst of proliferation, which is defective in mice with a specific deletion of NKAP in the iNKT cell lineage, leading to severe reductions in thymic and peripheral iNKT cell numbers. The decreased cell number is not due to defective homeostasis or increased apoptosis, and is not rescued by Bcl-xL overexpression. NKAP is also required for differentiation into NKT17 cells, but NKT1 and NKT2 cell development and function are unaffected. This failure in NKT17 development is rescued by transgenic expression of PLZF; however, the PLZF transgene does not restore iNKT cell numbers or the block in positive selection into the iNKT cell lineage in CD4-cre NKAP cKO mice. Therefore, NKAP regulates multiple steps in iNKT cell development and differentiation.