DESCRIPTION (provided by applicant): Single positive thymocytes that successfully complete positive and negative selection must still undergo one final step, called T cell maturation, before they gain functional competency and enter the long- lived T cell pool. Recent thymic emigrants (RTEs) must complete T cell maturation in the periphery, and although this process is poorly understood, in part due to lack of mouse models blocked at this developmental checkpoint, it is not dependent on either antigen-specific signals through the TCR or homeostatic signals through IL-7R?Previously, we cloned the transcriptional repressor NKAP on a genetic complementation screen to identify novel regulators of T cell activation. T cells from CD4-cre NKAP cKO mice cannot undergo T cell maturation. Almost all naive T cells in the periphery are phenotypically and functionally immature RTEs. We have used this model to better understand the events that occur during T cell maturation. Interestingly, we find that NKAP-deficient RTEs do not die by apoptosis, but rather are eliminated by complement, as demonstrated by C3 deposition on the cell surface. C4 and C1q are also bound to NKAP-deficient T cells, indicating activation of the classical arm of the complement pathway. As thymocytes complete development before export to the periphery, they increase incorporation of sialic acid into glycoproteins and glycolipids at the cell surface. This addition of sialic acid is critical to lymphocyte survival in the periphery, as stripping of cell surface sialic acids by neuraminidase leads to the binding of natural IgM and complement fixation. We find that NKAP-deficient T cells have a defect in sialylation, as demonstrated by increased binding of peanut agglutinin (PNA), leading to IgM recruitment. As T cells mature, they also upregulate expression of the complement inhibitor DAF/CD55 on the cell surface, but this is defective in NKAP-deficient RTEs which exacerbates complement-mediated elimination. Specifically, we find that the incorporation of ?8-linked sialic acids occurs concurrently with T cell maturation, and that this is altered in the absence of NKAP. There are six related proteins that mediate ?8 sialylation, and we find that one of them, ST8Sia6, is turned on specifically with maturation and that its expression is decreased in the absence of NKAP. Our preliminary results demonstrate that one substrate of ST8Sia is CD45, where it controls CD45 function through regulating dimerization, and therefore may regulate T cell responsiveness during T cell maturation. We believe that NKAP may work with Hdac3 and Runx1 to regulate T cell maturation, because CD4-cre knockout of either gene results in a phenotype similar to the NKAP knockout with respect to IgM/complement deposition, decreased ?8 sialylation and CD55 expression, which will be explored in this proposal.
|Effective start/end date||1/15/10 → 3/31/20|
- National Institute of Allergy and Infectious Diseases: $3,838,703.00
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