T cell activation involves the integration of several signals. In addition to antigen specific signaling through the T cell receptor (TCR) and costimulatory signaling through molecules such as CD28, signaling through additional molecules including the transmembrane receptor Notch are critical at many stages of T cell activation and development. To identify novel molecules that regulate T cell activation, we used a genetic approach to rescue the T cell activation defect in a Jurkat mutant cell line by retroviral expression of a leukocyte library. In our initial screen, we found that retroviral expression of NKAP complemented the defect in one Jurkat mutant cell line and restored T cell activation. We have demonstrated that NKAP is a novel negative regulator of Notch signaling that associates with CIR, which is part of the Notch co-repressor complex. To determine the function of NKAP in vivo, we generated mice with a floxed NKAP allele. Lck-cre NKAP conditional knockout mice have a severe block in ??T cell development at the DN3/2-selection checkpoint, although ??T cell development proceeds normally. Interestingly, mice deficient in either NcoR or mSin3a, two generic components of transcriptional corepressor complexes, also have a DN3 block during T cell development, indicating that epigenetic changes are required as cells pass the ?-selection checkpoint to become DP T cells. As predicted, examination of three Notch target genes, CD25, Deltex1 and Hes1 by QPCR demonstrated that NKAP lck-cre cKO DP T cells had increases in gene expression by 8- to 20-fold, proving that the NKAP functions as a negative regulator of Notch signaling in vivo, and is required for T cell development. Analysis of T cell development in CD4-cre NKAP cKO mice demonstrates a block within SP thymocytes as they progress from semimature to mature. In addition, there are decreased numbers of T cells in the periphery in CD4-cre NKAP cKO mice, and these naive T cells express markers consistent with being recent thymic emigrants, indicating that NKAP also plays a critical role in T cell maturation. This proposal will focus on understanding the function of NKAP during T cell development and maturation, to uncover how altered gene regulation in the absence of NKAP alters T cell development and to define the mechanism whereby loss of NKAP leads to upregulation of Notch target genes.
Specific Aim #1 : Regulation of T cell development and maturation by NKAP Specific Aim #2: Mechanism of altered gene expression in the absence of NKAP
T cells are critical to proper generation of the immune response, as failure to produce T cells results in severe immune deficiency. Mice deficient in the protein NKAP have a severe block in the generation of T cells. This proposal will focus on understanding how NKAP regulates T cell development and maturation.
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| Thapa, Puspa; Romero Arocha, Sinibaldo; Chung, Ji Young et al. (2017) Histone deacetylase 3 is required for iNKT cell development. Sci Rep 7:5784 |
| Thapa, Puspa; Manso, Bryce; Chung, Ji Young et al. (2017) The differentiation of ROR-?t expressing iNKT17 cells is orchestrated by Runx1. Sci Rep 7:7018 |
| Hsu, Fan-Chi; Shapiro, Michael J; Dash, Barsha et al. (2016) An Essential Role for the Transcription Factor Runx1 in T Cell Maturation. Sci Rep 6:23533 |
| Philips, Rachael L; Chen, Meibo W; McWilliams, Douglas C et al. (2016) HDAC3 Is Required for the Downregulation of ROR?t during Thymocyte Positive Selection. J Immunol 197:541-54 |
| Hsu, Fan-Chi; Belmonte, Paul J; Constans, Megan M et al. (2015) Histone Deacetylase 3 Is Required for T Cell Maturation. J Immunol 195:1578-90 |
| Hogquist, Kristin A; Xing, Yan; Hsu, Fan-Chi et al. (2015) T Cell Adolescence: Maturation Events Beyond Positive Selection. J Immunol 195:1351-7 |
| Dolence, Joseph J; Gwin, Kimberly A; Shapiro, Mariya B et al. (2015) Cell extrinsic alterations in splenic B cell maturation in Flt3-ligand knockout mice. Immun Inflamm Dis 3:103-17 |
| Hsu, Fan-Chi; Shapiro, Michael J; Chen, Meibo W et al. (2014) Immature recent thymic emigrants are eliminated by complement. J Immunol 193:6005-15 |
| Medina, Kay L; Tangen, Sarah N; Seaburg, Lauren M et al. (2013) Separation of plasmacytoid dendritic cells from B-cell-biased lymphoid progenitor (BLP) and Pre-pro B cells using PDCA-1. PLoS One 8:e78408 |
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