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

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083279-05
Application #
8602802
Study Section
Special Emphasis Panel (ZRG1-IMM-H (02))
Program Officer
Prabhudas, Mercy R
Project Start
2010-01-15
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
5
Fiscal Year
2014
Total Cost
$336,575
Indirect Cost
$113,825
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Shapiro, Michael J; Shapiro, Virginia Smith (2011) Transcriptional repressors, corepressors and chromatin modifying enzymes in T cell development. Cytokine 53:271-81
Hsu, Fan-Chi; Pajerowski, Anthony G; Nelson-Holte, Molly et al. (2011) NKAP is required for T cell maturation and acquisition of functional competency. J Exp Med 208:1291-304
Pajerowski, Anthony G; Shapiro, Michael J; Gwin, Kimberly et al. (2010) Adult hematopoietic stem cells require NKAP for maintenance and survival. Blood 116:2684-93