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.

Public Health Relevance

The maintenance of a diverse naive T cell pool is critical for generating novel immune responses to pathogens. Although most T cell development occurs in the thymus, peripheral T cell maturation is the final step which is required for entry of newly generated immature thymocytes into the long-lived naive T cell pool. Very little is known about this stage of T cell development, in part due to a lack of genetic models blocked at this stage. This proposal is focused on understanding the molecular events that occur during T cell maturation, using new conditional knockout mice generated by our lab.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083279-10
Application #
9613768
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Prabhudas, Mercy R
Project Start
2010-01-15
Project End
2019-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
10
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Dash, Barsha; Shapiro, Michael J; Chung, Ji Young et al. (2018) Treg-specific deletion of NKAP results in severe, systemic autoimmunity due to peripheral loss of Tregs. J Autoimmun 89:139-148
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
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; Belmonte, Paul J; Constans, Megan M et al. (2015) Histone Deacetylase 3 Is Required for T Cell Maturation. J Immunol 195:1578-90
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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