T cell-intrinsic and -extrinsic contributions of the IFNAR/STAT1-axis to thymocyte survival

Abstract

STAT1 is an essential part of interferon signaling, and STAT1-deficiency results in heightened susceptibility to infections or autoimmunity in both mice and humans. Here we report that mice lacking the IFNα/β-receptor (IFNAR1) or STAT1 display impaired deletion of autoreactive CD4(+)CD8(+)-T-cells. Strikingly, co-existence of WT T cells restored thymic elimination of self-reactive STAT1-deficient CD4(+)CD8(+)-T cells. Analysis of STAT1-deficient thymocytes further revealed reduced Bim expression, which was restored in the presence of WT T cells. These results indicate that type I interferons and STAT1 play an important role in the survival of MHC class I-restricted T cells in a T cell intrinsic and non-cell intrinsic manner that involves regulation of Bim expression through feedback provided by mature STAT1-competent T cells.

Figure 1. STAT1 and IFNAR are required for elimination of self-reactive TCR^HY thymocytes.

(a) Thymocytes from male TCR^HY, TCR^HYSTAT1^−/−, TCR^HYIFNAR^−/−, and TCR^HYIFNGR^−/− mice were gated on TCR^HY positive cells and analyzed for CD4 and CD8 expressing subpopulations. (b) Graph represents percentages of CD8⁺ single-positive (SP) cells (black) and CD4⁺CD8⁺ double-positive (DP) cells (gray) derived from thymi of TCR^HY transgenic male mice. Results shown represent averages and standard deviations of three to five experiments. (c) Total numbers of CD8⁻ and CD8⁺TCR^HY thymocytes from the indicated mice.

Figure 2. STAT1-deficient T cells fail to undergo deletion in the presence of wild type TEC.

BM derived from TCR^HY or TCR^HYSTAT1^−/− animals was transferred into lethally irradiated male 129WT (a) or STAT1^−/− mice (b). Thymi of recipient mice were analyzed 4 weeks post transfer. Upper panel: TCR^HY thymocytes were gated and analyzed for CD4 and CD8 expression, and percentages of T cell subpopulations are indicated. Lower panel: histograms indicate percentages of TCR^HYCD8⁺ cells. Representatives of at least three experiments are shown.

Figure 3. STAT1-deficient thymocytes fail to undergo TCR induced apoptosis.

(a) Mice were injected i.p. with 20 µg anti-CD3 antibody, and analyzed 48 hours post injection. The percentages of CD4⁺CD8⁺ double-positive (DP) cells in WT and STAT1^−/− mice are shown with and without anti-CD3 antibody injection. Results shown represent three mice each. (b) Flow-cytometry of PI-stained WT or STAT1^−/− thymocytes in culture. Double positive thymic T cells were stimulated with plate bound anti-CD3 for 24 hours, or treated with dexamethasone for 24 hours, and the sub-G₀G₁ DNA content of nuclei measured by flow cytometry. Results shown represent averages at least three experiments. c) Double positive thymocytes from female TCR^HY and TCR^HYSTAT1^−/− mice were incubated with indicated concentrations of smcy peptide and analyzed 18 hours later for CD69 expression. Average and standard deviations from 3–4 mice are shown.

Figure 4. WT T cells promote deletion of autoreactive STAT1-deficient TCR^HY thymocytes.

(a) Flow-cytometry of thymic T cell subsets of chimeric mice. Thymocytes from the indicated BM chimeras were gated on TCR^HY cells, and analyzed for CD4- and CD8-expressing subpopulations. The percentages of T cell subpopulations are indicated. (b) Graph represents percentages of total TCR^HYCD8⁺ T cells from the indicated BM chimeras (Average and SD of 4–6 mice each).

Figure 5. Bim expression correlates with altered elimination of STAT1-deficient thymocytes.

(a) Immunoblot analysis of Bcl family members in WT and STAT1^−/− thymocytes. Cells were isolated from thymi of WT and STAT1^−/− mice and cultured in the presence or absence of plate bound anti-CD3 (10 µg/ml). Following 6 hr incubation cells were lysed, subjected to SDS-PAGE and analyzed by immunoblot for the indicated proteins. Lysates were also prepared from freshly isolated thymocytes without further incubation (lanes 1 and 4). Data is representative of at least three independent experiments. (b) Flow cytometric analysis of intracellular Bim expression levels in thymocytes from chimeric mice. Chimeric mice were generated as described in Fig. 4, and Bim expression in TCR^HY-gated thymocytes was determined by intracellular staining using flow-cytometry.