Thymus-specific serine protease contributes to the diversification of the functional endogenous CD4 T cell receptor repertoire

Abstract

Thymus-specific serine protease (TSSP) is a novel protease that may contribute to the generation of the peptide repertoire presented by MHC class II molecules in the thymus. Although TSSP deficiency has no quantitative impact on the development of CD4 T cells expressing a polyclonal T cell receptor (TCR) repertoire, the development of CD4 T cells expressing the OTII and Marilyn transgenic TCRs is impaired in TSSP-deficient mice. In this study, we assess the role of TSSP in shaping the functional endogenous polyclonal CD4 T cell repertoire by analyzing the response of TSSP-deficient mice to several protein antigens (Ags). Although TSSP-deficient mice responded normally to most of the Ags tested, they responded poorly to hen egg lysozyme (HEL). The impaired CD4 T cell response of TSSP-deficient mice to HEL correlated with significant alteration of the dominant TCR-β chain repertoire expressed by HEL-specific CD4 T cells, suggesting that TSSP is necessary for the intrathymic development of cells expressing these TCRs. Thus, TSSP contributes to the diversification of the functional endogenous CD4 T cell TCR repertoire in the thymus.

Figure 1.

Reduced CD4 T cell proliferative response to HEL in Tssp^−/− mice. Tssp^−/− (KO) and WT control mice were immunized with the indicated protein. 10–11 d later, CD4 T cells were isolated from the draining LN and restimulated in vitro with WT spleen APCs and graded concentrations of the immunizing protein. For each Ag, one representative experiment out of at least two performed with one or two mice of each genotype is shown. Each symbol corresponds to one individual mouse. CAB, conalbumin.

Figure 2.

The reduced response of Tssp^−/− mice to HEL is a CD4 T cell property. (A) IL-2 production by the HEL-specific T cell transfectant BW-HEL upon stimulation with splenic APCs from Tssp^−/− (KO) or WT control mice loaded with graded concentrations of HEL_12–27 peptide or HEL protein, as indicated. One representative experiment out of four performed is shown. (B) WT or Tssp^−/− (KO) mice were primed with HEL protein (left) or HEL_12–27 peptide (right). 11 d later, CD4 T cells were isolated from the draining LN and restimulated in vitro with WT spleen APCs and graded concentrations of HEL_12–27 peptide. (C) WT and Tssp^−/− mice were primed in vivo, and their CD4 T cells were restimulated in vitro with WT spleen APCs and either HEL protein or HEL_12–27 peptide, as indicated. Results are expressed as the ratio of the proliferation of TSSP-deficient T cells divided by that of WT control. The figure compiles the results of three to five independent experiments. Each symbol corresponds to one mouse. Horizontal bars represent the mean. One-sample t test showed that the responses of WT and Tssp^−/− mice are significantly different for all stimulation conditions (P < 0.002). (D) WT and Tssp^−/− (KO) mice were primed in vivo with either WT or TSSP-deficient DCs pulsed with 40 ng HEL_12–27 peptide, and their CD4 T cells were restimulated in vitro with WT spleen APCs and a titrated amount of HEL_12–27 peptide. One representative experiment is presented in the left panel. The right panel combines results of three independent experiments expressed as the ratio of the proliferation of WT or TSSP-deficient T cells (X) divided by the mean value of the WT controls in the same experiment. Each symbol corresponds to one mouse. Horizontal bars represent the mean. Significant p-values are shown. (E) Purified LN CD4 T cells from HEL- or OVA-immunized WT and Tssp^−/− mice were labeled with CFSE and stimulated with WT splenocytes together with HEL_12–27 peptide, OVA protein, or anti-CD3/CD28 antibodies, as indicated. Cell division was evaluated by analyzing CFSE dilution on gated CD4 T cells at day 3 and 6 of activation. The percentage of divided WT or TSSP-deficient (KO) CD4 T cells is shown. One representative experiment out of four performed is shown.

Figure 3.

Impaired thymic selection of HEL-specific CD4 T cells in Tssp^−/− mice. WT or Tssp^−/− (KO) mice were lethally irradiated before reconstitution with WT BM cells (WT) or a 1:1 mix of WT and NOD-Cα° TSSP-deficient (Cα°-KO) BM cells, as indicated (BM → host). 8–10 wk after reconstitution, chimeras were immunized with HEL protein, and 7 d later, their CD4 T cell proliferative response to 10 µg/ml HEL_12–27 pulsed WT APCs was analyzed. The results of three independent experiments are expressed as the ratio of the proliferation of WT or TSSP-deficient CD4 T cells (X) divided by the mean value of the WT control chimeras in the same experiment. Each symbol corresponds to one individual chimera. Horizontal bars represent the mean. Significant p-values are shown.

Figure 4.

Vβ gene segment usage by naive and HEL_12–27-responsive CD4 T cells from Tssp^−/− and WT control mice. CD4 T cells isolated from HEL-primed WT or Tssp^−/− mice were CFSE labeled and stimulated with HEL_12–27 peptide in the presence of irradiated WT splenocytes. After 5–6 d of culture, dividing CD4 T cells were FACS sorted before RT-coupled real-time PCR analysis. Alternatively, LN CD4 T cells were isolated from 9–13-wk-old naive mice. Box and whiskers graph shows the Vβ gene usage for naive or HEL-specific CD4 T cells isolated from WT or Tssp^−/− (KO) mice (n = number of mice). Horizontal bars show the median values, boxes show the 25th and 75th percentile, and bars show the minimal and maximal values. In WT mice, Vβ6 usage was significantly higher than that of all other Vβ genes, except Vβ7 (P < 0.05). The p-value of those Vβ with an increased representation in CD4 T cells from primed mice as compared with that of naive mice is shown (*, P < 0.05; **, P < 0.01).