Positive selection of extrathymically developed T cells by self-antigens

Abstract

Most T cells develop through the thymus, where they undergo positive and negative selection. Some peripheral T cells are known to develop in the absence of thymus, but there is insufficient information about their selection. To analyze the selection of extrathymically developed T cells, we reconstituted thymectomized male or female recipient mice with bone marrow cells of mice transgenic for male H-Y antigen-specific T cell receptor (TCR). It was revealed that the T cells bearing self-antigen-specific TCR were not deleted in thymectomized male recipients. More importantly, the absence of H-Y antigen-specific T cells in thymectomized female recipients suggests positive selection of extrathymically developed T cells by the self-antigen. The extrathymically developed T cells in male mice expressed interleukin (IL)-2 receptor beta chain (IL-2Rbeta) and intermediate levels of CD3 (CD3(int)) but were natural killer cell (NK)1.1(-). They rapidly produced interferon gamma but not IL-4 after TCR cross-linking. Furthermore, a similar pattern of cytokine production was observed in CD3(int)IL-2Rbeta+NK1.1(-) cells in normal mice which have been shown to develop extrathymically. These results suggest that extrathymically developed CD3(int)IL-2Rbeta+NK1. 1(-) cells in normal mice are also positively selected by self-antigens.

Figure 1

(A) Development of H-Y antigen–specific T cells in male or female thymectomized mice. C57BL/6 mice (H-2^b) with or without adult thymectomy (ATX ) were lethally irradiated (11 Gy) and injected intravenously with 5 × 10⁶ T cell– depleted bone marrow cells from female H-Y transgenic mice (H-2^b). 7 wk after bone marrow transfer, development of T cells with transgenic TCR α chain (T3.70⁺) in the spleen and liver was examined by flow cytometric analysis. Four mice are examined, and representative data are shown. The same experiments were repeated, and similar results were obtained. (B) Expression levels of CD8 on T3.70⁺ cells developed in the bone marrow chimera mice. Expression of CD8 on T3.70⁺ cells in the spleen (  filled histograms) and liver (open histograms) were analyzed by overlaying histograms.

Figure 1

(A) Development of H-Y antigen–specific T cells in male or female thymectomized mice. C57BL/6 mice (H-2^b) with or without adult thymectomy (ATX ) were lethally irradiated (11 Gy) and injected intravenously with 5 × 10⁶ T cell– depleted bone marrow cells from female H-Y transgenic mice (H-2^b). 7 wk after bone marrow transfer, development of T cells with transgenic TCR α chain (T3.70⁺) in the spleen and liver was examined by flow cytometric analysis. Four mice are examined, and representative data are shown. The same experiments were repeated, and similar results were obtained. (B) Expression levels of CD8 on T3.70⁺ cells developed in the bone marrow chimera mice. Expression of CD8 on T3.70⁺ cells in the spleen (  filled histograms) and liver (open histograms) were analyzed by overlaying histograms.

Figure 2

Proliferative response of extrathymically developed H-Y antigen–specific T cells. Proliferative response of the spleen cells, which developed in female nonthymectomized mice (white bars) or male thymectomized mice (hatched bars), in response to male stimulator cells in the absence (A) or presence of 20 U/ml of IL-2 (B) or to immobilized T3.70 antibodies (C  ) were examined. Data are represented as stimulation index.

Figure 3

IFN-γ production of extrathymically developed H-Y antigen–specific T cells. Spleen cells of female nonthymectomized or male thymectomized recipient mice were cultured in the presence (hatched bars) or absence (white bars) of immobilized T3.70 antibodies. 24 h (left) or 96 h (right) after incubation, culture supernatants were harvested, and IFN-γ was measured by an ELISA assay. ATX, Adult thymectomy. n.d., Not detectable.

Figure 4

Expression of surface molecules on H-Y antigen–specific T cells. Expression of various surface molecules on T3.70⁺CD8⁺ cells developed in female nonthymectomized (top) or male thymectomized recipient mice (bottom) were analyzed using a flow cytometer. ATX, Adult thymectomy.

Figure 5

Production of IFN-γ or IL-4 by various subsets of T cells after intravenous injection of anti-TCR mAb in normal mice. C57BL/6 mice were injected intravenously with 3 μg of anti-CD3 mAb (2C11). After 90 min, spleen cells were harvested and cultured for 3 h at 37°C in the presence of 10 μg/ml Brefeldin A. Intracellular cytokine-producing cells were examined by flow cytometry and analyzed by gating on either CD3^highIL-2Rβ⁻ (top) or CD3^intIL-2Rβ⁺ (bottom) cells. Note that all of the CD3^int cells but none of the CD3^high cells express IL-2Rβ.