A role for Fas in negative selection of thymocytes in vivo

Abstract

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1-100 microg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vbeta8+ cells at the level of the semi-mature population of HSAhi CD4+ 8- cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal. SEB injection also caused marked elimination of Vbeta8+ HSAhi CD4+ 8- thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (</=1 microg) but not by high doses (100 microg). Similar findings applied when T cell receptor transgenic mice were injected with specific peptide. These findings suggest that clonal elimination of semi-mature medullary T cells is Fas independent at low doses of antigen but Fas dependent at high doses. Previous reports documenting that negative selection is not obviously impaired in lpr/lpr mice could thus reflect that the antigens studied were expressed at only a low level.

Figure 1

SEB-mediated negative selection of Vβ8⁺ CD4⁺ 8⁻ thymocytes in adult and neonatal C3H mice. Adult (10-wk-old) or newborn (4-d-old) mice were injected intraperitoneally with PBS or the indicated amount of SEB, and thymuses were harvested 2 d after injection; thymocyte suspensions were four-color stained for CD4, CD8, HSA, and Vβ8 or Vβ6 and were FACS^® analyzed as described in Materials and Methods. The following FACS^® plots are shown: CD4 versus CD8 on all thymocytes (left); Vβ8 and Vβ6 expression on gated CD4⁺ 8⁻ cells (middle); and HSA expression on gated Vβ8⁺ CD4⁺ 8⁻ and Vβ6⁺ CD4⁺ 8⁻ cells (right). The average number of total thymocytes recovered from each mouse is shown at the left. The FACS^® plots refer to thymocytes from individual mice and are representative of data from 2 to 3 mice per group.

Figure 2

The kinetics of negative selection of Vβ8⁺ CD4⁺ 8⁻ cells induced by injection of SEB. Neonatal (4-d-old) C3H mice were injected intraperitoneally with SEB (25 μg) at 1, 2, or 3 d before mice were killed. Thymocytes from SEB-injected mice were then stained with mAbs specific for Vβ8 or Vβ6, CD4, CD8, and HSA. (A) Left: Total cell numbers (mean data from 5–8 mice per group) and CD4 versus CD8 expression (data from one representative mouse). Middle and right: (a) Vβ8 expression on gated CD4⁺ 8⁻ cells; (b) HSA expression on gated Vβ8⁺ CD4⁺ 8⁻ cells; (c) Vβ8 expression on gated HSA^hi CD4⁺ 8⁻ cells; (d) Vβ8 expression on gated HSA^lo CD4⁺ 8⁻ cells. (B) Total numbers of CD4⁺ 8⁻ thymocyte subsets per mouse recovered from SEB-injected mice at the time points shown. Total numbers of Vβ8⁺ HSA^hi versus Vβ8⁺ HSA^lo cells (left) and Vβ6⁺ HSA^hi versus Vβ6⁺ HSA^lo cells (right) are shown. The data represent the mean values obtained from two different experiments involving 5–8 mice per group.

Figure 3

SEB-induced negative selection of CD4⁺ 8^hi, CD4⁺ 8^lo, and CD4⁺ 8⁻ thymocytes. The data show Vβ8 (bottom left) and Vβ6 (bottom right) expression on gated CD4⁺ 8^hi, CD4⁺ 8^lo, and CD4⁺ 8⁻ thymocytes from neonatal C3H mice injected with PBS or SEB 1 or 2 d earlier. The FACS^® gates used to define CD4/CD8 expression are shown at the top. For Vβ expression, the percentages shown refer to Vβ^lo and Vβ^hi cells for CD4⁺ 8^hi cells and to Vβ^hi cells for CD4⁺ 8^lo and CD4⁺ 8⁻ cells. The data illustrated are from a single representative from each group; the numbers in parentheses refer to the mean values obtained from three mice per group.

Figure 4

SEB-induced negative selection of CD4⁺ 8⁻ thymocytes in vitro. Purified HSA^hi or HSA^lo CD4⁺ 8⁻ thymocytes from neonatal C3H mice were cultured in vitro for 20 h with or without SEB (10 μg/ml) in the presence of splenic APCs. After culture, cells were surface stained for HSA, Vβ8, and Vβ6 expression; after washing, cells were fixed and stained by TUNEL for apoptotic cells. (A) Proportion of TUNEL⁺ (apoptotic) cells in gated Vβ8⁺ cells (left) and Vβ6⁺ cells (right) in HSA^hi versus HSA^lo subsets; TUNEL staining is plotted against forward scatter (FSC). (B) Representation of the data shown in A after subtraction of the background values for apoptosis found for cells cultured with APCs without SEB. (C) HSA expression on viable (TUNEL⁻) Vβ8⁺ cells (left) and Vβ6⁺ cells (right) after culture with APCs ± SEB. (D) Proportion of Vβ8⁺ cells in the viable (TUNEL⁻) subsets of HSA^hi versus HSA^lo cells after culture with APCs ± SEB. The data shown are representative of three separate experiments.

Figure 5

Susceptibility of HSA^hi versus HSA^lo subsets of CD4⁺ 8⁻ thymocytes to apoptosis in vitro after exposure to SEB in vivo. HSA^hi versus HSA^lo CD4⁺ 8⁻ thymocytes from neonatal C3H mice injected with PBS or SEB (50 μg/mouse) 28 h earlier were purified by panning and mAb + C (see Materials and Methods). The purified cells were cultured in normal tissue culture medium in vitro for 20 h at 37°C without SEB or APCs and then stained for Vβ8 or Vβ6 followed by TUNEL staining after fixation. Levels of apoptosis found for comparable cultured populations of thymocytes prepared from uninjected control mice have been subtracted from the data shown. The data represent the mean values obtained from three separate experiments.

Figure 6

BrdU incorporation by CD4⁺ 8⁻ thymocyte subsets after SEB injection. Neonatal C3H mice were injected with PBS or SEB and killed 1, 2, or 3 d later (1 d later for PBS); starting at 20 h before death, the mice received two intraperitoneal injections of BrdU 8 h apart (−20 h and −12 h). Purified CD8⁻ cells (cells treated with anti-CD8 mAb + C) were surface stained for Vβ8 or Vβ6, CD4, and CD8, then fixed and stained for BrdU. The data show BrdU versus HSA expression on gated Vβ8⁺ CD4⁺ 8⁻ (left) and Vβ6⁺ CD4⁺ 8⁻ (right) cells. The data are representative of three separate experiments.

Figure 7

Relative sensitivity of normal B6 and B6^lpr/lpr HSA^hi CD4⁺ 8⁻ thymocytes to SEB-induced negative selection in vitro. Purified HSA^hi CD4⁺ 8⁻ thymocytes prepared from adult (6-wk-old) mice were cultured in vitro for 20 h in wells coated with a fixed concentration of anti-CD28 mAb (10 μg/ml) plus a low or high concentration of anti-TCR mAb (0.1 μg/ml or 10 μg/ml). After culture, cells were stained for TUNEL to detect apoptotic cells. The data show percentage of apoptotic (TUNEL⁺) cells after subtraction of the background levels for cells cultured alone. The data represent the mean of four separate experiments.

Figure 8

Negative selection of HSA^hi CD4⁺ 8⁻ thymocytes from C3H versus MRL^lpr/lpr mice injected with graded doses of SEB or anti-TCR mAb. Thymocytes were prepared at 2 d after intraperitoneal injection of various doses of SEB or anti-TCR mAb and stained. (A) Neonatal (4-d-old) C3H mice injected with SEB. The data represent mean values from four separate experiments with two to three mice per group. (B) Adult (7-wk-old) mice injected with SEB. The data represent mean values from four separate experiments with two mice/group. (C) Neonatal (4-d-old) mice injected with anti-TCR mAb. The data show total numbers of CD4⁺ 8⁻ subsets per thymus. The data represent mean values from two separate experiments with two mice per group.

Figure 8

Negative selection of HSA^hi CD4⁺ 8⁻ thymocytes from C3H versus MRL^lpr/lpr mice injected with graded doses of SEB or anti-TCR mAb. Thymocytes were prepared at 2 d after intraperitoneal injection of various doses of SEB or anti-TCR mAb and stained. (A) Neonatal (4-d-old) C3H mice injected with SEB. The data represent mean values from four separate experiments with two to three mice per group. (B) Adult (7-wk-old) mice injected with SEB. The data represent mean values from four separate experiments with two mice/group. (C) Neonatal (4-d-old) mice injected with anti-TCR mAb. The data show total numbers of CD4⁺ 8⁻ subsets per thymus. The data represent mean values from two separate experiments with two mice per group.

Figure 9

Negative selection of HSA^hi CD4⁺ 8⁻ thymocytes from neonatal (4-d-old) Fas^+/− versus Fas^−/− D011 TCR transgenic mice injected with graded doses of ova 323–339 peptide. Thymocytes were prepared at 2 d after intraperitoneal injection of peptide or PBS as a control and stained for CD4, CD8, HSA, and idiotypic (Id) TCR expression. (A) Phenotype of normal (uninjected) Fas^+/− D011 thymocytes showing CD4 versus CD8 expression (top), TCR expression on CD4⁺ 8⁻ cells detected by the anticlonotypic mAb KJ1-26 (middle), and HSA expression on Id⁺ CD4⁺ 8⁻ cells (bottom); Id and HSA expression on normal nontransgenic CD4⁺ 8⁻ thymocytes is shown in the heavy lines. (B) Representative data on CD4 versus CD8 expression on thymocytes from Fas^+/− versus Fas^−/− D011 mice injected with PBS and 1 or 100 μg of ova peptide. (C) Total numbers of HSA^hi CD4⁺ 8⁻, HSA^lo CD4⁺ 8⁻, and CD4⁺ 8⁺ thymocytes/mouse from Fas^+/− versus Fas^−/− D011 mice injected with various doses of ova peptide; the data represent mean values from four separate experiments with one to two mice per group.