Murine thymic selection quantified using a unique method to capture deleted T cells

Abstract

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77(GFP) transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim(-/-) mice had an increased number of GFP(hi) cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.

Fig. 1.

Highly self-reactive thymocytes accumulate in Bim^−/− mice. (A and B) Thymocytes from Nur77^GFP mice that were either Bim deficient (Bim^−/−) or control (Bim^+/+ or Bim^+/−), were analyzed by flow cytometry. (C) GFP level was assessed on DP thymocytes from Bim^+/+ Nur77^GFP mice stained and gated on CD69loCD5lo (presel), CD69+CD5+ (postsel), or CD69+CD5+Active caspase 3+ (postsel apoptotic). (D) Bim^−/− or control thymi were analyzed using immunofluoresence. β5t was stained to determine the cortex region. (E) The mean fluorescence intensity of the inner and outer cortex of Bim^−/− mice. In B, cells were gated as indicated on the left: preselection CD69⁻,TCRβ^lo; postselection, CD69⁺,TCRβ^hi. Data are representative of more than eight (A and B), 5 (C), or 4 (D and E) animals from two to six independent analyses. Asterisks indicated P < 0.05 using a Student t test.

Fig. 2.

Enumeration of progenitors with intermediate or high GFP expression. Thymocytes from Nur77^GFP mice that were either Bim deficient (Bim^−/−) or control (Bim^+/+ or Bim^+/−), were analyzed by flow cytometry. (Upper) Gates used to define GFP intermediate (int) and GFP high (hi) populations. The intermediate gate, which defines the level of GFP expressed in positively selected naïve T cells, was established by gating on the GFP level of mature (HSA^lo) CD4SP or CD8SP, CD25^- thymocytes from the same experiment (black lines). The high gate was extended from the upper end of the intermediate gate. (Lower) The total numbers of thymocytes in the indicated gates. Data are representative or calculated from eight mice from six independent analyses. Error bars indicated SD. Asterisks indicated P < 0.05 using a Student t test.

Fig. 3.

Analysis of active caspase 3 yields similar DP to SP deletion ratios. Thymi from Bim control Nur77^GFP mice were analyzed by flow cytometry. (A) (Left) The gating scheme for active caspase 3. (Center) CD69 and CD5 expression on active-caspase 3+ cells. The red gate defines death by neglect; the blue gate defines clonal deletion. (Right) GFP levels in cells undergoing death by neglect versus clonal deletion is shown in the histogram overlay. (B) (Left and Center) The CD4/8 profiles of cells undergoing death by neglect or clonal deletion. (Right) Comparison of the ratio of DP to SP deletion using the methods used in Figs. 2 and 3 (Bim^−/− Nur77^GFP and active caspase 3, respectively). Data are representative from five to eight mice from at least three independent analyses.

Fig. 4.

Cells rescued from clonal deletion express a similar level of GFP compared with T_reg cells. Cells from thymi of control and Bim^−/− Nur77^GFP mice were analyzed by flow cytometry. (A) (Top) Cells were gated on Foxp3⁺ (dotted oval gate) or Foxp3⁻ (solid polygonal gate). (Middle and Bottom) Overlays of GFP on the different cell populations. (B) The percent and number of T_reg cells in control and Bim^−/− thymi. (C) GFP expression on T_reg cells and non-T_reg cells from Bim^−/− thymi. (D) Normalized mean fluorescence intensity of GFP expression on Bim^−/− cell populations. Data are representative from seven animals from at least four independent analyses. Asterisks indicated P < 0.05 using a Student t test.

Fig. 5.

Bim^−/− peripheral T cells are enriched for anergic phenotype cells. Peripheral CD4⁺ (non-T_reg) T cells and CD8 T cells from Bim^−/− and control mice were analyzed by flow cytometry. (A) The level of GFP in CD4 and CD8 T cells from spleen and lymph nodes. (B) (Left) The gating strategy used to define “anergic phenotype” cells in the peripheral lymphoid tissue. (Right) Nur77^GFP expression of the indicated cell populations. (C) Dot plots show the percentage of CD4⁺ CD44hi cells from Bim^−/− or control spleens that expressed an anergic phenotype. (D) CD8⁺ T cells from the spleen or lymph nodes of Bim^−/− and Bim^+/− were stained for CD62L and CD44. Data shown are representative of at least four animals from at least three independent analyses.