Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells

Abstract

CD4(+)25(+) T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-A(beta)(b) mice, major histocompatibility complex (MHC) class II I-A(b) expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMalpha-deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4(+)25(+) T cells are present in the thymus and periphery of K14-A(beta)(b) and H2-DMalpha-deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4(+)25(-) effector T cells. In contrast, CD4(+)25(+) T cells from MHC class II-deficient mice do not suppress responder CD4(+) T cells in vitro or in vivo. Thus, development of regulatory CD4(+)25(+) T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4(+)25(+) T cells in K14-A(beta)(b) and H2-DMalpha-deficient mice suggests that a subset of CD4(+)25(+) T cells is subject to negative selection on hematopoietic antigen-presenting cells.

Figure 1

K14-A_β ^b mice have CD4⁺25⁺ T cells. (A) Total LN cells from K14-A_β ^b or B6 (WT) were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25 as in Materials and Methods. Percentages indicated in histograms are of live CD4⁺ lymphocytes. (B) Total thymocytes from K14-A_β ^b or B6 (WT) mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and biotin-conjugated anti-CD25 followed by PE-conjugated streptavidin. Histograms are gated through live CD4-single positive thymocytes. Percentages indicated are of live CD4-single positive thymocytes.

Figure 1

K14-A_β ^b mice have CD4⁺25⁺ T cells. (A) Total LN cells from K14-A_β ^b or B6 (WT) were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25 as in Materials and Methods. Percentages indicated in histograms are of live CD4⁺ lymphocytes. (B) Total thymocytes from K14-A_β ^b or B6 (WT) mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and biotin-conjugated anti-CD25 followed by PE-conjugated streptavidin. Histograms are gated through live CD4-single positive thymocytes. Percentages indicated are of live CD4-single positive thymocytes.

Figure 3

MHC class II–deficient CD4⁺25⁺ T cells are not functional suppressors. (A) Total LN cells from MHC class II–deficient mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25 as in Materials and Methods. Percentages indicated in histograms are of live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from B6 mice were incubated with B6 APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of CD4⁺25⁺ cells from MHC class II–deficient or B6 mice for 72 h, pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of two experiments. (C and D) Class II–deficient CD4⁺25⁺ cells can not prevent wasting and inflammatory bowel disease. B6-Rag-2° recipients received 3 × 10⁵ B6 CD4⁺45RB^high cells either alone or with 3 × 10⁵ CD4⁺25⁺ cells from K14-A_β ^b, MHC class II–deficient, or B6 CD4⁺25⁺ cells. Results are representative of two experiments. (C) Shown are the percentages of initial body weight for individual mice on the day of killing. (D) H&E histology of colonic sections from individual animals which received effector cells and either MHC class II–deficient, K14-A_β ^b, or B6 CD4⁺25⁺ cells. Note the extensive mononuclear cell infiltrates, mucosal hyperplasia, and crypt abscesses in the recipients of MHC class II–deficient CD4⁺25⁺ cells.

Figure 3

MHC class II–deficient CD4⁺25⁺ T cells are not functional suppressors. (A) Total LN cells from MHC class II–deficient mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25 as in Materials and Methods. Percentages indicated in histograms are of live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from B6 mice were incubated with B6 APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of CD4⁺25⁺ cells from MHC class II–deficient or B6 mice for 72 h, pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of two experiments. (C and D) Class II–deficient CD4⁺25⁺ cells can not prevent wasting and inflammatory bowel disease. B6-Rag-2° recipients received 3 × 10⁵ B6 CD4⁺45RB^high cells either alone or with 3 × 10⁵ CD4⁺25⁺ cells from K14-A_β ^b, MHC class II–deficient, or B6 CD4⁺25⁺ cells. Results are representative of two experiments. (C) Shown are the percentages of initial body weight for individual mice on the day of killing. (D) H&E histology of colonic sections from individual animals which received effector cells and either MHC class II–deficient, K14-A_β ^b, or B6 CD4⁺25⁺ cells. Note the extensive mononuclear cell infiltrates, mucosal hyperplasia, and crypt abscesses in the recipients of MHC class II–deficient CD4⁺25⁺ cells.

Figure 3

MHC class II–deficient CD4⁺25⁺ T cells are not functional suppressors. (A) Total LN cells from MHC class II–deficient mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25 as in Materials and Methods. Percentages indicated in histograms are of live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from B6 mice were incubated with B6 APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of CD4⁺25⁺ cells from MHC class II–deficient or B6 mice for 72 h, pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of two experiments. (C and D) Class II–deficient CD4⁺25⁺ cells can not prevent wasting and inflammatory bowel disease. B6-Rag-2° recipients received 3 × 10⁵ B6 CD4⁺45RB^high cells either alone or with 3 × 10⁵ CD4⁺25⁺ cells from K14-A_β ^b, MHC class II–deficient, or B6 CD4⁺25⁺ cells. Results are representative of two experiments. (C) Shown are the percentages of initial body weight for individual mice on the day of killing. (D) H&E histology of colonic sections from individual animals which received effector cells and either MHC class II–deficient, K14-A_β ^b, or B6 CD4⁺25⁺ cells. Note the extensive mononuclear cell infiltrates, mucosal hyperplasia, and crypt abscesses in the recipients of MHC class II–deficient CD4⁺25⁺ cells.

Figure 3

MHC class II–deficient CD4⁺25⁺ T cells are not functional suppressors. (A) Total LN cells from MHC class II–deficient mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25 as in Materials and Methods. Percentages indicated in histograms are of live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from B6 mice were incubated with B6 APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of CD4⁺25⁺ cells from MHC class II–deficient or B6 mice for 72 h, pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of two experiments. (C and D) Class II–deficient CD4⁺25⁺ cells can not prevent wasting and inflammatory bowel disease. B6-Rag-2° recipients received 3 × 10⁵ B6 CD4⁺45RB^high cells either alone or with 3 × 10⁵ CD4⁺25⁺ cells from K14-A_β ^b, MHC class II–deficient, or B6 CD4⁺25⁺ cells. Results are representative of two experiments. (C) Shown are the percentages of initial body weight for individual mice on the day of killing. (D) H&E histology of colonic sections from individual animals which received effector cells and either MHC class II–deficient, K14-A_β ^b, or B6 CD4⁺25⁺ cells. Note the extensive mononuclear cell infiltrates, mucosal hyperplasia, and crypt abscesses in the recipients of MHC class II–deficient CD4⁺25⁺ cells.

Figure 2

K14-A_β ^b CD4⁺25⁺ cells are anergic and suppressive. (A) CD4⁺25⁺ cells (5 × 10⁴) from K14-A_β ^b or B6 (WT) were cultured with MHC class II-negative APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and rIL-2 (10 U/ml) for 72 h, pulsed with [³H]thymidine for 16 h and harvested. (B) CD4⁺25⁻ cells (5 × 10⁴) from K14-A_β ^b or B6 mice were incubated with MHC class II-negative APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of CD4⁺25⁺ cells from K14-A_β ^b or B6 mice for 72 h, pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of three experiments.

Figure 2

K14-A_β ^b CD4⁺25⁺ cells are anergic and suppressive. (A) CD4⁺25⁺ cells (5 × 10⁴) from K14-A_β ^b or B6 (WT) were cultured with MHC class II-negative APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and rIL-2 (10 U/ml) for 72 h, pulsed with [³H]thymidine for 16 h and harvested. (B) CD4⁺25⁻ cells (5 × 10⁴) from K14-A_β ^b or B6 mice were incubated with MHC class II-negative APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of CD4⁺25⁺ cells from K14-A_β ^b or B6 mice for 72 h, pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of three experiments.

Figure 4

K14-A_β ^b CD4⁺25⁺ T cells are generated in the thymus. (A) CD4⁺25⁻ cells (5 × 10⁴) from K14-A_β ^b mice were incubated with MHC class II-negative APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of K14-A_β ^b CD4⁺25⁺ thymocytes or peripheral K14-A_β ^b CD4⁺25⁺ cells for 72 h, pulsed with [³H]thymidine for 16 h, and harvested. Results are representative of three experiments. (B) CFSE-labeled, alloreactive H-2^bm12CD4⁺ cells (10⁷) were intravenously injected into indicated hosts. After 72 h animals were killed. LN and spleen cells were stained with APC-conjugated anti-CD4, and analyzed by FACS^®. Histograms are gated on live CD4⁺ splenocytes. This figure is representative of four independent experiments.

Figure 4

K14-A_β ^b CD4⁺25⁺ T cells are generated in the thymus. (A) CD4⁺25⁻ cells (5 × 10⁴) from K14-A_β ^b mice were incubated with MHC class II-negative APCs (10⁵), soluble anti-CD3ε (1 μg/ml), and the indicated number of K14-A_β ^b CD4⁺25⁺ thymocytes or peripheral K14-A_β ^b CD4⁺25⁺ cells for 72 h, pulsed with [³H]thymidine for 16 h, and harvested. Results are representative of three experiments. (B) CFSE-labeled, alloreactive H-2^bm12CD4⁺ cells (10⁷) were intravenously injected into indicated hosts. After 72 h animals were killed. LN and spleen cells were stained with APC-conjugated anti-CD4, and analyzed by FACS^®. Histograms are gated on live CD4⁺ splenocytes. This figure is representative of four independent experiments.

Figure 5

K14-A_β ^b CD4⁺25⁺ cells are activated by I-A^b-positive APCs. (A) CD4⁺25⁻ T cells (5 × 10⁴) from K14-A_β ^b mice were incubated with T cell–depleted B6 splenocytes. The indicated wells also received either K14-A_β ^b CD4⁺25⁺ cells (5 × 10⁴), B6 CD4⁺25⁺ cells (5 × 10⁴), or WT CD4⁺25⁺ cells (5 × 10⁴), and anti-CD3ε (1 μg/ml). Wells were cultured for 96 h and pulsed with [³H]thymidine for 16 h and harvested. (B) CD4⁺25⁺ cells (5 × 10⁴) from K14-A_β ^b or B6 mice were cultured with B6 splenocytes (10⁵) and rIL-2 (10 U/ml) in the indicated wells for 96 h, pulsed with [³H]thymidine for the final 16 h, and harvested. Results are representative of three experiments.

Figure 5

K14-A_β ^b CD4⁺25⁺ cells are activated by I-A^b-positive APCs. (A) CD4⁺25⁻ T cells (5 × 10⁴) from K14-A_β ^b mice were incubated with T cell–depleted B6 splenocytes. The indicated wells also received either K14-A_β ^b CD4⁺25⁺ cells (5 × 10⁴), B6 CD4⁺25⁺ cells (5 × 10⁴), or WT CD4⁺25⁺ cells (5 × 10⁴), and anti-CD3ε (1 μg/ml). Wells were cultured for 96 h and pulsed with [³H]thymidine for 16 h and harvested. (B) CD4⁺25⁺ cells (5 × 10⁴) from K14-A_β ^b or B6 mice were cultured with B6 splenocytes (10⁵) and rIL-2 (10 U/ml) in the indicated wells for 96 h, pulsed with [³H]thymidine for the final 16 h, and harvested. Results are representative of three experiments.

Figure 6

DMα-deficient CD4⁺25⁺ T cells also contain I-A^b reactive specificities. (A) Total LN cells from DMα-deficient or B6 mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25. Percentages in histogram are live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from DMα-deficient or B6 mice were incubated with T cell–depleted B6 splenocytes (2 × 10⁵). Additionally, indicated wells received either DMα-deficient CD4⁺25⁺ cells (5 × 10⁴), B6 CD4⁺25⁺ cells (5 × 10⁴), or B6 CD4⁺25⁺ cells (5 × 10⁴) and anti-CD3ε (1 μg/ml). Wells were cultured for 96 h and pulsed with [³H]thymidine for the final 16 h and harvested. (C) CD4⁺25⁺ cells (5 × 10⁴) from DMα-deficient or B6 mice were cultured with either DMα-deficient or B6, T cell–depleted splenocytes (2 × 10⁵), and rIL-2 (10 U/ml) as indicated, for 96 h. Wells were pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of three experiments.

Figure 6

DMα-deficient CD4⁺25⁺ T cells also contain I-A^b reactive specificities. (A) Total LN cells from DMα-deficient or B6 mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25. Percentages in histogram are live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from DMα-deficient or B6 mice were incubated with T cell–depleted B6 splenocytes (2 × 10⁵). Additionally, indicated wells received either DMα-deficient CD4⁺25⁺ cells (5 × 10⁴), B6 CD4⁺25⁺ cells (5 × 10⁴), or B6 CD4⁺25⁺ cells (5 × 10⁴) and anti-CD3ε (1 μg/ml). Wells were cultured for 96 h and pulsed with [³H]thymidine for the final 16 h and harvested. (C) CD4⁺25⁺ cells (5 × 10⁴) from DMα-deficient or B6 mice were cultured with either DMα-deficient or B6, T cell–depleted splenocytes (2 × 10⁵), and rIL-2 (10 U/ml) as indicated, for 96 h. Wells were pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of three experiments.

Figure 6

DMα-deficient CD4⁺25⁺ T cells also contain I-A^b reactive specificities. (A) Total LN cells from DMα-deficient or B6 mice were stained with APC-conjugated anti-CD4, Percp-conjugated anti-CD8, and FITC-conjugated anti-CD25. Percentages in histogram are live CD4⁺ lymphocytes. (B) CD4⁺25⁻ cells (5 × 10⁴) from DMα-deficient or B6 mice were incubated with T cell–depleted B6 splenocytes (2 × 10⁵). Additionally, indicated wells received either DMα-deficient CD4⁺25⁺ cells (5 × 10⁴), B6 CD4⁺25⁺ cells (5 × 10⁴), or B6 CD4⁺25⁺ cells (5 × 10⁴) and anti-CD3ε (1 μg/ml). Wells were cultured for 96 h and pulsed with [³H]thymidine for the final 16 h and harvested. (C) CD4⁺25⁺ cells (5 × 10⁴) from DMα-deficient or B6 mice were cultured with either DMα-deficient or B6, T cell–depleted splenocytes (2 × 10⁵), and rIL-2 (10 U/ml) as indicated, for 96 h. Wells were pulsed with [³H]thymidine for the final 16 h and harvested. Results are representative of three experiments.