CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graft-versus-host disease

Abstract

CD4(+)CD25(+) immunoregulatory T cells play a pivotal role in preventing organ-specific autoimmune diseases and in tolerance induction to allogeneic organ transplants. We investigated whether these cells could also control graft-versus-host disease (GVHD), the main complication after allogeneic hematopoietic stem cell transplantation (HSCT). Here, we show that the few CD4(+)CD25(+) T cells naturally present in the transplant regulate GVHD because their removal from the graft dramatically accelerates this disease. Furthermore, the addition of freshly isolated CD4(+)CD25(+) T cells at time of grafting significantly delays or even prevents GVHD. Ex vivo-expanded CD4(+)CD25(+) regulatory T cells obtained after stimulation by allogeneic recipient-type antigen-presenting cells can also modulate GVHD. Thus, CD4(+)CD25(+) regulatory T cells represent a new therapeutic tool for controlling GVHD in allogeneic HSCT. More generally, these results outline the tremendous potential of regulatory T cells as therapeutics.

Figure 1.

CD4⁺CD25⁺ regulatory T cells naturally present in the transplant modulate GVHD. Lethally irradiated (B6 × D2)F1 mice were grafted with semiallogeneic B6 BM cells supplemented with either 10 × 10⁶ B6 T cells (○; n = 10) or 10 × 10⁶ CD25-depleted B6 T cells (•; n = 10). Cumulative results from two independent experiments are shown. Kaplan-Meier survival curves were established for each group with the indicated P-values.

Figure 2.

Prevention of GVHD by the addition of fresh CD4⁺CD25⁺ regulatory T cells. Lethally irradiated mice were grafted with allogeneic BM cells supplemented with either 10 × 10⁶ T cells (○; n = 5) or 10 × 10⁶ T cells and 5 × 10⁶ freshly isolated CD4⁺CD25⁺ T cells (▴; n = 4). (A) Survival of (B6 × D2)F1 recipients transplanted with semiallogeneic B6 cells. (B) Survival of C3H recipients transplanted with fully allogeneic BALB/c cells. Kaplan-Meier survival curves were established with the indicated P-values.

Figure 3.

Phenotypic characterization and in vitro properties of ex vivo–expanded CD4⁺CD25⁺ T cells. (A) 0.2 × 10⁶ B6 (○) or 5.5 × 10⁶ BALB/c (•) purified CD4⁺CD25⁺CD62L^high T cells were stimulated with IL-2 and irradiated splenocytes from (B6 × D2)F1 or C3H mice, respectively. The graph depicts the expansion of living cells. (B) Flow cytometry analyses for the expression of CD4, CD25, and CD62L (inset) on total cells and CD4⁺CD25⁺CD62L^high T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4⁺CD25⁺CD62L^high T cells from BALB/c mice were stimulated with C3H APCs (left) or B6 APCs (right). After 2 wk of culture, T cells were restimulated with either the same allogeneic APCs (•) or third-party allogeneic APCs (○; B6 on the left and C3H on the right). Proliferation was assessed after 2, 2.5, or 3 d of stimulation. In both assays, T cell proliferation to third-party allogeneic APCs in the presence of IL-2, and the one obtained in the culture without APCs in the presence of IL-2, was comparable and below 10,000 cpm. (D) A constant number of BALB/c CD25-depleted cells (effector T cells) was stimulated by C3H APCs. Cells were cocultured with different numbers of BALB/c-expanded CD4⁺CD25⁺ T cells to assess their suppressive activity at different ratios between regulatory T cells and effector cells. Inhibition of the proliferation of effector T cells as compared with the culture without regulatory T cells (10,125 cpm) is shown.

Figure 3.

Phenotypic characterization and in vitro properties of ex vivo–expanded CD4⁺CD25⁺ T cells. (A) 0.2 × 10⁶ B6 (○) or 5.5 × 10⁶ BALB/c (•) purified CD4⁺CD25⁺CD62L^high T cells were stimulated with IL-2 and irradiated splenocytes from (B6 × D2)F1 or C3H mice, respectively. The graph depicts the expansion of living cells. (B) Flow cytometry analyses for the expression of CD4, CD25, and CD62L (inset) on total cells and CD4⁺CD25⁺CD62L^high T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4⁺CD25⁺CD62L^high T cells from BALB/c mice were stimulated with C3H APCs (left) or B6 APCs (right). After 2 wk of culture, T cells were restimulated with either the same allogeneic APCs (•) or third-party allogeneic APCs (○; B6 on the left and C3H on the right). Proliferation was assessed after 2, 2.5, or 3 d of stimulation. In both assays, T cell proliferation to third-party allogeneic APCs in the presence of IL-2, and the one obtained in the culture without APCs in the presence of IL-2, was comparable and below 10,000 cpm. (D) A constant number of BALB/c CD25-depleted cells (effector T cells) was stimulated by C3H APCs. Cells were cocultured with different numbers of BALB/c-expanded CD4⁺CD25⁺ T cells to assess their suppressive activity at different ratios between regulatory T cells and effector cells. Inhibition of the proliferation of effector T cells as compared with the culture without regulatory T cells (10,125 cpm) is shown.

Figure 3.

Phenotypic characterization and in vitro properties of ex vivo–expanded CD4⁺CD25⁺ T cells. (A) 0.2 × 10⁶ B6 (○) or 5.5 × 10⁶ BALB/c (•) purified CD4⁺CD25⁺CD62L^high T cells were stimulated with IL-2 and irradiated splenocytes from (B6 × D2)F1 or C3H mice, respectively. The graph depicts the expansion of living cells. (B) Flow cytometry analyses for the expression of CD4, CD25, and CD62L (inset) on total cells and CD4⁺CD25⁺CD62L^high T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4⁺CD25⁺CD62L^high T cells from BALB/c mice were stimulated with C3H APCs (left) or B6 APCs (right). After 2 wk of culture, T cells were restimulated with either the same allogeneic APCs (•) or third-party allogeneic APCs (○; B6 on the left and C3H on the right). Proliferation was assessed after 2, 2.5, or 3 d of stimulation. In both assays, T cell proliferation to third-party allogeneic APCs in the presence of IL-2, and the one obtained in the culture without APCs in the presence of IL-2, was comparable and below 10,000 cpm. (D) A constant number of BALB/c CD25-depleted cells (effector T cells) was stimulated by C3H APCs. Cells were cocultured with different numbers of BALB/c-expanded CD4⁺CD25⁺ T cells to assess their suppressive activity at different ratios between regulatory T cells and effector cells. Inhibition of the proliferation of effector T cells as compared with the culture without regulatory T cells (10,125 cpm) is shown.

Figure 4.

Regulation of GVHD by the addition of expanded CD4⁺CD25⁺ regulatory T cells. At the end of the culture (days 15 and 28 for regulatory T cells from BALB/c and B6 mice, respectively), expanded regulatory T cells were tested for their capacity to control GVHD. (A) Lethally irradiated mice were grafted with allogeneic BM cells supplemented with either 10 × 10⁶ fresh T cells (○; n = 5 per group) or 10 × 10⁶ fresh T cells and 7 × 10⁶ expanded CD4⁺CD25⁺ T cells (▴; n = 5 per group). For both genetic combinations, the addition of expanded CD4⁺CD25⁺ T cells statistically increased the survival of mice. (B) Lethally irradiated B6 mice were grafted with BALB/c BM cells and 10 × 10⁶ fresh BABL/c T cells (○, GVHD control group; n = 5) supplemented with 7 × 10⁶ expanded regulatory T cells derived from cultured CD4⁺CD25⁺ T cells stimulated by C3H splenocytes (▪, nonspecific regulatory T cells; n = 5), or B6 splenocytes (•, specific regulatory T cells; n = 5). The difference in survival between the GVHD control group and mice receiving nonspecific CD4⁺CD25⁺ T cells is statistically insignificant. When statistically significant, Kaplan-Meier survival curves were established with the indicated P-values.

Figure 4.

Regulation of GVHD by the addition of expanded CD4⁺CD25⁺ regulatory T cells. At the end of the culture (days 15 and 28 for regulatory T cells from BALB/c and B6 mice, respectively), expanded regulatory T cells were tested for their capacity to control GVHD. (A) Lethally irradiated mice were grafted with allogeneic BM cells supplemented with either 10 × 10⁶ fresh T cells (○; n = 5 per group) or 10 × 10⁶ fresh T cells and 7 × 10⁶ expanded CD4⁺CD25⁺ T cells (▴; n = 5 per group). For both genetic combinations, the addition of expanded CD4⁺CD25⁺ T cells statistically increased the survival of mice. (B) Lethally irradiated B6 mice were grafted with BALB/c BM cells and 10 × 10⁶ fresh BABL/c T cells (○, GVHD control group; n = 5) supplemented with 7 × 10⁶ expanded regulatory T cells derived from cultured CD4⁺CD25⁺ T cells stimulated by C3H splenocytes (▪, nonspecific regulatory T cells; n = 5), or B6 splenocytes (•, specific regulatory T cells; n = 5). The difference in survival between the GVHD control group and mice receiving nonspecific CD4⁺CD25⁺ T cells is statistically insignificant. When statistically significant, Kaplan-Meier survival curves were established with the indicated P-values.