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

Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL - J. Exp. Med. (2002)

Bottom Line: CD4(+)CD25(+) immunoregulatory T cells play a pivotal role in preventing organ-specific autoimmune diseases and in tolerance induction to allogeneic organ transplants.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.More generally, these results outline the tremendous potential of regulatory T cells as therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Centre National de la Recherche Scientifique UMR 7087, Hôpital Pitié-Salpêtrière, 756651 Paris, France.

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.

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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+CD62Lhigh 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+CD62Lhigh T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4+CD25+CD62Lhigh 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.
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fig3: 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+CD62Lhigh 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+CD62Lhigh T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4+CD25+CD62Lhigh 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.

Mentions: A major limitation in the potential use of regulatory T cells for preventing GVHD is the difficulty in obtaining a sufficient number of these relatively rare cells. Therefore, we tested whether they could be expanded while retaining their functional properties. We chose to stimulate these cells by allogeneic APCs in the presence of IL-2 with the aim to increase their number (24–27) and specificity to recipient-type alloantigens. We started with highly purified populations of CD4+CD25+CD62Lhigh T cells constituting the major fraction of the CD4+CD25+ regulatory T cells (26) to limit the contamination with conventional activated CD4+CD25+CD62Llow T cells (28). The cells purified from BALB/c or B6 mice were then cocultured with irradiated C3H or (B6 × D2)F1 splenocytes, respectively. In both cultures, regulatory T cells rapidly expanded. From 5.5 × 106 BALB/c CD4+CD25+ T cells, we were able to produce 100 × 106 regulatory T cells (20-fold expansion) after 15 d of culture. In the same manner, the number of B6 CD4+CD25+ T cells was increased 10-fold during the first 2 wk and 100-fold during the next 2 wk of culture (Fig. 3 A). Similar expansion was observed in another genetic combination, in which BALB/c CD4+CD25+ T cells were stimulated by B6 splenocytes (unpublished data). Importantly, these cells kept the phenotype of regulatory T cells because they expressed even higher levels of CD25 and most of them maintained high levels of CD62L expression (Fig. 3 B). Interestingly, the absence of down-regulation of CD62L expression after repeated activation could be an intrinsic characteristic of these regulatory T cells. Because regulatory T cells were stimulated by allogeneic splenocytes, we tested whether this population was enriched in cells responding preferentially to these alloantigens. After 2 wk of culture of BALB/c regulatory T cells stimulated by irradiated C3H APCs, these cells did not respond to B6 APCs after short-term stimulation, although they continued to proliferate to C3H APCs. Similar findings were observed when using B6 APCs instead of C3H APCs (Fig. 3 C). We then analyzed whether these ex vivo–expanded regulatory T cells maintained their in vitro–suppressive properties. When added to a culture of fresh CD25− T cells stimulated by allogeneic APCs, regulatory T cells strongly inhibited T cell proliferation (Fig. 3 D).


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

Cohen JL, Trenado A, Vasey D, Klatzmann D, Salomon BL - J. Exp. Med. (2002)

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+CD62Lhigh 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+CD62Lhigh T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4+CD25+CD62Lhigh 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.
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fig3: 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+CD62Lhigh 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+CD62Lhigh T cells after cell sorting (fresh) and after 2 wk of stimulation with allogeneic irradiated splenocytes and IL-2 (cultured). (C) CD4+CD25+CD62Lhigh 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.
Mentions: A major limitation in the potential use of regulatory T cells for preventing GVHD is the difficulty in obtaining a sufficient number of these relatively rare cells. Therefore, we tested whether they could be expanded while retaining their functional properties. We chose to stimulate these cells by allogeneic APCs in the presence of IL-2 with the aim to increase their number (24–27) and specificity to recipient-type alloantigens. We started with highly purified populations of CD4+CD25+CD62Lhigh T cells constituting the major fraction of the CD4+CD25+ regulatory T cells (26) to limit the contamination with conventional activated CD4+CD25+CD62Llow T cells (28). The cells purified from BALB/c or B6 mice were then cocultured with irradiated C3H or (B6 × D2)F1 splenocytes, respectively. In both cultures, regulatory T cells rapidly expanded. From 5.5 × 106 BALB/c CD4+CD25+ T cells, we were able to produce 100 × 106 regulatory T cells (20-fold expansion) after 15 d of culture. In the same manner, the number of B6 CD4+CD25+ T cells was increased 10-fold during the first 2 wk and 100-fold during the next 2 wk of culture (Fig. 3 A). Similar expansion was observed in another genetic combination, in which BALB/c CD4+CD25+ T cells were stimulated by B6 splenocytes (unpublished data). Importantly, these cells kept the phenotype of regulatory T cells because they expressed even higher levels of CD25 and most of them maintained high levels of CD62L expression (Fig. 3 B). Interestingly, the absence of down-regulation of CD62L expression after repeated activation could be an intrinsic characteristic of these regulatory T cells. Because regulatory T cells were stimulated by allogeneic splenocytes, we tested whether this population was enriched in cells responding preferentially to these alloantigens. After 2 wk of culture of BALB/c regulatory T cells stimulated by irradiated C3H APCs, these cells did not respond to B6 APCs after short-term stimulation, although they continued to proliferate to C3H APCs. Similar findings were observed when using B6 APCs instead of C3H APCs (Fig. 3 C). We then analyzed whether these ex vivo–expanded regulatory T cells maintained their in vitro–suppressive properties. When added to a culture of fresh CD25− T cells stimulated by allogeneic APCs, regulatory T cells strongly inhibited T cell proliferation (Fig. 3 D).

Bottom Line: CD4(+)CD25(+) immunoregulatory T cells play a pivotal role in preventing organ-specific autoimmune diseases and in tolerance induction to allogeneic organ transplants.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.More generally, these results outline the tremendous potential of regulatory T cells as therapeutics.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Centre National de la Recherche Scientifique UMR 7087, Hôpital Pitié-Salpêtrière, 756651 Paris, France.

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.

Show MeSH
Related in: MedlinePlus