Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia

Abstract

CD4+CD25+ regulatory T cells (Treg's) play a pivotal role in preventing organ-specific autoimmune diseases and in inducing tolerance to allogeneic organ transplants. We and others recently demonstrated that high numbers of Treg's can also modulate graft-versus-host disease (GVHD) if administered in conjunction with allogeneic hematopoietic stem cell transplantation in mice. In a clinical setting, it would be impossible to obtain enough freshly purified Treg's from a single donor to have a therapeutic effect. Thus, we performed regulatory T cell expansion ex vivo by stimulation with allogeneic APCs, which has the additional effect of producing alloantigen-specific regulatory T cells. Here we show that regulatory T cells specific for recipient-type alloantigens control GVHD while favoring immune reconstitution. Irrelevant regulatory T cells only mediate a partial protection from GVHD. Preferential survival of specific regulatory T cells, but not of irrelevant regulatory T cells, was observed in grafted animals. Additionally, the use of specific regulatory T cells was compatible with some form of graft-versus-tumor activity. These data suggest that recipient-type specific Treg's could be preferentially used in the control of GVHD in future clinical trials.

Figure 1

Regulation of GVHD by the addition of ex vivo–expanded Treg’s. At the end of the culture, Treg’s were tested for their capacity to control GVHD in the BALB/c → [BALB/c × C3H]F1 combination. (a) The picture illustrates the skin lesions and general status of grafted [BALB/c × C3H]F1 mice undergoing GVHD (upper pairs) or mice protected from GVHD by adding sTreg’s (lower pairs). (b) Mice were weighed at different time points prior to sacrifice at day 45. Mean weight curves were established for mice receiving BM cells alone (dashed line, n = 3), BM cells supplemented with 10 × 10⁶ conventional T cells (open circles, n = 5) in addition to either 10 × 10⁶ sTreg’s (filled squares, n = 15) or irTreg’s (filled circles, n = 14). P < 0.05 between all groups except for BM cells alone versus sTreg’s. (c) Histopathologic score of liver and spleen after semiallogeneic BMT. Grading of GVHD was performed 45 days after transplantation in liver and spleen. BM control mice infused with BM cells alone did not develop GVHD (n = 3). ND, not done. GVHD control mice received BM cells plus T cells and represented the maximum intensity of GVHD in this model (n = 4). Experimental mice received BM cells plus T cells and either sTreg’s (n = 8) or irTreg’s (n = 7). Points correspond to histopathological scores of individual mice; histograms show the mean histopathological score for each group. P < 0.05 between all groups for all tissues except for BM cells alone versus sTreg’s and BM cells alone versus irTreg’s in the liver.

Figure 2

Regulation of GVHD by sTreg’s is associated with good immune reconstitution. Immune reconstitution was evaluated 45 days after transplantation in the spleen of mice grafted as described in Figure 1. Total splenocytes were counted and stained with appropriate mAb’s. The number of B220⁺, CD3⁺, CD4⁺, and CD8⁺ cells was evaluated after analysis by flow cytometry for BM control mice (white bars; n = 3), GVHD control mice (black bars; n = 4), mice receiving sTreg’s (dark gray bars; n = 5), or irTreg’s (light gray bars; n = 5). Histograms indicate the mean number ± SEM of cells for each group. P < 0.05 between all groups and for all cell populations except for BM alone versus irTreg’s for CD8⁺ cells.

Figure 3

Increased survival of sTreg’s as compared to irTreg’s in the spleen of grafted mice. The injected Treg’s were detected in the spleen of animals grafted as described in Figure 1 by the expression of the Thy-1.1 congenic marker 45 days after transplantation. (a) Upper panels show proportions of Thy-1.1⁺ cells after they received either sTreg’s (n = 5) or irTreg’s (n = 5). Values indicate mean ± SEM of the absolute number of Thy-1.1⁺ cells. P < 0.05 between the two groups. Lower panels show the CD4 CD25 phenotype of cells gated on Thy-1.1⁺ cells. FSC, forward scatter. (b and c) The presence of injected Thy-1.1⁺ Treg’s was also evaluated in the spleen of grafted animals by immunohistochemistry. (b) Arrows indicate Thy-1.1–positive cells. (c) Each spleen was scored for the presence of injected Treg’s (Thy-1.1) or other T cells (Thy-1.2) in grafted mice receiving either sTreg’s (n = 8) or irTreg’s (n = 7). The y-axis indicates the intensity of staining ranging from 0 to 3. Each point corresponds to the histopathological score of an individual mouse; histograms indicate the mean histopathological score for each group. P < 0.05 between sTreg’s versus irTreg’s for Thy-1.1.

Figure 4

Comparison of in vitro and in vivo properties of cultured sTreg’s and irTreg’s. (a and b) 1 × 10⁶ sTreg’s or irTreg’s were labeled with CFSE and injected into semiallogeneic, nonirradiated [BALB/c × C3H]F1. At days 3, 10, and 28, splenocytes from grafted animals were collected. The injected Treg’s were detected in the spleen of grafted animals by the expression of the Thy-1.1 congenic marker. Cell proliferation was measured as the sequential loss of CFSE within the Thy-1.1⁺ cell population by flow cytometry (a) and by the count of the absolute number of Thy-1.1⁺ cells in the spleen (magnitude ×100) (b). (c) 1 × 10⁶ sTreg’s or irTreg’s were labeled with CFSE and injected into semiallogeneic irradiated [BALB/c × C3H]F1. At day 3, splenocytes from grafted animals were collected and cell division of donor cells was evaluated. (d) The in vitro suppressive properties of cultured Treg’s were tested after 3 weeks of culture. BALB/c CD25-depleted cells (effector T cells, white bar) were stimulated either by C3H APCs (left panel) or B6 APCs (right panel). Cells were cocultured with BALB/c sTreg’s (black bar) or irTreg’s (gray bar) in order to assess their suppressive activity. This figure is representative of three independent experiments.

Figure 5

GVL/GVT effects after control of GVHD by sTreg’s. (a) A20 leukemic cells were injected into irradiated mice at time of BMT. Results are presented as a Kaplan-Meier survival curve for mice receiving BM cells alone (dashed line, open squares, n = 5), BM cells supplemented with 0.5 × 10⁶ conventional T cells (open circles, n = 5), in addition to 0.5 × 10⁶ sTreg’s (filled squares, n = 5). P < 0.05 between the last two groups. GVL effect is also evaluated by the presence of A20 cells in the blood of mice detected by the coexpression of B220 and H-2K^d Ag, and also by their large size. (b) A similar experiment was reproduced using P815 cells. Results are presented as a Kaplan-Meier survival curve for mice receiving BM cells alone (dashed line, open squares, n = 5), or BM cells supplemented with 10 × 10⁶ conventional T cells (open circles, n = 5), in addition to 10 × 10⁶ sTreg’s (filled squares, n = 5). Because of severe morbidity due to the presence of tumor in all mice of the experimental group, the experiment was stopped at day 35.