Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia (original) (raw)
Ex vivo expansion of Treg’s. We first investigated, in a semiallogeneic BMT setting, the effect of Treg’s rendered specific for recipient allo-Ag’s (further referred to as sTreg’s) and of Treg’s rendered specific for third-party allo-Ag’s, which are thus irrelevant in the genetic combination of BMT used in this work (further referred to as irTreg’s). Because the irTreg population likely contained rare or no recipient-type, antigen-specific Treg’s (21), their use as a control should allow an evaluation of any potential nonspecific effect of Treg’s. Purified Treg’s from BALB/c mice were stimulated by allogeneic irradiated C3H APCs (sTreg’s) or B6 APCs (irTreg’s). In these conditions, numbers of Treg’s were dramatically increased by a factor of 4,500 for sTreg’s or 13,000 for irTreg’s, as previously described, using other genetic combinations and also maintained their CD4+CD25+CD62Lhigh phenotype after expansion (21). We tested cells that had been cultured for 42 days and that had not received stimulation by fresh APCs since day 35 for their in vivo functionality in a semiallogeneic BMT setting.
Comparison of the effect of sTreg’s and irTreg’s on clinical GVHD. In order to be able to analyze the effects of sTreg’s and irTreg’s on GVHD and immune reconstitution, we used a model of semiallogeneic BMT in which GVHD did not induce rapid mortality. When 9.5-Gy-irradiated [BALB/c × C3H]F1 mice were grafted with 5 × 106 bone marrow cells supplemented with 10 × 106 T cells from BALB/c mice, the mice developed severe clinical signs of GVHD, such as hunching, dull fur, skin lesions (Figure 1a, upper panel), weight loss (Figure 1b), and strong diarrhea but did not die during the first 45 days after transplantation. When 10 × 106 sTreg’s (BALB/c Treg’s cultured in the presence of C3H APC) were added to the inoculum, no clinical signs of GVHD were observed for the duration of the experiment (Figure 1a, lower panel). The mean weight curve of these mice was undistinguishable from that of control mice receiving BM cells alone and not developing GVHD. Thus in this experimental model, adding sTreg’s efficiently and durably prevented the occurrence of clinical GVHD. When mice received irTreg’s (BALB/c Treg’s cultured in the presence of B6 APC), the clinical outcome was different. After a short period of weight gain (days 5–10), weight curves declined rapidly and continually, as observed in mice of the GVHD control group (Figure 1b), although hunching, dull fur, skin lesions, and strong diarrhea were not observed (not shown).
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.
Mice were killed at day 45 after transplantation for histopathological studies, a time point at which all the mice of the GVHD control group developed strong clinical signs of GVHD. In control mice grafted with semiallogeneic BM cells and T cells, severe histological signs of GVHD were observed in the small and large bowel, skin, liver, and spleen. When sTreg’s were added to the transplant containing BM cells and T cells, no histological signs of GVHD (grade 0 or 1) were detected in the small bowel, skin, liver, and spleen Treg’s, although two of the eight mice exhibited mild signs of GVHD in the large bowel (Figure 1c and not shown). This confirmed, at the infraclinical level, the potent effect of sTreg’s for the prevention of GVHD. In contrast, in mice receiving irTreg’s instead of sTreg’s, histological analysis of target organs clearly showed signs of GVHD in the spleen for six out of seven mice, in the liver for four out of seven mice (Figure 1c), and in the large bowel but not in the skin for three out of seven mice (not shown). These histological signs of GVHD were of lower intensity than those seen in the GVHD control group but were significantly higher than in mice receiving sTreg’s or BM cells alone. Together, these results show that sTreg’s can efficiently prevent GVHD, whereas irTreg’s only provide a partial protection.
Enhanced immune reconstitution after semiallogeneic BMT with sTreg’s as compared with irTreg’s. The foregoing experiments were performed after infusion of similar proportions of total T cells and Treg’s to obtain a clinical effect. Since it has been demonstrated in vitro that Treg’s can mediate bystander suppression of conventional CD4+ or CD8+ T cells (15, 25), general immunosuppression could be a possible drawback of this strategy. We thus tested the effects of Treg injections on subsequent immune reconstitution by evaluating the number of total splenocytes, together with B and T cell reconstitution. In mice receiving BM cells alone, good immune reconstitution was achieved by 45 days after transplantation, with spleens containing about 80 × 106 cells, approximately 50% of which were B cells and 15% T cells. In contrast, control mice receiving BM cells plus T cells displayed strong lymphopenia characterized by profound splenic atrophy and an absence of both the B and T cell compartments (Figure 2), compatible with severe GVHD (26). Strikingly, adding sTreg’s efficiently prevented lymphopenia, since spleens contained about 150 × 106 cells, with approximately 55% B cells and 15% T cells. Interestingly, the numbers of B and T cells (both CD4+ and CD8+) were increased in comparison with mice receiving BM cells alone. Thus, adding sTreg’s in the transplant along with donor T cells favored immune reconstitution. In contrast, mice receiving irTreg’s displayed splenic atrophy compatible with an infraclinical GVHD and had slightly more total splenocytes and B and T cells (both CD4+ and CD8+ subpopulations) than mice of the GVHD control group. Together, these results revealed that sTreg’s favor immune reconstitution and irTreg’s do not.
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.
Increased number of sTreg’s as compared to irTreg’s in vivo. We next tested whether the differences observed between sTreg’s and irTreg’s in modulating GVHD and in promoting immune reconstitution were associated with their capacity to proliferate and/or to survive in vivo after their infusion into recipient mice. In our model, only Treg’s expressed the congenic marker Thy-1.1, which was thus used to trace them. When Thy-1.2 mice received 10 × 106 sTreg’s, 0.3 × 106 Thy-1.1+ cells were detected by flow cytometry in the spleen at day 45 after transplantation. Most of these Thy-1.1+ cells still expressed both CD4 and CD25 markers (Figure 3a). In contrast, when the same number of irTreg’s were used, only 0.006 × 106 Thy-1.1+ cells were detected in the spleen at day 45. This observation was confirmed by immunohistochemistry performed in the spleens and LNs of grafted mice. In mice treated with sTreg’s, numerous Thy-1.2+ T-cells were detected in the T cell zone of the spleen, attesting to the good T cell reconstitution in these mice protected from GVHD (Figure 3c) and consistent with the flow cytometry data (Figure 3a). In these mice, Thy-1.1+ Treg’s were easily detected in the spleen of protected mice (Figure 3b,c). In contrast, lower numbers of Thy-1.2+ T-cells were detected in the spleens of mice receiving irTreg’s (Figure 3c), attesting to poor T cell reconstitution and confirming flow cytometry data (Figure 3a). In these tissues, few or none of the Thy-1.1+–infused Treg’s were still present. Comparable results were also observed in the LNs of grafted animals (not shown).
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.
We tested whether the increased proportion of sTreg’s as compared with irTreg’s resulted from a difference in their proliferation to specific allo-Ag’s. We previously observed, using the same genetic combinations, that BALB/c sTreg’s, generated by a 2-week-culture in the presence of C3H APCs, proliferated very strongly when restimulated in vitro with C3H APCs, as compared with irrelevant B6 APCs (21). Here, we tested whether similar differences in the proliferation of cultured Treg’s to specific allo-Ag’s can also be observed in vivo. First, we addressed this point by injecting CFSE-stained Treg’s into nonirradiated, semiallogeneic recipient mice. The choice of using nonirradiated recipient mice was driven by previous reports showing that homeostatic expansion of Treg’s occurs in lymphopenic syngeneic hosts (27, 28). When C3H-specific cultured BALB/c Treg’s were transferred into [BALB/c × C3H]F1 recipients, they rapidly proliferated, with three to four rounds of division already evidenced by day 3 after infusion. In contrast, irTreg’s, which were in vitro selected for their capacity to respond to B6 APC, but not C3H APC, did not divide. An increased proportion of divided sTreg’s as compared with irTreg’s was still observed at day 14 but not at day 28 (Figure 4a). The presence of divided irTreg’s at day 28 was likely due to the extensive division of autoreactive Treg’s in the steady state, as we reported recently (29). Early proliferation of sTreg’s resulted in an increased absolute number of sTreg’s as compared with irTreg’s (Figure 4b). Then, we tested whether such observations could also be made in a BMT setting. Here again, the number of divided sTreg’s was increased at day 3 as compared with the number of irTreg’s when infused in lethally irradiated recipients (Figure 4c). These results likely explain the better potential of sTreg’s to durably control GVHD as compared with irTreg’s.
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.
Nevertheless, we also observed a partial beneficial effect against GVHD with irTreg’s, as illustrated by general clinical status and histopathological analysis observed in grafted mice (Figure 1). We thus tested whether irTreg’s could suppress activation of T cells stimulated by third-party allo-Ag’s in vitro. When BALB/c CD25– T cells were stimulated by irradiated allogeneic C3H splenocytes, adding sTreg’s strongly inhibited T cell proliferation, as previously described (21). Surprisingly, irTreg’s inhibited T cell proliferation as well. Similar findings were reproduced in another genetic combination (Figure 4d). These results indicate that irTreg’s specific for particular allo-Ag’s maintains suppressive activity on conventional T-cells stimulated by third-party allo-Ag’s.
GVL/GVT effects after prevention of GVHD using sTreg’s. We finally tested whether GVL/GVT effects could be maintained when sTreg’s were used to control GVHD. We first used the A20 leukemia cells of BALB/c origin. We thus developed a new model of GVHD in which lethally irradiated BALB/c mice were grafted with BM and T cells collected from B6 mice. In this experiment, sTreg’s were obtained after culture of purified B6 Treg’s stimulated by BALB/c-irradiated splenocytes. When mice received BM cells plus A20 cells, four out of five mice died from day 25 to day 42 from leukemia, as attested by the presence of A20 cells in the blood at days 15, 22 and 37. Mice receiving BM cells alone remained healthy (Figure 5a and not shown). Mice receiving BM and T cells together with A20 cells died with characteristic clinical signs of GVHD. Except for one mouse at day 22, A20 cells were never detected in the blood, attesting to an efficient GVL effect. In the experimental group in which sTreg’s were added to BM cells, T-cells, and A20 cells, four out of five mice were still alive at day 60. As compared with the two control groups, the presence of sTreg’s protected mice from lethal GVHD, whereas the GVL effect was maintained. Indeed, leukemic cells were not detected in these mice at days 15, 22, and 37, except for one mouse that died at day 34 (Figure 5a), probably from leukemia (not shown).
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-2Kd 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.
The GVT effect was also analyzed using another tumor cell line, the P815 mastocytoma, that we have previously used in an allogeneic BMT setting (30). Since P815 cells derived from DBA/2 mice, we tested the GVT effect in the B6→[B6 × D2]F1 combination of BMT, a situation in which sTreg’s significantly delayed GVHD (21). In the control group receiving BM and P815 cells, the presence of growing tumors at the site of injection was observed in all mice, and four out of five mice died between days 14 and 25. At the end of the experiment (day 35), a large tumor detected at the site of injection was still observed in the remaining mouse. In mice receiving allogeneic T cells in addition to BM and P815 cells, development of tumor cells was not detected at the site of injection, attesting to an efficient GVT effect. However, all mice developed clinical signs of severe GVHD, and two out of five died at days 19 and 29. As compared with these two control groups, the presence of sTreg’s in addition to BM cells, T cells, and P815 cells efficiently controlled GVHD. However, the GVT effect was lost, since all mice displayed tumors at the site of injection and four out of five mice died between days 19 and 28 (Figure 5b). The presence of tumors correlated with the detection of P815 cells in the blood (not shown). Inhibition by sTreg’s of GVT activity against P815 was also observed in another experiment in which mice received high numbers of P815 cells 2 days before irradiation.