Graft versus host disease (GVHD), mediated by donor T cells, is a significant source of morbidity and mortality following allogeneic stem cell transplantation. Mesenchymal stem cells (MSC) can successfully treat ongoing graft versus host disease, presumably due to their ability to suppress donor T cell proliferation. Little is known about the potential of MSC to prevent GVHD. Here we show that bone marrow-isolated MSC can suppress the development of GVHD if given after donor T cell recognition of antigen. IFN-c was required to initiate MSC efficacy. Recipients of IFN-c -/-T cells did not respond to MSC treatment and succumbed to GVHD. MSC, pre-treated with IFN-c, became immediately active and could suppress GVHD more efficiently than a fivefold-greater number of MSC that were not activated. When given at the time of bone marrow transplantation, activated MSC could prevent GVHD mortality (100% survival, p=0.006). MSC activation was dependent on the magnitude of IFN-c exposure, with increased IFN-c exposure leading to increased MSC suppression of GVHD. Activated MSC present a new strategy for preventing GVHD using fewer MSC. Key words: Mesenchymal stem cell Á GVH disease Á IFN-c See accompanying commentary by Dazzi and Marelli-Berg IntroductionAllogeneic hematopoietic stem cell transplants have the potential to play a significant curative role in the treatment of malignant and non-malignant hematopoietic disorders, autoimmune diseases, and immunological deficiencies, and in the induction of transplantation tolerance [1][2][3][4][5][6][7][8][9][10]. Widespread application of this therapeutic modality is limited due to the morbidity and mortality of graft versus host disease (GVHD), which affects 50% of stem cell transplant recipients [11][12][13][14][15][16]. While grafts highly matched to the recipient, young donors, donor/recipient sex match, and posttransplant immunosuppression are strategies used to reduce the risk of GVHD [17], thus far, the greatest preventative measure has been intentional underutilization of stem cell transplantation. Theoretically, strategies aimed at preventing GVHD would target early initiating factors either during the inflammatory milieu created in the wake of tissue damage from conditioning regimens [18,19] or during T cell antigen recognition and proliferation [20,21]. Once the efferent effector phase occurs, donor T cell-mediated destruction of host tissues occurs and preventive strategies are replaced with treatment regimens [19].Mesenchymal stem cells (MSC) have been used in the efferent phase of GVHD to successfully treat ongoing, acute, steroidresistant GVHD [22,23]. In contrast, when given at the time of BM transplant, for the prevention of GVHD, the incidence of grade III/ IV GVHD was not significantly improved [24], suggesting the [26,27,29,30]. In addition, MSC do not suppress the modest T cell proliferative response to recall antigens [31]. These findings suggest MSC may exert their optimal effects during the events surrounding larger scale T cell activation and proliferat...

Mesenchymal stem cells directly suppress ongoing immune responses. Through production of toleragenic cytokines, inhibition of lymphocyte proliferation, delivery of reparative and protective signals after reperfusion injury, and facilitation of hematopoietic chimerism, these cells demonstrate a wide-ranging potential for the development of multifaceted toleragenic strategies after transplantation.

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Mesecnhymal stem cells have been observed to inhibit graft versus host disease clinically, however the timing of infusion of these cells has not been well characterized. In previous studies, we have observed MSC to rescue lethally irradiated hosts that had received sub-optimal numbers of stem cells, permit the reduction of host conditioning while establishing equal or better levels of engraftment than the combination of intensive host conditioning and untreated HSC grafts, and enable xenogeneic engraftment (rat→ mouse) suggesting that administration of MSC in combination with an allogeneic transplant significantly alters host immune responses to enhance engraftment.. These findings could only be observed if MSC were given on the same day as the bone marrow stem cells. The purpose of this study was to determine to what extent MSC might affect donor immune responses involved in GVHD and to determine the optimal timing of these effects, in order to optimize the maximal beneficial effects of allogeneic stem cell grafts engineered with MSC. Since GVHD, mediated by donor T cells, requires host antigen presentation for initiation, we tested whether the effect of MSC occurred before or after this interaction. We used an established GVHD model in which 20x 10^6 Balb/c bone marrow cells in combination with 15 x 10^6 Balb/c splenocytes were administered to lethally irradiated B6 recipients to test whether MSC (1.0 x 105) could inhibit initiation of GVHD and to what extent these cells could mitigate or abrogate ongoing GVHD. In control animals, we observed donor T cell expansion to occur in the absence of B6 host T cells with corresponding destructive effects resulting in 100% lethality by day 48. Four experimental groups (n=10 per group) were used to test MSC administration at 4 time points: 1) on day 0 following co-culture with the graft to test whether cell contact between MSC and GVHD-producing splenocytes is necessary, 2) on day 2 to test whether donor T cell exposure to host antigen is required, 3) on day 20, to test the magnitude of effect of MSC on ongoing GVHD, and 4) on day 30 in which GVHD is severe and usually irreversible. Mice were weighed twice weekly and monitored daily for survival and clinical evidence of GVHD (ruffled fur, cachexia, alopecia, and diarrhea). When compared to survival of control animals, no statistically significant effect was observed when MSC were given with the stem cell grafts on day 0. Strikingly, survival was significantly increased to 60% when given on day 2 (p=0.01, log rank test), to 50% when given on day 20 (p=0.005), and to 40% for day 30 treated animals (p=0.009). Following MSC infusion, those animals that developed signs of GVHD such as ruffled fur and alopecia had dramatic improvement of these physical findings with most surviving animals experiencing a complete reversal to normal appearing fur. The observation that no effect occurred with MSC administered at the time of bone marrow transplantation suggests that the mechanism of effect requires host antigen presentation. We conclude that optimal timing for the infusion of donor specific MSC to abrogate GVHD begins after donor T cells have encountered host antigen and can be equally effective during early, late, and severe GVHD. Clinical strategies involving the use of allogeneic stem cell grafts engineered with MSC are likely to be more powerful in overcoming GVHD if the MSC infusion is administered in a delayed fashion.