IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease

Abstract

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-gamma was required to initiate MSC efficacy. Recipients of IFN-gamma(-/-) T cells did not respond to MSC treatment and succumbed to GVHD. MSC, pre-treated with IFN-gamma, 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-gamma exposure, with increased IFN-gamma exposure leading to increased MSC suppression of GVHD. Activated MSC present a new strategy for preventing GVHD using fewer MSC.

Figure 1

MSC phenotype following rapid immunodepletion. When compared to isotype control, MSC stained positive for MHC class I (H-2k^d), the VLA complex marker CD29, the cell adhesion receptor CD44, and the hematopoietic stem cell marker Sca-1. MSC stained negative for MHC class II (I-A^d), macrophage cell surface markers (CD11b, CD14), B cell marker (B220), lymphocytes (CD2, CD3, CD4, and CD8a), and the hematopoietic stem cell markers Thy-1 and c-kit. Histograms represent consistent findings of more than 30 independent experiments.

Figure 2

The effect of timing of MSC administration on 40-day survival following induction of GVHD. Following lethal irradiation, B6 recipients underwent transplantation with allogeneic BALB BMC and splenocytes on day 0 (control, dashed line) resulting in 30% survival, (experiment repeated six times). In experimental groups (solid line), BALB MSC were administered on days 0 (A), 2 (B), 20 (C) or 30 (D). MSC administered on days 2 and 20 significantly increased survival by 10–60% and 10–50%, respectively (p<0.05). n=10 for each experimental group.

Figure 3

Effect of MSC dose escalation. Low (0.1 × 10⁶) and high (0.5 × 10⁶) doses of MSC were administered on day 2 (A, n=10) or day 20 (B, n=10). Both low and high doses led to a significant improved survival when given on either days 2 or 20. High dose MSC given on day 20 significantly increased survival to 85% (p=0.006).

Figure 4

GVHD scores of lung, colon, spleen, and skin. Recipients of 0.5 × 10⁶ (high) or 0.1 × 10⁶ (low) MSC and control recipients were killed for histological examination. Sections taken from lung, colon, and spleen were scored on a scale of 1–4 with 4 being the most severe changes consisting of tissue destruction observed in GVHD. Skin was scored on a scale of 1–3, with 3 being the most severe. This experiment was repeated three times (n=3 per group).

Figure 5

Requirement of IFN-γ for MSC mitigation of GVHD. Splenocytes from IFN-γ knockout mice were used to induce GVHD (solid line, n=10). Addition of 0.1 × 10⁶ MSC on day 2 failed to affect survival (dashed line), indicating MSC required IFN-γ to initiate their suppressive effects.

Figure 6

Activation of MSC with IFN-γ. Either untreated (A, dashed gray line, n=10) or IFN-γ-treated BALB MSC were administered on day 0. MSC treated with 5 U (solid gray line, n=10) or 50 U (dashed black, n=10) IFN-γ showed no effect on GVHD-related mortality when compared to untreated MSC. MSC treated with 500 U (solid black line, n=10) were significantly more effective than untreated MSC (p=0.006) and MSC treated with lower doses of IFN-γ. IFN-γ treatment appeared to have a direct effect on MSC (B), increasing expression of MHC class II. No detection of CD45 or CD11b populations were noted after IFN-γ treatment (gray line) when compared to pre-treatment (black line), indicating that IFN-γ treatment did not expand an immunoregulatory dendritic cell population, (experiment performed >10 times prior to each transplant). Following transplantation and the development of GVHD, circulating IFN-γ measured in the serum by ELISA (C) surpassed 500 U by day 7 with a gradual drop below 500 by days 21 and 30, suggesting there was sufficient circulating IFN-γ to activate MSC by day 7, but not after day 30, (each time print represents analysis of three to five recipients measured in duplicate).