Graft-vs-host disease (GVHD) is a major source of morbidity in hematopoietic stem cell transplantation (HSCT), limiting its application to treat nonmalignant hematopoietic diseases and to induce tolerance to solid organ transplants. Cell-based immunotherapies, especially regulatory T cells (Tregs), have shown promise in GVHD prevention and treatment. The TGF-β-dependent conversion of naïve T cells to Tregs is regulated by retinoic acid (RA), a vitamin A metabolite. Furthermore, aldehyde dehydrogenase (ALDH) is a key enzyme to metabolize Vitamin A to RA. As bone marrow-derived immunoregulatory CD8⁺TCR^– facilitating cells (FC) induce conversion of antigen-specific Treg, we studied the role of FC in GVHD prevention through conversion of effector T cells (Teff) to Treg. We found that FC from B6 (H2^b) mice expressed significantly higher levels of ALDH (50.8±7.7%; P<0.0001) in comparison with CD8⁺TCR⁺ T cells (0.19±0.08%). Importantly, the coculture of sorted splenic CD4⁺CD25^– T effector cells and bone marrow-derived FC stimulated with CpG-ODN resulted in conversion of 2.5±1.2% of CD4⁺CD25^– cells to CD4⁺CD25⁺ Treg and 25.4±5.0% of these cells were Foxp3 positive. Significantly less CD4⁺CD25⁺ cell conversion (0.31±0.29%; P=0.013) was observed when CD4⁺CD25^– T cells were cultured without FC. Importantly, the addition of LE540, a RA receptor inhibitor, abrogated conversion of CD4⁺CD25^– Teff to Treg. We also investigated the role of FC in GVHD prevention in vivo. BALB/c (H2^d) recipients were conditioned with 950 cGy and reconstituted with 10,000 purified donor B6 HSC plus sorted splenic B6 CD4⁺CD25^– T cells (12,500 to 50,000) with or without B6 FC in a 1:2 ratio. Recipients of HSCs plus CD4⁺CD25^– T cells expired with an average survival time 15.1±9.7 days and exhibited features of GVHD including body weight loss, diarrhea, hunched posture, and dehydration. In contrast, when FC were added, the survival time was significantly increased (>68.9±29.7 days; P=0.0002) and the clinical GVHD signs were alleviated. These findings indicate that FC prevent GVHD in allogeneic HSCT. We hypothesize that the effect of FC in GVHD prophylaxis is through their function to promote Treg generation. Moreover, Treg promotion by FC is mediated by RA that is metabolized by ALDH enzyme as evidenced by the fact that FC express a high level of ALDH and inhibition of the RA receptor abrogates Treg generation by FC.

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