Vitamin C Stabilizes CD8iTregs and Enhances Their Therapeutic Potential in Controlling GvHD and Leukemia Relapse

Graft-versus-host disease (GvHD) remains a leading cause of non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT). The imbalance between T effector (Teffs) and regulatory cells (Tregs) is a defined characteristic of GvHD. Therefore, promoting Tregs for controlling GvHD has drawn an intense interest in preclinical and clinical studies of allo-HCT. Although CD4 Tregs (CD4⁺Foxp3⁺) have been shown to effectively prevent GvHD, they impaired graft-versus-leukemia (GVL) effect. Our lab has previously demonstrated that CD8 iTregs (CD8⁺Foxp3⁺) not only suppressed allogeneic T-cell responses, but also possessed the GVL activity themselves. However, instability of Foxp3 in CD8 iTregs is a major obstacle to translate this Treg population into clinic application.

Vitamin C has been reported to promote Foxp3 stability by accelerating its demethylation. In the current study, we asked whether vitamin C could stabilize CD8 iTregs and enhance their therapeutic potential in controlling GvHD. Addition of vitamin C in the culture significantly increased the generation of allo-reactive CD8 iTregs in vitro (Fig. 1A). These CD8 iTregs generated with vitamin C had strikingly increased demethylation on Foxp3 CpGs (Fig. 1B) and superior suppressive activity (data not shown) compared to control-treated CD8 iTregs. We further evaluated the capability of vitamin C-treated CD8 iTregs in preventing GvHD in allo-HCT. Using a MHC-mismatched murine BMT model, vitamin C-treated CD8 iTregs could significantly alleviate GvHD reflected by a marked reduction of recipient mortality (Fig. 1C) and clinical scores (data not shown).

To determine the Foxp3 stability in vivo, we analyzed the transferred CD8 iTregs and allogeneic Teffs in recipient spleens 7 days after allo-HCT. Vitamin C-treated CD8 iTregs (Ly5.2⁺) remarkably suppressed Teff (Ly5.1⁺) expansion and maintained higher Foxp3 expression compared to the control iTregs (Fig. 1D). To address a critical question whether vitamin C-treated CD8 iTregs can preserve the GVL activity, we performed a haploidentical transplant with a leukemia relapse model, and found that vitamin C-treated CD8 iTregs not only attenuated GvHD severity, but also prevented recipients from tumor relapse (Fig. 1E). In conclusion, our studies provide a rationale and mean to use stabilized CD8 iTregs with vitamin C for controlling GvHD and leukemia relapse in the clinic.

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