Alloantigen-reactive ITtregs Prevent GVHD While Preserving GVL In Mice

Background and Aim:Graft-versus-host disease is the most common and severe complication. Naturally occurring regulatory T cells (nTregs) may prevent graft-versus-host disease (GVHD). However, clinical application of nTregs has been severely hampered by their scarce availability and non-selective suppression. To overcome these limitations, we took an alternative approach to generate antigen-specific induced Tregs (iTregs), and tested their efficacy and selectivity in the prevention of GVHD in pre-clinical models of bone marrow transplantation (BMT), meanwhile we also observed the preservation of GVL effect.Method:We first selected HY as target antigen, because it is a naturally processed and ubiquitously expressed minor histocompatibility antigen (miHAg) with a proven role in GVHD and GVL effect. To generate HY-specific iTregs, CD4+CD25-T cells from TCR Tg Marilynn Foxp3gfp KI mice were stimulated with HY peptide in the presence of syngeneic T-cell depleted (TCD)-splenoctyes as APCs with TGF-β, IL-2and retinoic acid (RA), then tested their suppressive function in mixed lymphocyte reaction (MLR). We also studied HY-specific iTregs in three different BMT models in mice to understand the effect of iTregs in GVHD and then undermined the mechanism.To increase translational potential of our approach, we extended our studies to alloantigen-specific polyclonal iTregs. CD4+CD25-T cells from B6mice were stimulated with allogeneic DCs in the presence of TGF-β, IL-2and RA, then tested their suppressive function in MLR. We next examined whether alloreactive polyclonal iTregs were able to prevent GVHD while spare GVL activity by infusing A20lymphoma cells into BALB/c recipients upon BMT. Furthermore, we studied the mechanisms how iTregs can prevent GVHD while preserving GVL.Results:1.Ag-specific iTregs can be generated from CD4+CD25-T cells.2. HY-specific iTregs can strongly inhibit the proliferation of T cells in response to alloantigens in the presence of HY antigen in vitro. Only limited suppression can be observed in the absence of HY antigen.3. HY-specific iTregs were highly effective to prevent GVHD in male (HY+) but not female (HY-) recipients at relatively low ratios of Treg:Teff.4. HY-specific iTregs expanded in the recipients and suppressed the expansion and infiltration of donor T cells in spleen and liver.5. These BALB/c-iTregs could suppress the proliferation of B6T cells (donor type) in response to BALB/c alloantigens (recipient type) at high efficiency, but to FVB alloantigens (third party) at low efficiency in vitro.6. Allo-reactive iTregs generated with BALB/c DCs had superior efficacy than those generated with B10.BR DCs in preventing GVHD in BALB/c recipients.7. In B6â†'BALB/c BMT model, the majority of recipients with additional iTregs survived through the observation period with mild to modest GVHD but without tumor relapse.Conclusion:1. Large numbers of HY-specific iTregs can be generated in vitro, and these iTregs are highly suppressive to Teffs in response to allo-stimulation in vitro. Furthermore, these Ag-specific iTregs are highly effective in preventing GVHD by reducing the expansion, activation and infiltration of Teffs. The prevention of GVHD by iTregs is activation dependent.2.Allo-reactive iTregs were highly suppressive to Teff response to the alloantigens in vitro and in vivo in an antigen-dependent way. Allo-reactive Tregs can significantly reduce GVHD while preserving GVL activity.