High Frequency of Alloantigen-Specific Regulatory T-Cells In Human Blood Allow Rapid Ex Vivo Expansion for Adoptive Therapy.

Abstract 3747

Evidence from animal studies has demonstrated that allogeneic regulatory T (Treg) cells can be used as effective therapeutic tools in the prevention of allograft rejection and graft-vs-host disease (GVHD). However, translating Treg-based therapies from animal models to human clinical trials requires methods for the isolation and expansion of functional Treg. Loss of function has been a concern for Treg expanded broadly with anti-CD3 and CD28 antibody-coated beads. Based on preclinical data in rodents, allospecific Tregs are anticipated to be more potent and exert more selective immunoregulation than polyclonal Treg in the prevention or treatment of GVHD. Currently, no effective approach has been established for selective expansion of human allospecific Treg ex vivo. In this study, we show the selective direct and indirect recognition of alloantigen by specific human CD4⁺CD25⁺CD127⁻ Tregs. CD4⁺CD25⁺CD127⁻ Treg were freshly isolated from normal human blood, labeled with CFSE and stimulated by MHC (HLA)-mismatched APC's, in the presence of IL-2 and IL-15 to amplify the proliferative responses, and sirolimus to suppress other T cells. The precursor frequency of the antigen specific Tregs was calculated by Limiting Dilution Analysis and found to be 1/49 -1/138 among all Tregs. The expanded antigen specific Tregs with low CFSE content were in general greater than 90% by days 12–15 in culture. CFSE-low Tregs were sorted by flow cytometry and exhibited complete suppression against CD25-negative CD4 T cell responses up to ratios of 1:100, in contrast CFSE-high Tregs were not suppressive. Ex vivo expanded antigen specific Tregs maintained high Foxp3, retained lymphoid homing receptor CD62L and chemokine receptor CCR7 expression, suggesting that they are functional and able to migrate to lymphoid tissue in vivo. Among the APC's tested, DC was found to be better stimulus with potent suppressive potential when compared to T-cell stimulated PBMC's. Specific indirect allorecognition was elicited when Tregs were stimulated with autologous APC's pulsed with allogeneic cell lysate in the presence of sirolimus, IL-15 and IL-2. Precursor frequency of antigen specific Tregs with indirect allorecognition was 100 fold lower than the precursor frequency of Tregs with direct allospecificity. Highly suppressive antigen specific Tregs show a duplication time of approximately 24 hours in the presence alloantigen, cytokines and sirolimus, and can be expanded in vitro by 400 fold in a 12 day period. This magnitude of expansion predicts the feasibility of conducting translational clinical trials. Our data may provide a platform for the selective expansion of Tregs against minor histocompatibility antigens to prevent GVHD while sparing graft-vs.-leukemia effects.