The clinical translation of alloantigen-specific regulatory T cells (AS-Tregs) is constrained by their low frequency in peripheral blood, limited purity, and functional instability following prolonged ex vivo expansion. To address these hurdles, we developed a strategy to isolate and expand a functional CD25+CD27+CD70− AS-Treg population.
Initially, Tregs were co-cultured with allogeneic dendritic cells in the presence of IL-15, IL-2, and retinoic acid. Proliferating CD25+CD27+CD70− AS-Tregs were subsequently FACS-sorted and expanded via polyclonal anti-CD3/CD28 stimulation with IL-15, IL-2, rapamycin, and TGF-β.
Over three weeks, this protocol yielded a 434-fold expansion, achieving >95% purity (CD25+FOXP3+) while maintaining a substantial degree (>60%) of FOXP3-TSDR demethylation, a hallmark of stable Treg lineage commitment. The expanded CD27+ AS-Tregs exhibited a robust immunoregulatory phenotype, characterized by high expression of Helios, CTLA-4, and CD39, as well as chemokine receptors associated with allograft and lymphoid tissue homing (CXCR3, CCR4, and CCR7).
Functionally, CD27+ AS-Tregs suppressed T cell proliferation in an antigen-specific manner, even after exposure to inflammatory cytokines, and showed a concentration-dependent chemotactic response to CXCL10 in vitro.
Frontiers in Immunology published a clinical update in Infectious Disease on 10 Jun 2026.
The item focuses on CD25+CD27+CD70− alloantigen-specific Tregs: promising stable immunotherapy for transplantation.
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