Ex vivo inhibition of NF-kappaB signaling in alloreactive T-cells prevents graft-versus-host disease

Ex vivo induction of alloantigen-specific hyporesponsiveness through costimulatory blockade or exposure to immunoregulatory cytokines has been shown to suppress T-cell proliferation, IL-2 production, and graft-versus-host disease (GVHD) potential upon adoptive transfer. We hypothesized that targeting the intracellular NF-kappaB pathway—essential for T-cell activation and IL-2 transcription—could similarly induce alloantigen hyporesponsiveness and reduce GVHD. We demonstrate that treating mixed lymphocyte reaction PS-1145 (MLR) cultures with PS1145, a potent NF-kappaB inhibitor, induces T-cell hyporesponsiveness to alloantigen during both primary and secondary responses, while preserving responses to strong mitogenic stimuli in vitro. Importantly, GVHD lethality was significantly reduced in recipients of PS1145-treated T cells. When control or PS1145-treated MLR cells were transferred into syngeneic Rag(-/-) hosts, contact hypersensitivity (CHS) responses remained intact. However, GVHD lethality was restored, suggesting that lymphopenic expansion reverses alloantigen hyporesponsiveness. These findings highlight the NF-kappaB pathway as a key regulator of alloreactivity and suggest a novel small molecule-based strategy to prevent early post-transplant GVHD, while allowing recovery of donor T-cell function during lymphopenic expansion.