Efflux capacity and aldehyde dehydrogenase both contribute to CD8+ T-cell resistance to posttransplant cyclophosphamide.

Mechanisms of T-cell survival after cytotoxic chemotherapy, including posttransplantation cyclophosphamide (PTCy), are not well understood. Here, we explored the impact of PTCy on human CD8+ T-cell survival and reconstitution, including what cellular pathways drive PTCy resistance. In major histocompatibility complex (MHC)-mismatched mixed lymphocyte culture (MLC), treatment with mafosfamide, an in vitro active cyclophosphamide analog, preserved a relatively normal distribution of naïve and memory CD8+ T cells, whereas the percentages of mucosal-associated invariant T (MAIT) cells and phenotypically stem cell memory (Tscm) T-cell subsets were increased. Activated (CD25+) and proliferating CD8+ T cells were derived from both naïve and memory subsets and were reduced but still present after mafosfamide. By contrast, cyclosporine-A (CsA) or rapamycin treatment preferentially maintained nonproliferating CD25- naïve cells. Drug efflux capacity and aldehyde dehydrogenase-1A1 expression were increased in CD8+ T cells in allogeneic reactions in vitro and in patients, were modulated by common γ-chain cytokines and the proliferative state of the cell, and contributed to CD8+ T-cell survival after mafosfamide. The CD8+ T-cell composition early after hematopoietic cell transplantation (HCT) in PTCy-treated patients was dominated by CD25+ and phenotypically memory, including Tscm and MAIT, cells, consistent with MLC. Yet, MHC-mismatched murine HCT studies revealed that peripherally expanded, phenotypically memory T cells 1 to 3 months after transplant originated largely from naïve-derived rather than memory-derived T cells surviving PTCy, suggesting that initial resistance and subsequent immune reconstitution are distinct. These studies provide insight into the complex immune mechanisms active in CD8+ T-cell survival, differentiation, and reconstitution after cyclophosphamide, with relevance for post-HCT immune recovery, chemotherapy use in autologous settings, and adoptive cellular therapies.