Posttransplantation cyclophosphamide expands functional myeloid-derived suppressor cells and indirectly influences Tregs.

Posttransplantation cyclophosphamide (PTCy), given on days +3 and +4, reduces graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but its immunologic underpinnings are not fully understood. In a T-cell-replete, major histocompatibility complex-haploidentical murine HCT model (B6C3F1→B6D2F1), we previously showed that PTCy rapidly induces suppressive mechanisms sufficient to prevent GVHD induction by non-PTCy-exposed donor splenocytes infused on day +5. Here, in PTCy-treated mice, we found that depleting Foxp3+ regulatory T cells (Tregs) in the initial graft but not the day +5 splenocytes did not worsen GVHD, yet depleting Tregs in both cellular compartments led to fatal GVHD induced by the day +5 splenocytes. Hence, Tregs were necessary to control GVHD induced by new donor cells, but PTCy's impact on Tregs appeared to be indirect. Therefore, we hypothesized that myeloid-derived suppressor cells (MDSCs) play a complementary role. Functionally suppressive granulocytic and monocytic MDSCs were increased in percentages in PTCy-treated mice, and MDSC percentages were increased after administering PTCy to patients undergoing HLA-haploidentical HCT. PTCy increased colony-stimulating factors critical for MDSC development and rapidly promoted the generation of MDSCs from bone marrow precursors. MDSC reduction via anti-Gr1 treatment in murine HCT did not worsen histopathologic GVHD but resulted in decreased Tregs and inferior survival. The clinical implications of these findings, including the potential impact of expanded MDSCs after PTCy on engraftment and cytokine release syndrome, remain to be elucidated. Moreover, the indirect effect that PTCy has on Tregs, which in turn play a necessary role in GVHD prevention by initially transplanted or subsequently infused T cells, requires further investigation.

Murine allogeneic CAR T cells integrated before or early after posttransplant cyclophosphamide exert antitumor effects.

Relapse limits the therapeutic efficacy both of chimeric antigen receptor (CAR) T cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the 2 therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR T cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after posttransplant cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR T cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert antitumor effects. Indeed, allogeneic anti-CD19 CAR T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR T-cell treatment on day +9 or day +14 was safe but less effective, suggesting a limited therapeutic window. CAR T cells infused before PTCy were not eliminated, but surviving CAR T cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR T-cell expansion, higher phenotypic CAR T-cell activation, less CD4+CD25+Foxp3+ CAR T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR T cells and more cytotoxic CD8+ CAR T cells. This study provides mechanistic insight into PTCy's impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR T cells and allo-HCT that may compensate for deficiencies of each individual approach.

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.

Post-Transplantation Cyclophosphamide Uniquely Restrains Alloreactive CD4+ T-Cell Proliferation and Differentiation After Murine MHC-Haploidentical Hematopoietic Cell Transplantation.

Post-transplantation cyclophosphamide (PTCy) reduces the incidence and severity of graft-versus-host disease (GVHD), thereby improving the safety and accessibility of allogeneic hematopoietic cell transplantation (HCT). We have shown that PTCy works by inducing functional impairment and suppression of alloreactive T cells. We also have identified that reduced proliferation of alloreactive CD4+ T cells at day +7 and preferential recovery of CD4+CD25+Foxp3+ regulatory T cells (Tregs) at day +21 are potential biomarkers associated with optimal PTCy dosing and timing in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT model. To understand whether the effects of PTCy are unique and also to understand better the biology of GVHD prevention by PTCy, here we tested the relative impact of cyclophosphamide compared with five other optimally dosed chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) that vary in mechanisms of action and drug resistance. Only cyclophosphamide, methotrexate, and cytarabine were effective in preventing fatal GVHD, but cyclophosphamide was superior in ameliorating both clinical and histopathological GVHD. Flow cytometric analyses of blood and spleens revealed that these three chemotherapeutics were distinct in constraining conventional T-cell numerical recovery and facilitating preferential Treg recovery at day +21. However, cyclophosphamide was unique in consistently reducing proliferation and expression of the activation marker CD25 by alloreactive CD4+Foxp3- conventional T cells at day +7. Furthermore, cyclophosphamide restrained the differentiation of alloreactive CD4+Foxp3- conventional T cells at both days +7 and +21, whereas methotrexate and cytarabine only restrained differentiation at day +7. No chemotherapeutic selectively eliminated alloreactive T cells. These data suggest that constrained alloreactive CD4+Foxp3- conventional T-cell numerical recovery and associated preferential CD4+CD25+Foxp3+ Treg reconstitution at day +21 may be potential biomarkers of effective GVHD prevention. Additionally, these results reveal that PTCy uniquely restrains alloreactive CD4+Foxp3- conventional T-cell proliferation and differentiation, which may explain the superior effects of PTCy in preventing GVHD. Further study is needed to determine whether these findings also hold true in clinical HCT.