Abstract
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.
Highlights
Allogeneic hematopoietic cell transplantation (HCT) is the only potentially curative therapy for many life-threatening hematologic diseases, but historically was not accessible to many patients for lack of a suitable human leukocyte antigen (HLA)-matched donor
We have shown in our B6C3F1!B6D2F1 MHChaploidentical murine HCT model that optimal dosing and timing of PTCy are associated with reduced proliferation of alloreactive CD4+Foxp3- conventional T cells at day +7 and preferential recovery of CD4+CD25+Foxp3+ Tregs at day +21 [5, 6]
We sought to identify the best dose of each alternative chemotherapeutic (MTX, BEN, PTX, VCR, ARA-C) when given on days +3 and +4 in this HCT model; we explored a wide range of doses, spanning what were thought to be below effective doses to near-lethal ranges based on prior studies [15, 16, 19, 45–56]
Summary
Allogeneic hematopoietic cell transplantation (HCT) is the only potentially curative therapy for many life-threatening hematologic diseases, but historically was not accessible to many patients for lack of a suitable human leukocyte antigen (HLA)-matched donor. To assess whether the biological effects of PTCy may be unique and provide further insights into our mechanistic understanding of the immunological mechanisms by which PTCy prevents GVHD, we investigated in our murine MHChaploidentical HCT model [5] the relative efficacy of five other chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) We chose these drugs as they represent an array of mechanisms of action, metabolism, and drug resistance (Table 1) and include methotrexate, which has a long history of clinical use for GVHD prophylaxis, and bendamustine, which has recently been explored as an alternative to PTCy; topoisomerase inhibitors and other alkylators beyond bendamustine were intentionally excluded over theoretical concern for therapy-related myeloid neoplasms in any clinical application of these studies
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