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

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.

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

“…In their recent article Hadjis et al. investigated whether post-transplant cyclophosphamide (PT-CY) is unique in protecting against graft-versus-host disease (GvHD) by comparing it to multiple other “optimally dosed” chemotherapeutics ( 1 ). They concluded that PT-CY was superior to all other agents in ameliorating both clinical and histopathological GvHD.…”

“…report, a B6C3F1/Crl (H-2 b/k ) → B6D2F1/Crl (H-2 b/d ) mouse model was used. Recipient mice were conditioned with 10.5 Gy of single dose total body irradiation (TBI), were given 10 7 bone marrow cells along with 4x10 7 splenocytes (SC) on the same day (day 0), and received levofloxacin in their drinking water ( 1 ). We used a CB6F1 (H-2 d/b ) → CAF1/J (H-2 d/a ) model with recipient mice conditioned on day -1 with 6 or 10 Gy of TBI and infused 10 7 donor bone marrow cells along with 3x10 7 SC without prophylactic antibiotics ( 2 ).…”

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

“…39 The therapeutic efficacy of PTCy was previously attributed to the selective killing of alloreactive T cells that become activated and preferentially proliferate early post-HCT. 6 Although we have shown here and in prior studies that PTCy does broadly kill a substantial number of T cells, 6,7,14,16,17 this effect does not appear selective for alloreactive T cells. 6,14,16,17 Even so, the allure of the idea of cyclophosphamide selectively killing proliferating alloreactive T cells endures.…”

“…6 Although we have shown here and in prior studies that PTCy does broadly kill a substantial number of T cells, 6,7,14,16,17 this effect does not appear selective for alloreactive T cells. 6,14,16,17 Even so, the allure of the idea of cyclophosphamide selectively killing proliferating alloreactive T cells endures. We found that proliferating CD8 + T cells were reduced after PTCy but a sizable population remained, distinct from that seen with CsA or rapamycin treatment.…”

“…[3][4][5] Despite PTCy's recent burgeoning use worldwide, PTCy's immunologic impact and the immune landscape after PTCy are only beginning to be understood. [6][7][8][9][10][11][12][13][14][15][16][17] CD8 + T cells recover much more rapidly after PTCy than do CD4 + T cells, with the exception of CD4 + Foxp3 + regulatory T cells (T regs ) that are highly enriched after PTCy. 7,8,12,15 Early post-HCT in PTCy-treated patients, naïve CD8 + T cells rapidly acquire CD95-positivity, conferring a T-stem-cell-memory (Tscm)-like phenotype, and this population contributes substantially to T-cell reconstitution after PTCy.…”