Mechanisms of Graft-versus-Host Disease Prevention by Post-transplantation Cyclophosphamide: An Evolving Understanding

Abstract

Post-transplantation cyclophosphamide (PTCy) has been highly successful at preventing severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). The clinical application of this approach was based on extensive studies in major histocompatibility complex (MHC)-matched murine skin allografting models, in which cyclophosphamide was believed to act via three main mechanisms: (1) selective elimination of alloreactive T cells; (2) intrathymic clonal deletion of alloreactive T-cell precursors; and (3) induction of suppressor T cells. In these models, cyclophosphamide was only effective in very specific contexts, requiring particular cell dose, cell source, PTCy dose, and recipient age. Achievement of transient mixed chimerism also was required. Furthermore, these studies showed differences in the impact of cyclophosphamide on transplanted cells (tumor) versus tissue (skin grafts), including the ability of cyclophosphamide to prevent rejection of the former but not the latter after MHC-mismatched transplants. Yet, clinically PTCy has demonstrated efficacy in MHC-matched or partially-MHC-mismatched HCT across a wide array of patients and HCT platforms. Importantly, clinically significant acute GVHD occurs frequently after PTCy, inconsistent with alloreactive T-cell elimination, whereas PTCy is most active against severe acute GVHD and chronic GVHD. These differences between murine skin allografting and clinical HCT suggest that the above-mentioned mechanisms may not be responsible for GVHD prevention by PTCy. Indeed, recent work by our group in murine HCT has shown that PTCy does not eliminate alloreactive T cells nor is the thymus necessary for PTCy's efficacy. Instead, other mechanisms appear to be playing important roles, including: (1) reduction of alloreactive CD4⁺ effector T-cell proliferation; (2) induced functional impairment of surviving alloreactive CD4⁺ and CD8⁺ effector T cells; and (3) preferential recovery of CD4⁺ regulatory T cells. Herein, we review the history of cyclophosphamide's use in preventing murine skin allograft rejection and our evolving new understanding of the mechanisms underlying its efficacy in preventing GVHD after HCT. Efforts are ongoing to more fully refine and elaborate this proposed new working model. The completion of this effort will provide critical insight relevant for the rational design of novel approaches to improve outcomes for PTCy-treated patients and for the induction of tolerance in other clinical contexts.

Keywords: alloreactive T cells; graft-versus-host disease; haploidentical allogeneic hematopoietic cell transplantation; post-transplantation cyclophosphamide; regulatory T cells; skin allograft rejection; tolerance.

Figure 1

Proposed new working model of the mechanisms by which post-transplantation cyclophosphamide prevents graft-versus-host disease after allogeneic hematopoietic cell transplantation. After infusion into the recipient, host-alloreactive donor T cells become activated, highly proliferative, and more productive of inflammatory cytokines (depicted by the small red dots around the cells). Since PTCy, as an alkylator, is not a cell cycle-dependent cytotoxic agent, both host-alloreactive donor T cells (highly proliferative), and host-non-alloreactive donor T cells (lowly proliferative) are affected and there is some cell death in all subsets. It is unclear in vivo whether host-alloreactive donor T cells are more resistant to PTCy or just more rapidly reconstitute due to their ongoing rapid proliferation; survival (or death) after PTCy likely is due to a complex array of factors occurring in each individual cell and may be modulated by several interrelated factors (24). Between post-transplant days +3 and +7, there is continued high-level proliferation of host-alloreactive donor CD8⁺ effector T cells and reduced but continued proliferation of the surviving host-alloreactive donor CD4⁺ T cells (effector (Teff) and regulatory (Treg) cells). The reduced proliferation of host-alloreactive donor CD4⁺ effector T cells seems to be important for PTCy's efficacy as dosing schedules that fail to decrease host-alloreactive donor CD4⁺ effector T-cell proliferation are ineffective or suboptimal at preventing GVHD (79). Impaired functionality of surviving host-alloreactive donor effector T cells occurs early after PTCy (as early as post-transplant day +5) and appears to increase with time, as depicted with progressive decreases in cytokine production and changes in color of those subsets. This change in host-alloreactive donor effector T-cell functionality appears to be in part a direct (or at least rapid) effect of PTCy, but also is augmented by preferential reconstitution of donor CD4⁺ regulatory T cells between days +7 and +21, which suppress the host-alloreactive donor effector T cells, further contributing to their impaired functionality. Throughout this entire process, host-non-alloreactive donor T cells are not activated, maintain low-level proliferation, and consequently contract relative to host-alloreactive donor T cells. The dynamics of host-alloreactive vs. host-non-alloreactive donor T cells at later time points are unknown, but indirect data suggest that they may change with time and/or antigenic (e.g., viral) stimulation (80). This proposed working model is heavily based on experimental data in murine HCT (–23) and does not account for the integration of other immunosuppressant agents with PTCy as is commonly done clinically. However, the proposed model is expected to be imperfect and incomplete, and further study likely will increase its accuracy, comprehensiveness, and complexity.