Posttransplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression
Lucas P. Wachsmuth, Michael T. Patterson, Michael A. Eckhaus, David J. Venzon, Ronald E. Gress, Christopher G. Kanakry
Lucas P. Wachsmuth, Michael T. Patterson, Michael A. Eckhaus, David J. Venzon, Ronald E. Gress, Christopher G. Kanakry
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Research Article Immunology

Posttransplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression

Abstract

Posttransplantation cyclophosphamide (PTCy) recently has had a marked impact on human allogeneic hematopoietic cell transplantation (HCT). Yet our understanding of how PTCy prevents graft-versus-host disease (GVHD) largely has been extrapolated from MHC-matched murine skin-allografting models that were highly contextual in their efficacy. Herein, we developed a T cell–replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) to test the putative underlying mechanisms: alloreactive T cell elimination, alloreactive T cell intrathymic clonal deletion, and suppressor T cell induction. In this model and as confirmed in four others, PTCy did not eliminate alloreactive T cells identified using either specific Vβs or the 2C or 4C T cell receptors. Furthermore, the thymus was not necessary for PTCy’s efficacy. Rather, PTCy induced alloreactive T cell functional impairment, which was supported by highly active suppressive mechanisms established within one day after PTCy that were sufficient to prevent new donor T cells from causing GVHD. These suppressive mechanisms included the rapid, preferential recovery of CD4+CD25+Foxp3+ regulatory T cells, including those that were alloantigen specific, which served an increasingly critical function over time. Our results prompt a paradigm shift in our mechanistic understanding of PTCy. These results have direct clinical implications for understanding tolerance induction and for rationally developing novel strategies to improve patient outcomes.

Authors

Lucas P. Wachsmuth, Michael T. Patterson, Michael A. Eckhaus, David J. Venzon, Ronald E. Gress, Christopher G. Kanakry

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Figure 1

In a T cell–replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1), optimally dosed PTCy prevents severe GVHD.

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In a T cell–replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1...
On day 0, 10- to 12-week-old recipient female B6D2F1 mice were irradiated (10.5 Gy) and transplanted with 10 × 106 TCD BM cells ± 40 × 106 splenocytes (splen) from 10- to 12-week-old female B6C3F1 donors. PBS vehicle or PTCy was administered on days +3 and +4. (A) PTCy doses of 10–50 mg/kg/d prevented lethality and resulted in steadily increasing weights and normalization of clinical scores after days +90–100. (B) PTCy doses of 1 or 5 mg/kg/d were ineffective in preventing fatal GVHD. (C and D) Mice receiving T cell–replete grafts with or without PTCy became full donor T cell chimeras by day (C) +7 to (D) +21. (E and F) Mice treated with the optimal dose of PTCy (25 mg/kg) in this model had only mild histopathologic evidence of GVHD at either day (E) +7 or (F) +21. Combined results from (A, B, D–F) 2 or (C) 3 independent experiments are shown. n = 10/group for A and B; n = 6/group for C and E; and n = 8/group for D and F except for the 100 mg/kg PTCy groups in D (n = 5) and F (n = 7). ***P ≤ 0.001; ****P ≤ 0.0001, 1-way ANOVA followed by the Holm-Šidák post hoc test.