Posttransplantation cyclophosphamide prevents graft-versus-host disease by inducing alloreactive T cell dysfunction and suppression
Lucas P. Wachsmuth, … , Ronald E. Gress, Christopher G. Kanakry
Lucas P. Wachsmuth, … , Ronald E. Gress, Christopher G. Kanakry
Published March 26, 2019
Citation Information: J Clin Invest. 2019;129(6):2357-2373. https://doi.org/10.1172/JCI124218.
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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 3

Alloreactive 2C TCR+ CD8+ T cells proliferate and preferentially expand despite PTCy, while nonalloreactive 2C TCR+ CD8+ T cells remain phenotypically naive, minimally proliferative, and consequently contract.

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Alloreactive 2C TCR+ CD8+ T cells proliferate and preferentially expand ...
(A–C) Splenocytes from 2C TCR+ B6C3F1 transgenic mice were admixed with WT B6C3F1 splenocytes to manufacture grafts in which approximately 8% (acceptable range 7.2%–8.8%) of infused CD8+ T cells were 2C TCR+ (Vβ8.1/8.2+1B2+) and then used as donor splenocytes to transplant mice as shown in Figure 1. (A) GVHD remained universally severe and fatal in vehicle-treated mice, while PTCy remained effective. (B) Left: high-level proliferation (Ki-67+) of 2C TCR+ T cells was observed in vehicle-treated mice at days +3 and +7. This proliferation was not reduced by 25 mg/kg PTCy on days +3 and +4. Right: 2C TCR+ T cells expanded from the 8% (dotted blue line) percentage in the allograft to dominate the T cell repertoire at day +7. This relative expansion was not blocked by PTCy on days +3 and +4. (C) 2C TCR+ T cells (left) persisted and (right) remained proliferative at day +200. The proliferation data shown are for the liver. (D–F) An admixed graft approach identical to that used in A–C was used, except that the model was B6→B6C3F1. 2C TCR+ T cells, nonalloreactive in this model, (D) proliferated at low levels, in contrast with alloreactive Vβ3+ and Vβ5+ T cells, (E) maintained a naive phenotype, and (F) consequently contracted by day +7 from the 8% (dotted blue line) percentage in the allograft. Combined results from 2 independent experiments are shown. n = 8/group. *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001, unpaired t test with Welch’s correction for the mice treated with or without PTCy assessed on day +7.