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.
View: Text | PDF
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

×

Figure 2

Optimally dosed PTCy neither induces pan–T cell depletion nor eliminates alloreactive T cells, but does reduce CD4+ T cell proliferation.

Options: View larger image (or click on image) Download as PowerPoint
Optimally dosed PTCy neither induces pan–T cell depletion nor eliminates...
Mice were transplanted as in Figure 1 and assessed at day +7, +21, or +200. All groups were allogeneic (Allo, B6C3F1→B6D2F1) unless specifically labeled as syngeneic (B6D2F1→B6D2F1). (A and B) T cell numbers in different tissue compartments at day (A) +7 or (B) +21. (C) High global T cell proliferation (Ki-67+) was observed at day +7. CD4+ T cell proliferation was reduced with increasing doses of PTCy, while CD8+ T cells continued to proliferate robustly despite treatment with 25 mg/kg PTCy. Only after 100 mg/kg PTCy was CD8+ T cell proliferation significantly reduced. (D–F) Regardless of PTCy treatment or dose, alloreactive Vβ6+ T cells persisted at levels similar to those seen in donors (dotted blue line; median values from Supplemental Figure 7: CD8+, 11.9%; CD4+CD25–Foxp3– conventional T cells [CD4+ Tcons], 10.5%) and even exceeded donor levels in the spleen and liver at day +7 after 100 mg/kg PTCy. These effects contrast with those in mice treated with TCD BM without PTCy in which Vβ6+ T cells were deleted over time. Combined results from (A, C, D) 3 (n = 6/group) or (B, E) 2 (n = 8/group except 100 mg/kg PTCy [n = 5]) independent experiments are shown. F shows data from all mice from Figure 1A surviving to day +200. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, 1-way ANOVA followed by the Holm-Šidák post hoc test using the vehicle-treated splenocyte group as the control. LN, peripheral lymph nodes.