Post-Transplantation Cyclophosphamide Modulates the Gut Microbiota Towards GVHD Protection

Post-transplantation cyclophosphamide (PTCy) reduces severe graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Additionally, the gut microbiota taxa and diversity have been associated with occurrence of GVHD and mortality in HCT patients. To understand better the impact of PTCy on the gut microbiota and the importance of the gut microbiota for PTCy's mechanisms of GVHD prevention, we used our T-cell-replete MHC-haploidentical murine HCT model (B6C3F1→B6D2F1). PTCy 25mg/kg/day or vehicle (PBS) was given on days +3/+4. PTCy treatment was associated with increased frequencies of bacterial taxa known to be protective against GVHD, such as ruminococcacae, clostridiales, lachnospiraceae , and blautia . This change coincided with increased levels of plasma butyrate, a metabolite known to protect against GVHD; butyrate levels positively correlated with percentages of regulatory T cells (Tregs) at day +21 in mice treated with or without PTCy. Many of the bacterial taxa modulations induced by PTCy were not affected by prophylactic treatment with levofloxacin. PTCy also led to faster numerical recovery of bacteria over time. To assess the mechanistic importance of PTCy modulation of the gut microbiota, we performed fecal microbiota transplants (FMT), in which mice did not receive post-HCT treatment themselves, but rather pre-HCT received FMT via oral gavage using stool from mice treated with PTCy or PBS. Results showed that mice receiving stool from PTCy-treated mice had significantly better weights at early post-HCT time points and a trend towards better overall survival. We repeated these experiments with germ-free (GF) recipient mice reconstituted with FMT via oral and rectal gavage using stool from donors transplanted under specific pathogen-free conditions; GF mice that received PTCy-modified microbiota also had significantly higher weights in the first few weeks after HCT. Nevertheless, PTCy maintained its efficacy in preventing GVHD in GF recipients that were not FMT reconstituted. Moreover, neither levofloxacin treatment from days 0 to +14 nor aggressive depletion of the gut microbiota via continual treatment using oral broad-spectrum antibiotic cocktail (imipenem, vancomycin, neomycin) starting 2 weeks prior to HCT worsened clinical or histopathologic GVHD in PTCy-treated mice. PTCy modulates the microbiome towards taxa protective against GVHD, which may contribute to Treg recovery via increased butyrate levels. Even so, antibiotic treatment does not hinder GVHD prevention by PTCy, and consequently concerns regarding antibiotic exposure perhaps may be of less concern for affecting post-HCT outcomes in PTCy-treated recipients. Overall, PTCy's modulation of the microbiota contributes to GVHD prevention, but is not necessary for the mechanisms by which PTCy prevents severe GVHD.