Post‐transplant cyclophosphamide limits reactive donor T cells and delays the development of graft‐versus‐host disease in a humanized mouse model

Abstract

Graft-versus-host disease (GVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT) that develops when donor T cells in the graft become reactive against the host. Post-transplant cyclophosphamide (PTCy) is increasingly used in mismatched allo-HSCT, but how PTCy impacts donor T cells and reduces GVHD is unclear. This study aimed to determine the effect of PTCy on reactive human donor T cells and GVHD development in a preclinical humanized mouse model. Immunodeficient NODscid-IL2Rγ null mice were injected intraperitoneally (i.p.) with 20 × 10 6 human peripheral blood mononuclear cells stained with carboxyfluorescein succinimidyl ester (CFSE) (day 0). Mice were subsequently injected (i.p.) with PTCy (33 mg kg −1 ) (PTCymice) or saline (saline-mice) (days 3 and 4). Mice were assessed for T-cell depletion on day 6 and monitored for GVHD for up to 10 weeks. Flow cytometric analysis of livers at day 6 revealed lower proportions of reactive (CFSE low ) human (h) CD3 + T cells in PTCy-mice compared with saline-mice. Over 10 weeks, PTCy-mice showed reduced weight loss and clinical GVHD, with prolonged survival and reduced histological liver GVHD compared with saline-mice. PTCy-mice also demonstrated increased splenic hCD4 + :hCD8 + T-cell ratios and reduced splenic Tregs (hCD4 + hCD25 + hCD127 lo ) compared with saline-mice. This study demonstrates that PTCy reduces GVHD in a preclinical humanized mouse model. This corresponded to depletion of reactive human donor T cells, but fewer human Tregs.

The P2X7 Receptor Antagonist AZ10606120 Does Not Alter Graft-Versus-Host Disease Development and Increases Serum Human Interferon-γ in a Humanized Mouse Model

“…Moreover, intracellular staining of human T cells at endpoint revealed the presence of both IFN-γ + CD4 + and IFN-γ + CD8 + T cells and that the relative proportion of IFN-γ + cells in either subset was not affected by AZ10. Notably, the proportion of IFN-γ + CD8 + T cells was increased compared IFN-γ + CD4 + T cells in AZ10-treated but not saline-treated mice, suggesting that AZ10 may be increasing the total number of IFN-γ + CD8 + T cells, which are likely to be predominantly memory CD8 + T cells based on other observations in this model [45]. This finding however, appears at odds with the emerging role of P2X7 activation in promoting the long-term survival of memory human CD8 + T cells in vitro [46], with P2X7 blockade predicted to reduce CD8 + T cells.…”

Post-transplant cyclophosphamide prevents xenogeneic graft-versus-host disease while depleting proliferating regulatory T cells

“…Specifically, the role of Treg on GVHD mitigation by PTCy has been intensively studied. 13 , 14 , 34 Here, we extensively studied the mechanisms of GVHD prevention by PTCy in three humanized mouse models of GVHD. Several important observations were made.…”

Insights into mechanisms of graft-versus-host disease through humanised mouse models

“…Likewise, T-cell proliferation could be detected as early as 7 days post-human T-cell injection in non-irradiated NBSGW mice, with the proliferation serving as a reliable marker of GVHD development [ 78 ]. Human T-cell proliferation can also be detected 6 days post-hPBMC injection in non-irradiated NSG mice [ 79 ]. Thus, these studies further suggest that GVHD can be induced shortly after transplantation in the absence of radiation-associated tissue damage.…”

“…Various studies have used small molecule inhibitors or biologics to study the role of T cells in humanised mice ( Table 2 ). Use of anti-proliferative agents such as cyclophosphamide, azacytidine, anti-thymocyte globulin, alemtezumab [anti-CD52 monoclonal antibody (mAb)], OKT3 (anti-CD3 mAb), CsA, immunoglobulin-like transcript 3 (ILT3)-Fc, IT1208 (anti-CD4 mAb) and anti-CD45RC mAb to reduce GVHD in these models has highlighted the importance of T-cell proliferation in disease progression [ 79 , 89 , 96–100 ]. Moreover, co-administration of human CD83-targeted T cells into humanised mice demonstrated a role for activated CD83 + CD4 + T cells including Th1 and Th2 cells but not Th17 cells, in GVHD progression [ 101 ].…”