182 Regulatory T Cells Are Resistant to Cyclophosphamide (Cy) Through Expression of Aldehyde Dehydrogenase (ALDH) Upon Allogeneic Stimulation

Track: Contributed Abstracts
Wednesday, February 13, 2013, 6:45 PM-7:45 PM
Hall 1 (Salt Palace Convention Center)
Christopher G. Kanakry, MD , Department of Oncology, The Johns Hopkins University, Baltimore, MD
Sudipto Ganguly, PhD , Department of Oncology, The Johns Hopkins University, Baltimore, MD
Christopher Thoburn , Department of Oncology, The Johns Hopkins University, Baltimore, MD
Brandy Perkins , Department of Oncology, The Johns Hopkins University, Baltimore, MD
Richard J. Jones, MD , Department of Oncology, The Johns Hopkins University, Baltimore, MD
Allan D. Hess, PhD , Department of Oncology, The Johns Hopkins University, Baltimore, MD
Leo Luznik, MD , Department of Oncology, The Johns Hopkins University, Baltimore, MD
High-dose, post-transplantation Cy (PT/Cy) is a novel and effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT).  We previously reported that regulatory T cells (Tregs) are resistant to Cy in allogeneic reactions; activated Tregs (CD4+CD45RA-Foxp3+hi) are relatively increased 30 and 60 days after PT/Cy in vivo and both naïve (CD4+CD45RA+Foxp3+) and activated Tregs are resistant to cytotoxicity induced by activated Cy (mafosfamide) in mixed lymphocyte reactions (MLR) in vitro.  However, the mechanisms of Treg resistance to Cy have not been understood.  As resistance of hematopoietic stem cells to Cy is dependent on expression of the ALDH-1 family (retinaldehyde dehydrogenases), we hypothesized that Tregs also might express ALDH as a potential mechanism of resistance to Cy.  To examine ALDH expression, we used Aldefluor as an established and reproducible flow cytometric-based method.  There was minimal to no Aldefluor-positivity in unstimulated Tregs.  However, after allogeneic stimulation in MLR, significant subsets of both naïve and activated Tregs were Aldefluor-positive.  These findings were confirmed by PCR which showed undetectable mRNA transcript levels in unstimulated cells but significant upregulation of ALDH1A1 in naïve and activated Tregs at Days 3 and 7 of MLR.  These ALDH+ cells persisted after mafosfamide treatment but were not present in significant amounts in the presence of cyclosporine or rapamycin (activated Tregs, p=0.027).  ALDH expression correlated with expression of the activation markers CD69 and CD25 and with proliferation.  The clinical relevance of these findings was confirmed by showing Aldefluor+CD4+ T cells in peripheral blood retrieved from patients on Day 3 post-alloBMT at the time of receiving PT/Cy.  To dissect functional implications of these findings, we explored pharmacological inhibition of ALDH using diethylaminobenzaldehyde (DEAB).  DEAB treatment drastically reduced activation and proliferation of CD4+CD25+ T cells in MLR (p=0.031 for both comparisons) and abolished Treg resistance to mafosfamide-induced cytotoxicity (activated Tregs, p<0.001).  Therefore, resistance of Tregs to Cy is dependent at least in part on ALDH expression in allogeneic reactions.  Given the role of ALDH as the rate-limiting step in retinoic acid (RA) biosynthesis and the crucial contributions of RA to both immune tolerance and CD4+ T cell function, pharmacologic manipulation of this pathway may open a new avenue for modulating alloreactivity after alloBMT.
See more of: Poster Session 1: Immune Reconstitution
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