Aldehyde dehydrogenase expression drives human regulatory T cell resistance to posttransplantation cyclophosphamide

Abstract

High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4(+) T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4(+) T cells with relative preservation of memory CD4(+)Foxp3(+) T cells. In particular, CD4(+)CD45RA(-)Foxp3(+hi) effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4(+)Foxp3(+) T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4(+) T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.

Fig. 1. CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ T cell reconstitution is favorable after alloBMT using PTCy as single-agent GVHD prophylaxis

Freshly frozen PBMCs prospectively collected at predetermined time points from donors (n = 16) and patients (n = 47) before and after alloBMT were immunophenotyped. (A) T cell counts from donors and patients. (B) Percentages and counts of CD4⁺CD25⁺Foxp3⁺ T cells. Data are shown in box-and-whisker plots representing the median, quartiles, and 1.5 times the interquartile range. Open circles represent outliers beyond this range. Statistical comparisons were performed using GEEs, and detailed results are presented in Table 2.

Fig. 2. eT_regs are preserved after alloBMT using PTCy and are relatively increased in patients with aGVHD

(A) Representative flow cytometric plots of healthy donors (n = 16), patients without aGVHD (n = 16), and patients with aGVHD (n = 31) using a gating strategy that distinguishes five subsets among CD4⁺ T cells. (B and C) Percentages and counts of eT_regs (B) and CD4⁺CD45RA⁻Foxp3^+lo Fr III cells (C). (D) Pie charts showing the relative distribution of CD4⁺ T cell fractions for donors, patients before transplant, and patients after transplant. Median values of each fraction were used and normalized to 100% between all fractions for each group. Statistical comparisons were performed using GEEs, and detailed results are presented in Table 2.

Fig. 3. CD4⁺Foxp3⁺ T cell fractions, but not naïve T_cons, are resistant to mafosfamide in MLR

Human CD3⁺CD4⁺ T cells were flow cytometrically separated and cocultured at a 1:1 ratio in triplicate with irradiated allogeneic CD3⁻ PBMCs. Cells were either untreated, pulse-treated with mafosfamide (Maf) on day 3, or treated with CsA or rapamycin (Rap) from days 0 to 7. (A) Total numbers of viable CD4⁺ T cell counts at day 7 of MLR. (B and C) Percentages (top) and total numbers (bottom) of various CD4⁺ T_con (B) and Foxp3⁺ (C) fractions. n = 10 for all groups except rapamycin (n = 4). Data are shown in box-and-whisker plots representing the median, quartiles, and range. *P < 0.05, **P ≤ 0.01, ***P≤0.001 compared with control groups by RM-ANOVAs followed by Holm-Sidak post hoc tests.

Fig. 4. CD4⁺Foxp3⁺ T cell fractions express ALDH1 after allogeneic stimulation

(A to D) Human MLR cultures were set up using immuno-magnetically bead-separated CD3⁺ and irradiated allogeneic CD3⁻ PBMCs ata 1:1 ratio in sextuplicate. (A) Representative histogramsof Aldefluor flow cytometric analysis from MLR day 7 using a gating strategy that distinguishes six CD4⁺ T cell subsets on the basis of the expression of CD25, CD45RA, and CD127. The threshold of Aldefluor positivity is based on the DEAB negative control. (B) Aldefluor positivity within various CD4⁺ fractions from unstimulated cells (n = 8) and cells stimulated in MLR for 3 or 7 days (n = 6). (C) Aldefluor positivity in MLR cultures that were either untreated or treated with mafosfamide, CsA, or rapamycin. The example shown is representative of six independent experiments. (D) ALDH1A1 expression by CD4⁺ T cell fractions for unstimulated cells or cells stimulated in MLR. The expression is shown relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Error bars show SEMs. Lines depict data from the same individual over time. (E) ALDH activity in PB CD4⁺ T cells retrieved from patients on day 3 after alloBMT (n = 6) and the PB or BM of their healthy BM donors (n = 2).

Fig. 5. ALDH inhibition with DEAB blocks proliferation and activation of CD4⁺ T cells in MLR and sensitizes T_regs to mafosfamide

Human MLR cultures were set up as in Fig. 4. (A to C) ALDH activity and alloantigen-induced activation and proliferation in MLR cultures were assessed by combining Aldefluor testing and the CellTrace Violet proliferation marker. Cells were first gated for viability before other analyses. n = 6. (A) Proliferation patterns of six CD4⁺ T cell subsets based on the expression of CD25, CD45RA, and CD127 for untreated cells or cells treated with 50 μM DEAB from days 0 to 3. The percentages of proliferating cells within each fraction are shown. (B) Expression of the early activation marker CD69 by Aldefluor⁺ cells of various CD4⁺ T cell fractions. (C) CD69 expression at days 3 and 7 of MLR for CD4⁺ cells that were either untreated or treated with DEAB from days 0 to 3. Lines connect paired samples. (D) DEAB was added to designated MLR cultures from days 0 to 3 followed by either no additional treatment or treatment with mafosfamide (7.5 μg/ml) on day 3. Cells were analyzed with the gating strategy using Foxp3 and CD45RA expression before assessment for viability. n = 6 for all treatments except DEAB alone (n = 4). *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001 by paired t tests (C) or RM-ANOVAs (D).

Fig. 6. GVHD protection conferred by PTCy is dependent on the presence of T_regs

NSG mice were irradiated (2.5 Gy) followed by infusion of 5 × 10⁶ human PBMC grafts or RPMI. Human PBMC grafts were flow cytometrically sorted to include all PBMCs or PBMCs selectively depleted of nT_regs and eT_regs. Designated groups received PTCy (100 mg/kg) on day +3. All animals were followed daily for survival. Combined results of four separate experiments are shown.