Mechanisms of CD8+ T-Cell Resistance to Post-Transplantation Cyclophosphamide (PTCy)

Despite the lymphocyte reducing effects of PTCy after hematopoietic cell transplantation (HCT), CD8+ T-cell numerical reconstitution is rapid and diverges from CD4+ T-cell reconstitution. To understand this rapid reconstitution and explore possible mechanisms of CD8+ T-cell resistance to PTCy, we used mixed lymphocyte culture (MLC) of healthy donor CD3+ T cells with irradiated HLA-mismatched CD3− peripheral blood mononuclear cells as previously published (Kanakry CG et al., STM, 2013). Accordingly, cells in MLCs were treated on day 3 with the active Cy analog mafosfamide (Maf) 7.5 ug/ml or from days 0-7 with cyclosporine (CsA) 600 ng/ml or rapamycin (Rap) 15 ng/ml. We found that all drugs reduced CD8+ T-cell numbers at day 7. However, the relative impact on CD8+ subsets differed by treatment. Maf preserved a similar distribution of memory and naive subsets compared with untreated MLCs, unlike CsA and Rap which preferentially spared naives. Percentages of mucosal associated invariant T (MAIT) and phenotypically stem cell memory CD8+ T cells were increased after Maf. Proliferation was reduced after Maf but persisted at lower levels, distinct from CsA or Rap, which abolished proliferation. Mechanisms of resistance to Cy include expression of aldehyde dehydrogenase (ALDH), the major in vivo detoxifying enzyme for Cy. Multidrug transporter (MDR) effluxing also may play a role. At day 3 of MLC, all assessed CD8+ T-cell subsets increased expression of ALDH1A1 from undetectable expression in donor T cells. In a Rhodamine-123 efflux assay, all CD8+ T-cell subsets also increased MDR activity at day 3 compared with donor cells. Both ALDH1A1 expression and Rhodamine-123 effluxing declined at day 7. Increased effluxing by CD8+ T cells also was seen in clinical HCT from donor and recipient day 0 to recipient day +3 blood samples. In MLC, pharmacologic inhibition of ALDH with diethylaminobenzaldehyde or MDR with PK11195 sensitized CD8+ T cells to Maf, with combined inhibition leading to death of nearly all cells, suggesting both mechanisms may contribute to CD8+ T-cell resistance. To dissect the underlying molecular mechanisms, we explored the relative impact of proliferative state and cytokines on MDR activity. Effluxing was largely restricted to those cells that either had not proliferated or had proliferated in MLC only a single generation; most T cells that had proliferated ≥2 generations were not effluxing. Exogenous IL-4, IL-7, or IL-15 reduced effluxing, but IL-2, IL-6, IL-9, IL-17, IL-21, or IL-23 did not affect it. Conversely, exogenous IL-2 modulated ALDH1A1 expression in a dose-dependent manner: low doses (0.1-1 IU/ml) increased expression, while high doses (≥100 IU/ml) reduced it. Our work provides insight into the impact of PTCy on CD8+ T-cell subsets and the biology of chemotherapy resistance pathways relevant for understanding the dynamics of CD8+ T-cell immune reconstitution after PTCy.