Photodepletion differentially affects CD4+ Tregs versus CD4+ effector T cells from patients with chronic graft-versus-host disease.

Even the most potent immunosuppressive drugs often fail to control graft-versus-host disease (GVHD), the most frequent and deleterious posttransplantation complication. We previously reported that photodepletion using dibromorhodamine (TH9402) eliminates T cells from healthy donors activated against major histocompatibility complex-incompatible cells and spares resting T cells. In the present study, we identified photodepletion conditions selectively eradicating endogenous proliferating T cells from chronic GVHD patients, with the concomittant sparing and expansion of CD4(+)CD25(+) forkhead box protein 3-positive T cells. The regulatory T-cell (Treg) nature and function of these photodepletion-resistant cells was demonstrated in coculture and depletion/repletion experiments. The mechanism by which Tregs escape photodepletion involves active P-glycoprotein-mediated drug efflux. This Treg-inhibitory activity is attributable to interleukin-10 secretion, requires cell-cell contact, and implies binding with cytotoxic T-lymphocyte antigen 4 (CTLA-4). Preventing CTLA-4 ligation abrogated the in vitro generation of Tregs, thus identifying CTLA-4-mediated cell-cell contact as a crucial priming event for Treg function. Moreover, the frequency of circulating Tregs increased in chronic GVHD patients treated with TH9402 photodepleted cells. In conclusion, these results identify a novel approach to both preserve and expand Tregs while selectively eliminating CD4(+) effector T cells. They also uncover effector pathways that could be used advantageously for the treatment of patients with refractory GVHD.

A clinical-scale selective allodepletion approach for the treatment of HLA-mismatched and matched donor-recipient pairs using expanded T lymphocytes as antigen-presenting cells and a TH9402-based photodepletion technique.

Selective allodepletion is a strategy to eliminate host-reactive donor T cells from hematopoietic stem cell allografts to prevent graft-versus-host disease while conserving useful donor immune functions. To overcome fluctuations in activation-based surface marker expression and achieve a more consistent and effective allodepletion, we investigated a photodepletion process targeting activation-based changes in p-glycoprotein that result in an altered efflux of the photosensitizer TH9402. Expanded lymphocytes, generated using anti-CD3 and IL-2, were cocultured with responder cells from HLA-matched or -mismatched donors. Optimal results were achieved when cocultured cells were incubated with 7.5 muM TH9402, followed by dye extrusion and exposure to 5 Joule/cm(2) light energy at 5 x 10(6) cells/mL. In mismatched stimulator-responder pairs, the median reduction of alloreactivity was 474-fold (range, 43-fold to 864-fold) compared with the unmanipulated responder. Third-party responses were maintained with a median 1.4-fold (range, 0.9-fold to 3.3-fold) reduction. In matched pairs, alloreactive helper T-lymphocyte precursors were reduced to lower than 1:100 000, while third-party responses remained higher than 1:10 000. This establishes a clinical-scale process capable of highly efficient, reproducible, selective removal of alloreactive lymphocytes from lymphocyte transplant products performed under current Good Manufacturing Practice. This procedure is currently being investigated in a clinical trial of allotransplantation.