Graft-versus-host disease (GVHD) is the principal cause of morbidity and mortality after hematopoietic stem cell transplantation. Even the most potent immunosuppressive agents often fail to control GVHD. Because of its unique cell-mediated approach, photopheresis represents an appealing alternative for the treatment of GVHD, particularly in its chronic form. Photodynamic therapy (PDT) using TH9402 (4,5-dibromorhodamine methyl ester), a photosensitizer, which upon activation with visible light, exhibits specific toxicity against activated T lymphocytes, while preserving resting T cells, has emerged as a potentially interesting alternative treatment modality for GVHD patients. However, the immunologic mechanisms involved in GVHD modulation by PDT still remain obscure. Since CD4+CD25+FoxP3+ regulatory T cells (Tregs) have an inhibitory effect on GVHD, we sought to determine the role of Tregs in the context of photopheresis using TH9402 for GVHD modulation. We first evaluated, using flow cytometry, the impact of PDT on activated T cells and Tregs obtained from steroid-refractory chronic GVHD patients. High (10 uM) and low (1,32 uM) TH9402 concentrations were compared to measure their ability preserve Tregs. Interestingly, low intensity TH9402 treatment demontrated particularly interesting features, resulting in the elimination of more than 90% of activated CD4+CD25+FoxP3- and CD4+CD44high T cells, while preserving 95% of CD4+CD25+ FoxP3+ cells (p<0.001; n=5 pts). The proportion of live CD4+CD25+ FoxP3+ cells increased from 51.8±5.2% to 89.0±5.9% (mean±SD; pre and post PDT, respectively; p<0.01) thus enriching the graft in Tregs. Next, we evaluated the ability of PDT treated mononuclear cells to inhibit the proliferation of untreated MNCs from cGVHD pts. The addition of PDT cells reduced the proliferation of cGVHD T lymphocytes by 41–76% (p<0.001, n=6 pts). This inhibitory effect disappeared following inhibition of Pgp-171 by verapamil, which promoted TH9402 intracellular retention and effector cell elimination upon light exposure, indicating that Tregs must not only be present but viable to exert their suppressive activity. In addition, higher levels of IL-10, but not TGF-β, were secreted when cGVHD cells were exposed to PDT-treated cells (34.7±5.1ng/mL) than when exposed to untreated cells (20.3±3.2ng/mL, p<0.05). Furthermore, the inhibitory effect decreased 5-fold when cGVHD cells were co-cultured for 6 days with CD4+CD25+ depleted PDT cells (p<0.001). Addition of anti-IL-10 monoclonal antibody (mAb) to the co-culture (PDT-treated cells with cGVHD cells) resulted in a 3-fold decrease in the inhibition of cGVHD cell proliferation mediated by PDT-treated cells (p<0.01) and a 1.5-fold decrease when anti-TGF-β mAb was added to the co-culture (p<0.05). In conclusion, our results demonstrate that TH9402 PDT not only eliminates activated T cells, but preserves Tregs, with the functional ability to inhibit residual alloreactive cGVHD cells. This inhibitory effect requires live effector Tregs, secretion of IL-10 and TGF-β expression. With such dual effector mechanisms, photopheresis using TH9402 should translate into improved treatment efficacy and enhanced quality of life for patients with chronic GVHD.

Author notes

Disclosure:Research Funding: Research support by Kiadis Pharma.