Novel Photodepletion Strategy to Preserve and Expand Tregs While Eliminating CD4+ Effector T Cells From Patients with Chronic Graft-Versus-Host Disease
Authors: Bastien Jean-Philippe , Krosl Gorazd , Therien Cynthia , Rashkovan Marissa , Scotto Christian , Cohen Sandra , Allan David S. , Hogge Donna E. , Egeler R. Maarten , Perreault Claude , Roy Denis-Claude 
Published: October 2019

Abstract 353

Even the most potent immunosuppressive drugs often fail to control graft-versus-host disease (GVHD), the most frequent and deleterious post-transplantation adverse condition. The development of a strategy to eliminate alloreactive T cells and spare regulatory T cells (Tregs) would provide a direly needed therapeutic option for patients with refractory GVHD. We previously reported that photodepletion using dibromorhodamine (TH9402) eliminates T cells from healthy donors activated against major histocompatibility complex (MHC)-incompatible cells and spares resting T cells. In the present study, we identified novel photodepletion conditions (1.32 μM TH9402, 45 minutes incubation, and 5 J/cm2 light delivery at 514 nm) selectively eradicating 60–90% of endogenous proliferating host T cells as measured by 3H-thymidine incorporation, and CD4+CD25+FOXP3- effector memory phenotype cells (p<0.001) from chronic GVHD patient cells, with concomitant sparing of the majority of CD4+CD25+FOXP3+ Tregs.

All GVHD patients tested (n=10) had severe extensive chronic disease and were refractory to at least 2 immunosuppressive agents. Preservation of Treg function after photodepletion was confirmed by demonstrating reduction in the proliferation of GVHD T lymphocytes by 66.6±7.1% to 75.8±7.0% (mean±SEM; p<0.001, n=6) after their exposure to photodepletion lymphocytes at ratios of 8:1 to 1:1, respectively. The fact that immunomagnetic depletion of CD4+CD25+ T cells from lymphocytes exposed to photodepletion abrogated the inhibitory activity of both GVHD patient and healthy donor cells indicated that these Tregs harbored a CD4+CD25+ phenotype. Moreover, Treg activity was recovered upon CD4+CD25+ cell repletion. Treg survival to photodepletion relied on P-glycoprotein induced efflux of TH9402 since inhibition of this membrane transporter by verapamil (50 μM) induced TH9402 accumulation and Treg demise upon illumination. We found that IL-10 secretion increased when photodepletion cells were cultured with GVHD untreated cells (p<0.01) and anti-IL-10 monoclonal antibodies (mAbs) blocked their suppressive activity. TGF-β was not found to be implicated in the suppression of proliferation. Allowing cell-cell contact between photodepletion exposed lymphocytes and cGVHD cells in 3- to 6-day cultures led to doubling of Treg numbers (p<0.01, n=5) while separating photodepletion treated and untreated GVHD cells abrogated both IL-10 secretion and Treg suppressive activity. Additionally, blocking CTLA-4 ligation with anti-CTLA-4 mAbs abrogated Treg function and prevented the generation of Tregs ex vivo (p<0.05), thus identifying CTLA-4-mediated cell-cell contact as a crucial priming event for Treg function.

This increase in Tregs was not caused by increased proliferation as measured by EdU labelling. Exposing isolated CD4+CD25- cells to TH9402 photodepletion itself also failed to increase the number of FOXP3 expressing cells. However Treg numbers increased when CD4+CD25- cells were incubated with CD4+CD25+ cells previously exposed to photodepletion, suggesting that photodepletion cells promote the acquisition of Treg features in CD4+CD25- T cells.

This photodepletion strategy was then evaluated in 5 patients suffering from refractory chronic GVHD. The patients had their cells collected by lymphopheresis, exposed to photodepletion and reinfused back into them on a weekly basis. Interestingly, the frequency of circulating Tregs in cGVHD patients undergoing TH9402 photodepletion increased in comparison to healthy donors (p<0.05), with a doubling in their numbers in comparison to pre-treatment levels (p<0.05) as early as 2 weeks after initiating treatment and these patients' conditions improved. In conclusion, these results identify a new 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.

Disclosures:

Egeler: Kiadis Pharma: Employment. Roy:Kiadis Pharma: Research Funding.