Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+CD62L+ functional regulatory T-cells in patients with graft-versus-host disease

Abstract

Background: Extracorporeal photochemotherapy (ECP) produces clinical improvements in refractory/resistant graft-versus-host disease (GvHD). Immunological mechanisms of ECP are still under investigation.

Methods: We have evaluated the changes in frequency and immunophenotype of circulating regulatory T cells (T-regs) in 10 patients undergoing allogeneic hematopoietic stem cell transplantation, receiving ECP for acute (n=4) or chronic (n=6) GvHD. T-regs were monitored for expression of surface CD4, CD25, GITR, CD45RO, CD62L and intracytoplasmic Foxp3. T-regs were sorted by fluorescence-activated cell sorting to perform functional assays by interferon (IFN)-gamma enzyme-linked immunospot and real-time quantitative polymerase chain reaction (RQ-PCR) to measure Foxp3, transforming growth factor (TGF)-beta, and interleukin (IL)-10 mRNA.

Results: ECP was accompanied by a significant increase of CD4+CD25+ T-regs after six procedures, increasing from 8.9% to 29.1% of total CD4 (P<0.05), with a simultaneous increase of glucocorticoid induced tumor necrosis factor receptor expression on CD4+CD25+ cells (from 15% to 40.8%, P<0.05). This increase was sustained after 12 procedures. T-regs expressed high levels of CD62L, CD45RO, and Foxp3. Sorted CD4+CD25+ T-regs were potently inhibitory toward the CD4+CD25- fraction, when matched with an allogeneic target (IFN-gamma secretion was reduced by 79%). Trans-well experiments showed that cell-to-cell contact was necessary to exert inhibitory activity. RQ-PCR revealed a significant expression of Foxp3 in CD4+CD25+ T-regs, but there was virtually no detection of TGF-beta and IL-10. GvHD improved in all patients, allowing tapering or discontinuation of immunosuppressive drugs.

Conclusion: Our study shows a time correlation between ECP and increasing percentages of circulating functional T-regs. Albeit suggestive, our results need to be confirmed on larger series to determine the actual role of T-reg in mediating the clinical effect of ECP.