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.
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.


CONCLUSIONS
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.
Biagi
Ettore
E
Pediatric Department, University of Milano-Bicocca, San Gerardo Hospital, Milan, Italy. ettore.biagi@pediatriamonza.it
Di Biaso
Iolanda
I
Leoni
Veronica
V
Gaipa
Giuseppe
G
Rossi
Vincenzo
V
Bugarin
Cristina
C
Renoldi
Giuliano
G
Parma
Matteo
M
Balduzzi
Adriana
A
Perseghin
Paolo
P
Biondi
Andrea
A
D000293:Adolescent; D000328:Adult; D002450:Cell Communication; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism; D053450:Glucocorticoid-Induced TNFR-Related Protein; D006086:Graft vs Host Disease / Q000097:blood* / Q000188:drug therapy* / Q000276:immunology; D006801:Humans; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D019041:L-Selectin / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D017893:Photopheresis; D012333:RNA, Messenger / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects / Q000276:immunology / Q000378:metabolism* / Q000473:pathology*; D014018:Tissue Distribution; D014019:Tissue Donors; D016378:Tissue Transplantation; D014184:Transplantation, Homologous
C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053450:Glucocorticoid-Induced TNFR-Related Protein; D053645:Interleukin-2 Receptor alpha Subunit; D012333:RNA, Messenger; D017475:Receptors, Nerve Growth Factor; D018124:Receptors, Tumor Necrosis Factor; C491405:TNFRSF18 protein, human; D019041:L-Selectin; D017493:Leukocyte Common Antigens