Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2).
Human CD4(+)CD25(high)FOXP3(+) T regulatory cells (Treg) can suppress responder T cell (RC) functions by various mechanisms. In co-cultures of Treg and autologous activated RC, both cell subsets up-regulate the expression of granzymes and perforin, which might contribute to Treg-mediated suppression. Here, we investigate the sensitivity and resistance of Treg and RC to granzyme/perforin-mediated death. CD4(+)CD25(neg) RC were single cell-sorted from the peripheral blood of 25 cancer patients and 15 normal controls. These RC were carboxyfluorescein diacetate succinimidyl ester (CFSE) labeled and co-cultured with autologous CD4(+)CD25(high)FOXP3(+) Treg +/- 150 or +/-1,000 IU/mL of interleukin-2 (IL-2) to evaluate suppression of RC proliferation. In addition, survival of the cells co-cultured for 24 h and 5 days was measured using a flow-based cytotoxicity assay. Freshly isolated Treg and RC expressed granzyme A (GrA), granzyme B (GrB), and perforin. Percentages of positive cells were higher in cancer patients than controls (p < 0.01) and increased upon OKT3 and IL-2 stimulation. Treg, co-cultured with RC at 150 IU/mL of IL-2, no longer expressed cytotoxins and became susceptible to RC-mediated, granzyme/perforin-dependent death. However, in co-cultures with 1,000 IU/mL of IL-2, Treg became resistant to apoptosis and induced GrB-dependent, perforin-independent death of RC. When the GrB inhibitor I or GrB-specific and GrA-specific small inhibitory ribonucleic acids were used to block the granzyme pathway in Treg, RC death, and Treg-mediated suppression of RC, proliferation were significantly inhibited. Human CD4(+)CD25(high) Treg and CD4(+)CD25(neg) RC reciprocally regulate death/growth arrest by differentially utilizing the granzyme-perforin pathway depending on IL-2 concentrations.
Department of Pathology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
Malgorzata
M
Czystowska
Laura
L
Strauss
Christoph
C
Bergmann
Marta
M
Szajnik
Hannah
H
Rabinowich
Theresa L
TL
Whiteside
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015704:CD4 Antigens / Q000276:immunology; D002478:Cells, Cultured; D018920:Coculture Techniques; D005260:Female; D053804:Granzymes / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology*; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000276:immunology; D054353:Perforin; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology*; D055815:Young Adult
D015704:CD4 Antigens; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D034741:RNA, Small Interfering; D054353:Perforin; D053804:Granzymes