Reciprocal granzyme/perforin-mediated death of human regulatory and responder T cells is regulated by interleukin-2 (IL-2)

Abstract

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.

Fig. 1

Expression of GrA, GrB, and perforin in CD4⁺ CD25^high Treg and CD4⁺ CD25^neg RC. aRepresentative dot blots showing the expression of cytotoxins in unstimulated and stimulated Treg and RC obtained from the peripheral blood of a normal control (NC) and a patient with head and neck squamous cell carcinoma (HNSCC). Percentages of positive cells are indicated. b Mean percentages of positive cells ± SD for GrB, GrA, and perforin in Treg or RC of all NC and HNSCC patients enrolled in this study. Freshly harvested, single cell-sorted CD4⁺ CD25^neg or CD4⁺CD25^high T cells were examined immediately after isolation or were activated with OKT3 and IL-2 (150 IU/mL) for 24 h prior to staining with the labeled cytotoxin-specific Abs. Stained cells were studied by flow cytometry. Gates were set on CD4⁺CD25^high or CD4⁺CD25^neg T cells. The asterisks in b indicate significant differences (p<0.01) in the percentage of positive cells between fresh vs. stimulated T cell subsets

Fig. 2

Cell death and suppression function in early and late co-cultures of Treg with RC. CD4⁺CD25^high Treg and CD4⁺CD25^neg cells obtained from a HNSCC patient were either incubated separately or in co-cultures (1S:1RC ratios) for 24 h (a) or for 5 days (b) in the presence of either 150 IU/mL of IL-2 (left panels) or 1000 IU/mL of IL-2 (right panels). At the dose of 150 IU/mL of IL-2, Treg induced suppression of RC proliferation (C, left panel) but then underwent apoptosis, while RC remained viable in these co-cultures (B, left panel). At the doses of 1,000 IU/mL of IL-2, Treg were resistant to death and mediated apoptosis of RC (B, right panel), suppressing their proliferation (C, right panel). After 5-day co-culture (c), no suppression of RC proliferation is evident at the low IL-2 dose (left), while at the high IL-2 dose (right) a complete suppression of RC proliferation occurs. Results of a representative FLOCA of three independent experiments are shown

Fig. 3

Expression of GrA, GrB, or perforin measured in early (24 h) and late (5 days) co-cultures of Treg (S) with RC. CD4⁺ CD25^high Treg (a) and CD4⁺ CD25^neg RC (b) were either incubated separately or in co-cultures (1S:1RC ratios) in the presence of either 150 IU/mL of IL-2 (left panels) or 1000 IU/mL of IL-2 (right panels) for 24 h or 5 days. Cells were obtained from a patient with HNSCC. When cells were cultured alone, high dose of IL-2 increased the expression of granzymes and perforin both in Treg and RC. Treg had a higher overall expression of granzymes and perforin compared to RC. After the first 24 h of co-culture, dramatic changes in the expression of granzymes and perforin were observed and they intensified over time, depending on the IL-2 concentration. At 150 IU/mL of IL-2, Treg were losing the granule expression, while RC were increasing their expression. At 1000 IU/mL of IL-2, the situation was reversed. Representative data are shown out of three independent experiments performed

Fig. 4

Effects of GrB-specific or GrA-specific siRNAs on Treg suppressor function. a Treg (CD4⁺ CD25^high) were transfected with GrB-specific siRNA, GrA-specific siRNA, or irrelevant siRNA. Transfected Treg were stimulated with OKT3 and IL-2 as described in “Materials and methods” section, and incubated for 4 days. Expression of GrA and GrB, respectively, was determined by flow cytometry in fresh, non-transfected vs. siRNA-transfected Treg. The frequency of GrA⁺ or GrB⁺ cells is indicated in the upper right quadrantsb Suppression of RC proliferation after co-culture of non-transfected or transfected Treg with RC+1,000 IU/mL of IL-2 at different S:RC ratios. Note decreased levels of suppression mediated by Treg transfected with GrA- or GrB-specific siRNA. c Apoptosis of RC (percentage of 7-AAD⁺ RC) after co-culture of siRNA-transfected Treg with RC+1,000 IU/mL of IL-2. Co-cultures were established at different S:RC ratios. Note the decreased percent of 7-AAD+ RC in the presence of siRNA. The data are from a representative FLOCA of three independent experiments performed with T-cell subsets obtained from a HNSCC patient

Fig. 5

Effects of GrB-specific siRNAs on RC-mediated killing of Treg. a After co-culture of non-transfected RC with Treg+150 IU/mL of IL-2, 97% of RC expressed GrB and these RC were fully viable, while nearly all Treg were 7-AAD⁺. RC proliferation was not inhibited. b After transfection of GrB siRNA, only 4% of CD4⁺ CD25^neg RC expressed GrB. When these GrB-silenced RC were co-incubated with Treg +150 IU/mL of IL-2, both Treg and RC were 7-AAD^neg. These Treg completely suppressed RC proliferation (98%)