Involvement of CD27/CD70 interactions in antigen-specific cytotoxic T-lymphocyte (CTL) activity by perforin-mediated cytotoxicity

Abstract

CD27 molecules are shown to be essential in the regulation of the death, activation and differentiation of T and B cells. However, the influence of CD27 on cytotoxic T-cell function remains obscure. Autologous EBV transformed B-cell lines (LCL), which highly express CD27 ligand CD70, here stimulated T cells and induced the cytotoxic T-lymphocyte (CTL) activity via T-cell antigen receptors (TCR). The cytotoxicity against LCL was diminished when anti-CD70 blocking MoAb was added initially in the culture. Resting T cells killed more CD70-transfected P815 cells than wild type P815 cells in the presence of anti-CD3 MoAb as measured by a 4-h 51Cr release assay, and the cytotoxicity of both of the cell populations completely disappeared in the presence of concanamycin A (CMA). The expression of the perforin by the LCL-induced CTL in the presence of anti-CD70 blocking MoAb was diminished as compared with that without the blockage of CD27/CD70 interactions. The CTL induced by LCL did not kill Fas-transfected WR cells. CD27 signalling in the T cells did not affect Fas ligand (FasL) mRNA expression, LAK activity and IFN-gamma synthesis in humans. Our data demonstrate that CD27/CD70 interactions enhance the cytotoxicity of CTL in the induction phase through enhancement of killing activity induced via the perforin-dependent mechanism, but not via the Fas/FasL system.

Fig. 1

(a)CD27-mediated cytotoxicity by resting T cells. E⁺ cells were preincubated with or without CMA (1000 nm) for 30 min The pretreatment cells were then tested for the cytotoxicity against wild/P815 (upper) or CD70/P815 (lower) (1 × 10⁴/well) in the presence of 0·2 µg/ml anti-CD3 MoAb at E : T ratios of 40 : 1, 20 : 1, and 10 : 1 in a 4-h ⁵¹Cr-release assay. □ Medium, ▪ anti-CD3 MoAb, anti-CD3MoAb + CMA. (b) Effects of CD27/CD70 interactions on cytotoxic T-lymphocyte activity. E⁺ cells (2 × 10⁶/well) were cocultured with autologous LCL (8 × 10⁵/well) in the presence (▪, ) of 5 µg/ml anti-CD70 MoAb or in the absence (□, ) of anti-CD70 MoAb for 12 days. The pretreated cells were tested for the cytotoxicity against autologous LCL (1 × 10⁴/well) at E : T ratios of 40 : 1 and 20 : 1 with (, ) or without (□, ▪) CMA (1000 nm) in a standard 4-h ⁵¹Cr-release assay. Results are expressed as the mean percentage of cytotoxicity ± SD of triplicates. Representative data from three experiments using different donors are shown. Statistical analysis: two-sample t-test; n.s., not significant; *P < 0·01; **P < 0·05.

Fig. 2

The expression of perforin in cytotoxic T lymphocyte. E⁺ cells were cocultured with autologous LCL in the absence of or in the presence of (b) 5 µg/ml of anti-CD70 MoAb for 12 days. The pretreated cells were stained with antiperforin-PE (——) after permeabilization. Isotype-matched PE-labelled mouse IgG MoAb was used as the negative control (·········). Representative data from three experiments using different donors are shown.

Fig. 3

Correlation of the expression of CD27 and perforin on CTL. E⁺ cells were cocultured with autologous LCL in the absence of or in the presence of 5 µg/ml of anti-CD70 MoAb for 12 days. E⁺ cells were stained with anti-CD27-biotin plus streptavidin-PerCP and anti-CD4-FITC or anti-CD8-FITC before and after cocultured with LCL. Then, these cells were permeabilized as described in Materials and Methods, and stained with antiperforin-PE. Similar data were obtained in three independent experiments.

Fig. 4

FasL-mediated cytotoxicity of CTL and effects of CD27/CD70 interaction on LAK activity. (a) The expression of Fas antigen on Fas/WR. Wild/WR and Fas/WR were stained with anti-Fas-FITC (——) and analysed by a FACScan. Isotype-matched FITC-labelled mouse IgG MoAb was used as the negative control (··········). (b) FasL-mediated cytotoxicity of CTL. E⁺ cells (2 × 10⁶/well) were cocultured with autologous LCL (8 × 10⁵/well) in the presence (▪) of or in the absence (□) of 5 µg/ml anti-CD70 MoAb for 12 days. The pretreated cells were tested for the cytotoxicity against autologous LCL in a 4-h ⁵¹Cr-release assay or against Fas/WR in a 12-h ⁵¹Cr-release assay at E : T ratios of 40 : 1 and 20 : 1. Fas/WR () and LCL () were mixed with anti-Fas MoAb (7C11) which induced apotosis of the Fas-expressing cells in a 12- h ⁵¹Cr-release assay for positive controls. Results are expressed as the mean percentage of cytotoxicity ± SD of triplicates. Similar results were obtained in three independent experiments. (c) IL-2 (25 ng/ml) activated E⁺ cells (2 × 10⁶/well) were cocultured with mock/300–19 or CD70/300–19 (8 × 10⁵/well) in the absence (□) of anti-CD70 MoAb or in the presence (▪) for 5 days. The pretreated cells were tested for the cytotoxicity against Fas/WR at E : T ratios of 40 : 1 and 20 : 1 in a 12-h ⁵¹Cr-release assay. Results are expressed as the mean percentage of cytotoxicity ± SD of triplicates. Similar results were obtained in three independent experiments. (d) The expression of FasL and β₂-microglobulin were analysed by RT-PCR as described in the Materials and Methods. Lane 1: no DNA, lane 2 : 24 h IL-2 activated E⁺ cells with mock/300–19, lane 3 : 48 h IL-2 activated E⁺ cells with mock/300–19, lane 4 : 24 h IL-2 activated E⁺ cells with CD70/300–19, and lane 5 : 48 h IL-2 activated E⁺ cells with CD70/300–19. Representative data from three independent experiments are shown. Statistical analysis: two-sample t-test; n.s., not significant; *P < 0·01.