Time point zero was defined as first measurement after addition of T cells. Viability Assay Fifteen thousand cells were seeded/96-well and incubated for 24 h. cells was independent of TRAIL as it was not inhibited by the addition of neutralizing anti-TRAIL antibodies or TRAIL-R2-Fc fusion protein. In accordance, knockdown (KD) of death receptors TRAIL-R1 or TRAIL-R2 in Colo357 cells had no effect on T cell-mediated cytotoxicity. However, KD of decoy receptor TRAIL-R4, which robustly enhanced TRAIL-induced apoptosis, interestingly, almost completely abolished the T cell-mediated lysis of these tumor EG00229 cells. This effect was associated with a reduced secretion of granzyme B by T cells and enhanced PGE2 production as EG00229 a result of increased expression level of synthetase cyclooxygenase (COX)-2 by TRAIL-R4-KD cells. In contrast, knockin of TRAIL-R4 decreased COX-2 expression. Importantly, reduced release of granzyme B by T cells cocultured with TRAIL-R4-KD cells was partially reverted by bispecific antibody [HER2xCD3] and led in consequence to enhanced lysis of tumor cells. Likewise, inhibition of COX-1 and/or COX-2 partially enhanced T cell-mediated lysis of TRAIL-R4-KD cells. The combination of bispecific antibody and COX-inhibitor completely restored the lysis of TRAIL-R4-KD cells by T cells. In conclusion, we uncovered an unexpected novel role of TRAIL-R4 in tumor cells. In contrast to its known pro-tumoral, anti-apoptotic function, TRAIL-R4 augments the anti-tumoral cytotoxic activity of T cells. 0.05 and ** 0.01. Open in a separate window Figure 3 Neutralization of TRAIL does not reverse the inhibitory effects of the TRAIL-R4 KD in Colo357 cells on V1 T cell-mediated cytotoxicity. (A) Ten thousand control KDand TRAIL-R4 KD [TR4 KD (a) or TR4 KD (b)] Colo357 cells per well were cultured in complete medium overnight. Cell Index (CI) was analyzed in 5 min steps over ~ 32 h. After overnight adherence, Colo357 cells were cultured with additional complete medium [control: green, TR4 KD (a): blue dark or TR4 KD (b): orange line] or positive control Triton-X-100 (black line). After 32 h, Colo357 cells were cocultured with two V1 T cell lines of different donors (#2, red lines and #3, purple lines) with an E/T ratio of 25:1 and 12.5 IU/mL rIL-2 in the presence of medium (red or purple lines) or 1 g/mL TRAIL mAb (dark green lines). Lysis of tumor cells was measured after normalization to 1 1 in one min steps for 18 h as indicated. The average of three replicates with SD is represented for each tumor cell line with effector cells of one representative healthy donor (#2) and one pancreatic cancer patient (#3) in independent experiments. (B) The culture conditions were similar to the ones described in (A) with the difference that only control KDand TRAIL-R4 KD [TR4 KD (a)] Colo357 cells were applied as target cells. After 32 h, Colo357 cells were cocultured with five different V1 T cell lines of different donors with an E/T ratio of 25:1 and 12.5 IU/mL rIL-2 in the presence of medium or 20 M zVAD-fmk. Each symbol represents a different donor. Black bars represent mean of the five independent experiments. Cytotoxicity was analyzed by Real-Time Cell Analyzer and fold change in Cell Index (CI) was calculated using formula as follows: 0.01 and *** = 0.001. (C) After culturing EG00229 104 Colo357 cells (green line) in complete medium for 30 h, impedance of these adherent tumor cells EG00229 expressed as CI was measured in 5 min steps. The CI was normalized to 1 1 shortly before the addition of substances as follows: Triton-X-100 to induce maximal lysis (black line), medium (green line), 1 g/mL PGE2 (light blue line), V2 T cell line (brown line) or V2 T cell line plus PGE2 (dark blue lines) with an E/T ratio of 25:1 in the presence of 12.5 IU/mL rIL-2. CI was then measured in 1 min steps over additional 26 h. The loss of tumor cell impedance and thus a decrease of CI correlated with lysis of tumor cells. The average of triplicates and standard deviation were calculated; one representative experiment. Several replications of the experiments using four different V2 T cell lines and five different V1 T cell lines of different donors in independent experiments were performed (right panel). The cytotoxicity of T cell lines against the indicated tumor cells in the presence of medium or PGE2 was calculated 4 h after addition of T.The granzyme B release can be drastically enhanced by an addition of bsAb leading to a strong enhancement of the V2 T cell-cytotoxicity (34, 53). receptors TRAIL-R1 or TRAIL-R2 in Colo357 cells had no effect on T cell-mediated cytotoxicity. However, KD of decoy receptor TRAIL-R4, which robustly enhanced TRAIL-induced apoptosis, interestingly, almost completely abolished the T cell-mediated lysis of these tumor cells. This effect was associated with a reduced secretion of granzyme B by T cells and enhanced PGE2 production as a result of increased expression level of synthetase cyclooxygenase (COX)-2 by TRAIL-R4-KD cells. In contrast, knockin of TRAIL-R4 decreased COX-2 expression. Importantly, reduced release of granzyme B by T cells cocultured with TRAIL-R4-KD cells was partially reverted by bispecific antibody [HER2xCD3] and led in consequence to enhanced lysis of tumor cells. Likewise, inhibition of COX-1 and/or COX-2 partially enhanced T cell-mediated lysis of TRAIL-R4-KD cells. The combination of bispecific antibody and COX-inhibitor completely restored the lysis of TRAIL-R4-KD cells by T cells. In conclusion, we uncovered an unexpected novel role of TRAIL-R4 in tumor cells. In contrast to its known pro-tumoral, anti-apoptotic function, TRAIL-R4 augments the anti-tumoral cytotoxic activity of T cells. 0.05 and ** 0.01. Open in a separate window Figure 3 Neutralization of TRAIL does not reverse the inhibitory effects of the TRAIL-R4 KD in Colo357 cells on V1 T cell-mediated cytotoxicity. (A) Ten thousand control KDand TRAIL-R4 KD [TR4 KD (a) or TR4 KD (b)] Colo357 cells per well were cultured in complete medium overnight. Cell Index (CI) was analyzed in 5 min steps over ~ 32 h. After overnight adherence, Colo357 cells were cultured with additional complete medium [control: green, TR4 KD (a): blue dark or TR4 KD (b): orange line] or positive control Triton-X-100 (black line). After 32 h, Colo357 cells were cocultured with two V1 T cell lines of different donors (#2, red lines and #3, purple lines) with an E/T ratio of 25:1 and 12.5 IU/mL rIL-2 in the presence of medium (red or purple lines) or 1 g/mL TRAIL mAb (dark green lines). Lysis of tumor cells was measured after normalization to 1 1 in one min steps for 18 h as indicated. The average of three replicates with SD is represented for each tumor cell line with effector cells of one Kl representative healthy donor (#2) and one pancreatic cancer patient (#3) in independent experiments. (B) The culture conditions were similar to the ones described in (A) with the difference that only control KDand TRAIL-R4 KD [TR4 KD (a)] Colo357 cells were applied as target cells. After 32 h, Colo357 cells were cocultured with five different V1 T cell lines of different donors with an E/T ratio of 25:1 and 12.5 IU/mL rIL-2 in the presence of medium or 20 M zVAD-fmk. Each symbol represents a different donor. Black bars represent mean of the five independent experiments. Cytotoxicity was analyzed by Real-Time Cell Analyzer and fold change in Cell Index (CI) was calculated using formula as follows: 0.01 and *** = 0.001. (C) After culturing 104 Colo357 cells (green line) in complete medium for 30 h, impedance of these adherent tumor cells expressed as CI was measured in 5 min steps. The CI was normalized to 1 1 shortly before the addition of substances as follows: Triton-X-100 to induce maximal lysis (black line), medium (green line), 1 g/mL PGE2 (light blue line), V2 T cell line (brown line) or V2 T cell line plus PGE2 (dark blue lines) with an E/T ratio of 25:1 in the presence of 12.5 IU/mL rIL-2. CI was then measured in 1 min steps over additional 26 h. The loss of tumor cell impedance and thus a decrease of CI correlated with lysis of tumor cells. The average of triplicates and standard EG00229 deviation were calculated; one representative experiment. Several replications of the experiments using four different V2 T.