IRX-2, a novel immunotherapeutic, protects human T cells from tumor-induced cell death

Abstract

IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8(+) T cells. MV induced significant CD8(+) T-cell apoptosis, as evidenced by Annexin binding (64.4+/-6.4%), caspase activation (58.1+/-7.6%), a loss of mitochondrial membrane potential (82.9+/-3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCRzeta chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation (P<0.005-0.05), prevented cytochrome c release and Bid cleavage, and concurrently restored the expression of Bcl-2, Bcl-xL, FLIP and Mcl-1 (P<0.005-0.01) in T cells. In addition, IRX-2 reversed MV-induced inhibition of the PI3K/Akt pathway. An Akt inhibitor (Akti-1/2) abrogated protective effects of IRX-2, suggesting that Akt is a downstream target of IRX-2 signaling. Thus, ex vivo pretreatment of CD8(+) T cells with IRX-2 provided potent protection from tumor-induced apoptosis. IRX-2 application to future cancer biotherapies could improve their effectiveness by bolstering T-cell resistance to tumor-induced immunosuppression.

Figures

Figure 1

Preincubation with IRX-2 decreases CD8+ Jurkat cell apoptosis induced by various agents. CD8+ Jurkat cells were pretreated with IRX-2 (1 : 3 dilution ~4 ng/ml or 90 IU/ml IL-2) for 24 h and then treated with tumor-derived MV (10 μg), CH-11 Ab (400 ng/ml) or staurosporine (1 μg/ml) for 3 h. (a) Caspase activation was detected by FITC-VAD-FMK staining and flow cytometry. The gate was set on CD8+ Jurkat cells. Dot plots shown are representative of five independent experiments. (b). CD8+ Jurkat cells were analyzed for Annexin V binding by flow cytometry. Dead cells (7-AAD+) were excluded, and the gate was set on 7-AAD− CD8+ Jurkat cells. (c) Mean percentage ± S.D. of FITC-VAD-FMK+ Jurkat T cells following incubation with various apoptosis-inducing agents. (d) Mean percentage ± S.D. of Annexin-V-positive/7-AAD− Jurkat cells following incubation with various apoptosis-inducing agents. Results shown in c and d are representative of three independent experiments (*P<0.05; **P<0.002). In each case, apoptosis levels±IRX-2 pretreatment are compared. (e, f) Antiapoptotic effects of IRX-2 are time and concentration dependent. CD8+ Jurkat cells were pretreated with IRX-2 (1 : 3 dilution) before MV incubation for increasing periods of time (0–24 h) or with increasing IRX-2 concentrations for a 24 h period, and subsequently treated with MV (10 μg) for 3 h. Activation of caspases was analyzed by measuring FITC-VAD-FMK binding by flow cytometry. (e) Time-course analysis. (f) Concentration-course analysis. Results are mean percentages±S.D. of four independent experiments

Figure 2

IRX-2 blocks MV-induced activation of caspases-3 and -7 and a loss of mitochondrial membrane potential (MMP) in CD8+ Jurkat cells. (a) Activation of caspases-3 and -7 in CD8+ Jurkat cells was assessed by flow cytometry for caspase-3/-7-FAM binding. Results of a representative experiment out of five experiments performed are shown. (b) Mean fluorescence intensity (MFI) of caspase-3/-7-FAM expression was determined in CD8+ Jurkat cells that were either untreated, incubated with 10 μg MV for 3 h or pretreated for 24 h with IRX-2 (1 : 3 dilution) and then incubated with MV for 3 h. CD8+ Jurkat cells were also co-incubated with MV and 20–100 μM of the pan-caspase inhibitor Z-VAD-FMK (zVAD) or co-incubated with MV and the anti-Fas neutralizing mAb ZB4 (10 μg/ml) (controls). Results are the mean MFI± S.D. of three independent experiments. (c) Western immunoblots showing caspase-3 activation in CD8+ Jurkat cells. The cells were either untreated, treated with IRX-2 (1 : 3 dilution) for 24 h (+ IRX), treated with MV (10 μg) for 3 h (+ MV 3 h) or 24 h (+ MV 24 h) or preincubated with IRX-2 for 24 h and then treated with MV (10 μg) for 3 or 24 h (+ IRX→MV). Whole-cell lysates of the cells were separated on SDS-PAGE and transferred to PVDF membranes for subsequent western blotting. Activation of caspase-3 is shown as a decrease in the inactive proform and the appearance of the active subunits p17 and p10. Results are representative of three independent western blots. (d) CD8+ Jurkat cells were analyzed by flow cytometry for a decrease in red fluorescence of the cationic dye, JC-1, indicating a loss of MMP. Percentages of JC-1-red-negative cells were determined in untreated CD8+ Jurkat cells, after 3 h treatment with MV (10 μg) alone or after 24 h pretreatment with IRX-2 (1 : 3 dilution) +MV. Cells treated with 50 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a protonophore that dissipates the H+ gradient across the inner mitochondrial membrane, were used as a positive control. The data are from a representative flow cytometry experiment out of five experiments performed. (e) Effects of MV alone, IRX + MV or MV in combination with Z-VAD-FMK or ZB4 on a loss of MMP in Jurkat cells. The data are means±S.D. of five independent experiments (*P<0.005; **P<0.002 compared to samples without IRX-2)

Figure 3

Preincubation with IRX-2 protects CD8+ Jurkat cells from MV-induced DNA fragmentation. (A) Fluorescent microscopy of CD8+ Jurkat cells that were either untreated (a), incubated for 24 h with IRX-2 alone (b) or MV alone for 24 h (c) or preincubated with IRX-2 for 24 h and subsequently treated with MV for 24 h (d) and then stained by the TUNEL method to reveal DNA strand breaks (red nuclei) indicative of apoptosis. (B) Percentage of TUNEL-positive CD8+ Jurkat cells in the above cocultures. A minimum of 300 CD8+ Jurkat cells were counted for each treatment group. Results are expressed as the mean percentages±S.D. of two independent experiments (*P<0.0002 compared to MV-treated sample). (C) Antiapoptotic effects of IRX-2 require a pretreatment of T cells. CD8+ Jurkat cells were untreated, treated with IRX-2 (1 : 3 dilution) for 24 h (+ IRX), MV for 3 h (+ MV), preincubated with IRX-2 for 24 h and then treated with MV (10 μg) for 3 h (+ IRX→MV) or first incubated with MV and then treated with IRX-2 (+ MV→IRX-2) or incubated with both agents simultaneously (+ MV and IRX) for 3 h 24 + + or h, respectively. Activation of caspases was analyzed by flow cytometry. Results are mean percentages±S.D. from a representative experiment out of three experiments performed (*P<0.002 compared to MV-treated sample)

Figure 4

Expression of proapoptotic and antiapoptotic proteins in the cytosol and mitochondrial fractions of T cells treated with MV or IRX-2 + MV. Jurkat cells were treated with MV and IRX-2 as indicated. The cells and were then subjected to subcellular fractionation to obtain cytosol and heavy membrane (HM) fractions. The subcellular fractions were resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting. β-Actin and Cox IV served as controls for equal loading for the cytosol and HM fractions, respectively

Figure 5

MV and IRX-2 modulate the expression of signaling molecules in the cytokine and TCR pathway and the activation of the Akt/PI3K pathway. CD8+ Jurkat cells were untreated or treated with IRX-2 (1 : 3 dilution) and MV in different combinations. Whole-cell lysates of cells from each treatment group were separated on SDS-PAGE and transferred to PVDF membranes for subsequent western blotting. (a) The expression levels of JAK3, phosphorylated and total STAT5, CD3ζ and FLIP was analyzed by probing the membrane with the specific Abs. Reprobing with β-actin Ab confirmed equal protein loading. The results shown are representative of four independent experiments. (b) The activation of Akt-1/-2 was analyzed by immunoblotting with Ser473-specific anti-phospho Akt mAb. Reprobing with a total Akt Ab confirmed equal protein loading. Results shown are from a representative experiment out of three experiments performed. (c) CD8+ Jurkat cells were preincubated with IRX-2 for 24 h or left untreated. Cells were then treated with the Akt inhibitor, Akti-1/2, at different concentrations (0–5 μM) for 1 h before the addition of MV for additional 3 h. The level of apoptosis was measured by FITC-VAD-FMK staining and flow cytometry. Results are mean percentages of positive cells±S.D. obtained in three independent experiments (*P<0.05; **P<0.01 compared to MV-treated sample without IRX-2 and Akt inhibitor)