Mechanisms of T-cell protection from death by IRX-2: a new immunotherapeutic

Abstract

Objectives: IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV).

Methods: Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured.

Results: IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells (p < 0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection.

Conclusions: This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.

Figures

Fig. 1

TMV-induced apoptosis of CD8+ Jurkat-cells is caspase-dependent and modulates the pro- and anti-apoptotic protein expression in T cells. a CD8+ Jurkat cells were pre-incubated with different caspase inhibitors (10 μM) for 1 h and were left untreated or were treated with TMV (10 μg) for 4 h. Cells were analyzed for annexin V binding by flow cytometry. Dead cells (7-AAD+) were excluded, and the gate was set on 7-AADneg CD8+ Jurkat cells. Results are mean percentages ± SD of annexin V+/7-AADneg cells from three independent experiments. b Activated peripheral blood (PB) CD8+ cells were pre-incubated with IRX-2 (at 1:3 dilution) for 24 h and then treated with 10 μg TMV for additional 24 h. Expression levels of different pro- and anti-apoptotic protein were measured by quantitative flow cytometry. The data are the mean fluorescence intensity (MFI) ± SD of pro- and anti-apoptotic proteins in TMV- and IRX-2-treated activated primary CD8+ cells and were obtained in 3 independent experiments (*p < 0.005 compared to MV-treated samples and +, p < 0.001 compared to untreated samples). c Representative histograms showing the expression (MFI) of pro- and anti-apoptotic proteins in MV- and IRX-2-treated activated primary CD8+ cells

Fig. 2

Akt involvement in IRX-2 protective effects. CD8+ Jurkat cells were pre-incubated with IRX or left untreated. Then cells were treated with the Akt inhibitor Akti-1/2 (5 μM) for 1 h prior to the addition of TMV for additional 3 h. Activation of caspases in these cells was measured by flow cytometry (a) and Western blots (b). Results shown in a and b are representative of 3 independent experiments. Influence of the Akt inhibitor on the expression of pro- and anti-apoptotic proteins was measured by flow cytometry (c). The data are mean fluorescence intensities (MFI) ± SD of 3 independent experiments (+p < 0.05 in comparison to untreated samples without Akt inhibitor; *p < 0.02 in comparison to samples +IRX-2 and TMV without the Akt inhibitor)

Fig. 3

Cycloheximide abrogates IRX-2-induced protection from apoptosis. CD8+ Jurkat cells were pre-incubated with IRX-2, with CHX alone or with IRX-2 + CHX (0.1 μg/mL) for 24 h and then incubated with TV for 4 h and analyzed by quantitative flow cytometry. a Mean percentages ± SD of annexin V positive/7AAD-negative (gray bars) or Casp-zVAD-FMK positive (black bars) CD8+ Jurkat cells of 3 independent experiments. b Caspase-activation in MV/IRX-treated CD8+ Jurkat cells after CHX pre-incubation. Representative histograms of 3 independent experiments are shown. c Mean fluorescence intensities (MFI) ± SD of pro-and anti-apoptotic proteins in CD8+ Jurkat cells treated with MV and pre-incubated with IRX-2 ± CHX. Representative histograms out of 3 independent experiments are shown

Fig. 4

Expression of Fas in IRX-2 and/or TMV-treated T cells. a CD8+ Jurkat cells were left untreated or were incubated with different concentrations of ZB-4 prior to addition of TV for 3 h. Cells were analyzed for annexin V binding by flow cytometry. Results are mean percentages ± SD of annexin V+/7-AADneg cells from 3 independent experiments (*p < 0.001 in comparison with the TMV-treated samples). b CD8+ Jurkat cells or activated primary CD8+ or CD4+ T cells were pre-incubated with IRX-2 for 24 h and then treated with 10 μg TMV for additional 4 h or 24 h. Percentages of Fas+ cells (left panel) and MFI for Fas (right panel) were measured by flow cytometry. The data are means ± SD of 3 independent experiments (*p < 0.02 in comparison to untreated samples: **p < 0.02 in comparison to TMV-treated samples). c Representative histograms showing Fas-expression in activated CD8+ and CD4+ T cells after a TMV ± IRX-2 treatment

Fig. 5

FLIP overexpression in T cells enhances IRX-2 protective effects Jurkat cells stably transfected with human FLIP (FLIP) or control vector (mock) were pre-incubated with IRX-2 or left untreated and then incubated with MV for 4 h or 24 h. a Overexpression of c-FLIP in Jurkat-FLIP transfectants is shown by Western blot and flow cytometry. Percentages of annexin V+/7AADneg (b) and of Casp-zVAD-FMK+ (c) in Jurkat-FLIP transfectants and control cells after incubation with TMV and IRX-2. Results shown in a and b are means ± SD of 3 independent experiments (*p < 0.02 compared to mock-transfected Jurkat cells). d Representative histograms showing caspase-activation in TMV- and IRX-2-treated Jurkat cells transfected with FLIP or the control vector. e Caspase 8 and 9 activation and cytochrome c release as measured by Western blots in TMV/IRX-2 treated Jurkat-mock or Jurkat-FLIP transfectants. Cells treated with CH-11 antibody (400 ng/mL) were used as positive controls. Note the absence of caspase-cleavage in the Jurkat-FLIP transfectants in comparison to control cells after TMV or CH-11 Ab treatment

Fig. 6

Protective effects of IRX-2 and apoptosis-inducing effects of TMV involve NF-κB activation in Jurkat cells. Translocation of p65 subunit of NF-κB from cytoplasm into nuclei was measured using an ArrayScan microscope and quantitated as MFI of p65 subunit translocated to cell nuclei. Cells were treated with medium (control), cytokines or TMV as described in “Materials and methods”. The data are means ± SD from 3 independent experiments. *Significant differences from untreated controls at p < 0.05