Over-expressing Akt in T cells to resist tumor immunosuppression and increase anti-tumor activity

Abstract

Background: Tumor employs various means to escape immunosurveillance and inhibit immune attack, and strategies have been developed to counteract the inhibitory signals. However, due to the complex suppressive mechanisms in the tumor microenvironment, blocking one or a few inhibitory signals has only limited effects on therapeutic efficacy. Instead of targeting tumor immunosuppression, we considered from another point of view, and hypothesized that manipulating T cells to make them resist any known or unknown suppressive mechanism may be more effective for cancer treatment.

Methods: We used OT-1 cells transduced with retroviruses encoding Akt and human peripheral blood lymphocytes (PBLs) transduced with retroviruses encoding both Akt and a chimeric antigen receptor (CAR) specific for tumor antigen EpCAM to examine the effect of over-expressing Akt on tumor specific T cells in tumor environment.

Results: We show that Akt activity of T cells in the tumor environment was inhibited, and over-expressing Akt in OT-1 cells increased the cytokine production and cell proliferation in the presence of B16-OVA tumor cells. What's more, adoptive transfer of OT-1 cells over-expressing Akt inhibited B16-OVA tumor growth and prolonged mouse survival. To examine if over-expressing Akt could increase the anti-tumor activity of T cells in human cancer, PBLs co-expressing EpCAM specific CAR and Akt were cultured with EpCAM-expressing human prostate cancer cells PC3M, and less inhibition on cell proliferation and less apoptosis were observed. In addition, adoptive transfer of PC3M specific T cells over-expressing Akt resulted in more dramatic tumor inhibitory effects in PC3M bearing NOD/SCID mice.

Conclusions: These data indicates that over-expressing Akt in tumor specific T cells increases T cell proliferation and activity in the tumor environment, and enhances anti-tumor effects of adoptively transferred T cells. Our study provides a new strategy to improve the efficacy of adoptive T cell therapy, and serves as an important foundation for clinical translation.

Fig. 1

Akt activity of T cells is down-regulated in tumor environment. a CD8⁺ T cells isolated from OT-1 transgenic mice were stimulated with anti-CD3 and anti-CD28 antibodies for 2 days, and then incubated with or without B16-OVA tumor cell culture supernatant. 24 h later, cells were collected, and the levels of Akt, phosphorylated Akt, and β-actin were examined by western blot. b CD8⁺ T cells were isolated from tumor draining lymph nodes (TDLN) or distant lymph nodes of B16-OVA tumor bearing mice, the levels of Akt, phosphorylated Akt, and β-actin were examined by western blot

Fig. 2

OT-1 cells transduced with Akt or myr-Akt display increased IFN-γ production and proliferation in the presence of B16-OVA tumor cells. a OT-1 cells transduced with control retroviruses or retroviruses encoding Akt or myr-Akt were incubated with or without B16-OVA tumor cells at an E:T ratio of 2:1, 24 h later, the cells were stained with anti-CD8 and anti-IFN-γ antibodies, and analyzed by flow cytometry. The analysis was carried out with gated CD8⁺ population. b OT-1 cells transduced with control retroviruses or retroviruses encoding Akt or myr-Akt were labeled with CFSE, and co-cultured with or without B16-OVA tumor cells at an E:T ratio of 2:1, 3 days later, the cells were stained with anti-CD8 antibody, and analyzed by flow cytometry. The analysis was carried out with CD8⁺ population

Fig. 3

Adoptive transfer of OT-1 cells transduced with Akt can inhibit B16-OVA tumor growth and prolong mouse survival. OT-1 cells transduced with control retroviruses or retroviruses encoding Akt or myr-Akt were intratumorally injected into B16-OVA tumor bearing mice at 2x10⁶ cells/mouse at 11 days after tumor challenge, and the mice without treatment were used as control. Tumor growth was monitored every other day, and the death of mouse was defined when the tumor size reached 2 cm. a Line graph depicting the tumor volume of mice, statistical analysis was performed using two-way ANOVA. b Kaplan-Meier survival analysis of transduced OT-1 treated or untreated mice. Comparison of survival curves was made by Log-rank test

Fig. 4

Engineer human peripheral blood lymphocytes (PBLs) to target prostate cancer cells. a Immunosuppressive phenotypes of human prostate cancer cell line PC3M were examined by staining with HLA-DR, CD80, B7-H1, TGF-β, and IL-10. b Different constructs used to transduce PBLs, V_H, anti-human EpCAM immunoglobulin heavy chain variable region; V_L, anti-human EpCAM immunoglobulin light chain variable region; CD28, part of the extracellular domain and the entire transmembrane and intracellular domains of CD28; CD3ζ, the cytoplasmic domain of CD3ζ; IRES, internal ribosome entry sequence. c Human PBLs were transduced with retroviruses encoding EpCAM specific CAR, CAR-Akt, or CAR-myr-Akt, at 5 days after transduction, the mRNA levels of transduced CAR were determined by RT-PCR and real-time PCR, and the protein levels of Akt, phosphorylated Akt were determined by western blot

Fig. 5

Over-expressing Akt increases the activity of CAR expressing PBLs in the presence of PC3M. PBLs transduced with CAR, CAR-Akt, or CAR-myr-Akt were co-cultured with or without PC3M at an E:T ratio of 2:1, 3 days later, cell proliferation was examined by CFSE assay (a), and cell apoptosis was determined by Annexin V/PI staining (b), the analysis was performed with gated CD8⁺ population

Fig. 6

Over-expressing Akt enhances the anti-tumor efficacy of PBLs in vitro and in vivo. a PBLs transduced with CAR, CAR-Akt, or CAR-myr-Akt were incubated with PC3M-luc at an E:T ratio of 2:1, 24 h later, cell viability was determined by luminescence imaging, and the luminescence intensity was normalized to that of PC3M-luc cells without treatment. Statistical analysis was performed using student’s t-test. b NOD/SCID mice were subcutaneously injected with PC3M tumor cells at 6 × 10⁶ cells/mouse, at 21 days after tumor challenge, PBLs transduced with CAR, CAR-Akt, or CAR-myr-Akt were intratumorally injected into mice at 3 × 10⁶ cells/mouse, and the mice without treatment were used as control. Tumor volume was measured every few days, and statistical analysis was performed using two-way ANOVA