In vitro induction of T cells that are resistant to A2 adenosine receptor-mediated immunosuppression

Abstract

Background and purpose: The increased levels of extracellular adenosine in inflamed tissues down-regulate activated immune cells via the A(2A) adenosine receptor. This A(2A) adenosine receptor-mediated immunosuppression is a disqualifying obstacle in cancer immunotherapy as it protects cancerous tissues from adoptively transferred anti-tumour T cells. The aim of this study was to test whether the negative selection of T cells will produce T cells that are resistant to inhibition by extracellular adenosine.

Experimental approach: Cytotoxic T lymphocytes (CTL) were developed by mixed lymphocyte culture in the presence or absence of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA). The sensitivity of CTL to adenosine analogues was characterized by cAMP induction, interferon-gamma production and cytotoxicity.

Key results: CTL that could proliferate even in the presence of NECA were less susceptible to inhibition by A(2A) adenosine receptor agonists, as shown by a much smaller accumulation of cAMP and less inhibition of interferon-gamma production compared with control CTL. The successful protocol to produce CTL that are both resistant to adenosine-mediated immunosuppression and maintain strong cytotoxicity and interferon-gamma secretion required NECA to be added only during the expansion stage after the establishment of CTL. In contrast, the priming of resting T cells in the presence of NECA resulted in T cells with impaired effector functions.

Conclusions and implications: Adenosine-resistant effector T cells were successfully obtained by exposure of activated T cells to NECA. These in vitro studies form the basis for future attempts to produce anti-tumour T cells that are more effective in adoptive immunotherapy.

Figure 1

NECA suppressed the mixed lymphocyte reaction, but there was significant development of CTL. CTL were induced by mixed lymphocyte culture using spleen cells from C57BL/6 (H-2^b) and DBA/2 (H-2^d) mice. The cells were cultured for 5 days in the presence or absence of NECA (0.1–10 µmol L⁻¹). Resulting cells were examined for their proliferative activity (A), IFN-γ levels in the culture supernatant (B) and cytotoxicity against P815 mastocytoma (C). Effector–target ratio for cytotoxicity assay was 5:1. D. Proportion of CD4 and CD8 cells after 5 days. Large (activated) cells were gated for the analysis. Data from CTL induced with 10 µmol L⁻¹ NECA shown are representative results; there was no marked difference in CTL induced with other concentrations of NECA. E. CD8⁺ cell expansion during mixed lymphocyte culture. C57BL/6 cells were stimulated after being labelled with CFSE and its fluorescence in CD8⁺ cells was analysed after 3 days of culture. Large (activated) CD8-expressing cells were gated for the analysis. Numbers in the panels indicate percentage of population. Data shown here represent average ± s.d. of triplicate samples. The statistical significance was calculated by Student's t-test: a P < 0.05; b P < 0.01; c P < 0.001. CFSE, carboxyfluorescein succinimidyl ester; CTL, cytotoxic T lymphocytes; IFN-γ, interferon-γ; NECA, 5′-N-ethylcarboxamidoadenosine; NECA-CTL, CTL developed in the presence of NECA.

Figure 2

CTL developed with NECA show impaired response to A_2A/A_2B adenosine receptor agonists. CTL were induced as described in Figure 1. cAMP production from the CTL was determined after incubation with A_2A/A_2B adenosine receptor agonists (CGS and NECA) and adenylate cyclase activator (forskolin). The concentration of cAMP inducers was 10 µmol L⁻¹. Data shown here represent average ± s.d. of triplicate samples. The statistical significance was calculated by Student's t-test: a P < 0.05; b P < 0.01 versus control CTL. CGS, CGS21680; CTL, cytotoxic T lymphocytes; NECA, 5′-N-ethylcarboxamidoadenosine.

Figure 4

CTL treated with NECA for only the last 2 days were not only resistant to immunosuppression by A_2A/A_2B adenosine receptor agonists but also high IFN-γ producers comparable with control CTL. CTL were prepared as described for Figure 3. A and B. On day 7, the cells were re-stimulated by immobilized anti-CD3 and anti-CD28 mAbs for 24 h with CGS or NECA (10 µmol L⁻¹). A. IFN-γ levels in the culture supernatant were quantified by ELISA. B. Intracellular IFN-γ expression in stimulated CTL was evaluated after further incubation with brefeldin A for 2 h. Numbers in each panel show the percentage of IFN-γ producers. The data shown here represent two separate experiments. C. NECA-CTL treated with NECA for only the last 2 days also showed an impaired cAMP response to A_2A/A_2B adenosine receptor agonists. Data shown here represent average ± s.d. of triplicate samples. The statistical significance was calculated by Student's t-test: a P < 0.05; b P < 0.01; c P < 0.001 versus control CTL. CTL, cytotoxic T lymphocytes; IFN-γ, interferon-γ; NECA, 5′-N-ethylcarboxamidoadenosine; NECA-CTL, CTL developed in the presence of NECA.

Figure 3

Cytotoxicity of NECA-CTL was maintained well when NECA was added only during the secondary mixed lymphocyte culture. Primary CTL were induced by mixed lymphocyte culture for 5 days. After extensive washing, the cells were re-stimulated with spleen cells from DBA/2 mice for 2 more days. NECA (0.1–1 µmol L⁻¹) was withheld during the primary mixed lymphocyte culture and was added for 2 days after re-stimulation. The resulting cells were examined for their proliferative activity (A), IFN-γ levels in the culture supernatant (B), cytotoxicity against P815 mastocytoma (C) and flowcytometric analysis (D). Effector–target ratio for cytotoxicity assay was 5:1, 2.5:1 and 1.25:1. Data shown here represent average ± s.d. of triplicate samples. The statistical significance was calculated by Student's t-test: b P < 0.01; c P < 0.001. CTL, cytotoxic T lymphocytes; IFN-γ, interferon-γ; NECA, 5′-N-ethylcarboxamidoadenosine; NECA-CTL, CTL developed in the presence of NECA.

Figure 5

NECA-CTL maintained their resistance to A_2A adenosine receptor stimulation at least for 24 h. NECA was removed by extensive washing 2 days after re-stimulation. Cells were re-cultured with IL-2 for 24–48 h, and then IFN-γ production was induced by immobilized anti-CD3 and anti-CD28 mAbs. The inhibitory effect of CGS (10 µmol L⁻¹) is expressed as a percentage of IFN-γ levels remaining compared with control. Data shown represent average ± s.d. of triplicate samples. CTL, cytotoxic T lymphocytes; IFN-γ, interferon-γ; NECA, 5′-N-ethylcarboxamidoadenosine; NECA-CTL, CTL developed in the presence of NECA.

Figure 6

NECA-CTL could produce high levels of IFN-γ upon recognition of tumour cells even in the presence of A_2A/A_2B adenosine receptor agonists. CTL were induced as described for Figure 3. In order to activate antigen-specific anti-tumour responses, the same number of CTL was evaluated after co-culture with mitomycin C-treated P815 cells. The susceptibility of the CTL to A_2A/A_2B adenosine receptor activation was examined by stimulating them in the presence of CGS or NECA (10 µmol L⁻¹). IFN-γ levels in the supernatant were determined after 2 days. Data shown here represent average ± s.d. of triplicate samples. The statistical significance was calculated by Student's t-test: a P < 0.05; b P < 0.01; c P < 0.001 versus control CTL. CGS, CGS21680; CTL, cytotoxic T lymphocytes; IFN-γ, interferon-γ; NECA, 5′-N-ethylcarboxamidoadenosine; NECA-CTL, CTL developed in the presence of NECA.