Using GFP-positive immunomodulatory cells as biosensors to evaluate T cell costimulation and improvement of antitumor response.

Introduction

Immunotherapy strategies may modulate lymphocyte activation, by inhibiting mechanisms of immunosuppression, or inducing T cell costimulation. In this work, we developed an in vitro assay that allows evaluating the antitumor potential of immunomodulatory vaccines. The assay is based on the generation of a reporter cell line which simultaneously encodes expression of the eGFP marker and harbors an immunomodulator such as GM-CSF, 4-1BBL, OX40L. The GFP positive immunomodulatory cell lines are incubated with primary T cells, that can be costimulated, enhancing their activation and consequently boosting the elimination of tumor cells. When tumor cells are killed by activated T cells, they release the intracytoplasmic GFP, that can be monitored by flow cytometry.

Material and Method

Immunomodulatory cell lines were established by transducing B16 tumor cells, with a retroviral vector encoding the reporter gene eGFP, generating the B16-GFP lineage. Next, the B16-GFP line was transduced with a second retroviral vector that harbors an immunomodulator such as GM-CSF, 4-1BBL or OX40L. In this manner, the tumor-derived cell line harbors both the GFP reporter gene and one immunomodulator. These cell lines are incubated with CD4 T cells or CD8 T cells, followed by GFP analysis by flow cytometry. GFP leakage indicates an antitumor immunomodulatory effect. In vivo experiments were performed using C57B6 mice, injecting 5x10(4) sc B16F10 cells into the right flank and 1x10(6) irradiated vaccine cells on the left flank on days 1, 4 and 7 after administration of the parental B16F10 tumor cells.

Results and discussion

We observed that the GFP-positive tumor cell lines harboring the GM-CSF 4-1BBL and OX40L imunomodulators, induced in vitro costimulation of CD4 T cells and CD8 T cells, potentiating antitumor cytotoxicity, which was evidenced by a mean reduction of 80% in GFP positive cells, counted by flow cytometry. Performing in vivo assays, we observed as expected, an increase on the survival fraction of challenged mice which received immunomodulatory vaccines.

Conclusion

We have developed an in vitro assay to evaluate the therapeutic benefit of immunomodulatory vaccines. Results of the in vitro assay were recapitulated by in vivo assays, in which we observed an increase in the survival fraction of challenged animals. These in vitro assays will enable the evaluation of new therapeutic strategies that target T cell costimulatory pathways, to potentiate the antitumor response and may contribute to reduce animal experimentation.

Supported by FAPESP