Improved in vivo efficacy of tumor-infiltrating lymphocytes after restimulation with irradiated tumor cells in vitro.

Abstract

We investigated different culture conditions for tumor-infiltrating lymphocytes (TILs) with regard to proliferation, phenotypic changes, in vitro cytotoxicity, and in vivo therapeutic efficacy. After enzymatic digestion of the murine fibrosarcoma, MCA-105, TIL cultures were initiated as pure lymphocyte (groups 1 and 2) or mixed lymphocyte/tumor suspensions (groups 3 and 4). Group I TILs were grown in culture medium containing 100 IU/ml recombinant interleukin-2 (rIL-2). Group 2 TILs were stimulated with solid-phase anti-CD3 monoclonal antibody (mAb) for 48 h and cultured in rIL-2 (100 IU/ml)-containing medium. Group 3, which consisted initially of a surplus of tumor cells, received the same treatment as group 2. Group 4 was also activated with anti-CD3 mAb and rIL-2 but was additionally restimulated weekly with irradiated tumor cells (TILs to tumor, 20:1). Groups 1 and 2 showed up to twofold higher increases in TIL numbers compared with groups 3 and 4 by the end of culture week 5. Although the original lymphocyte/tumor cell suspension consisted of 12.0 +/- 3.8% CD4+ T cells and 5.3 +/- 3.3% CD8+ T cells, all four TIL cultures showed approximately 80% CD8+ TILs and no CD4+ TILs by the end of culture week 4. In vitro cytotoxicity did not correlate with in vivo efficacy of the examined TIL cultures. By using the MCA-105 pulmonary metastases model in C57BL/6 mice, only suboptimal doses of TILs (2 x 10(6)) from group 4, which had been restimulated weekly with irradiated tumor, showed significant tumor eradication compared with all other treatment groups (p < 0.01). We conclude that in vitro tumor restimulation of TILs improves in vivo efficacy, most likely through the education of tumor-reactive T cells.