Differentiation of antitumor-specific cytotoxic T lymphocytes from autologous tumor infiltrating lymphocytes in non-Hodgkin’s lymphomas

The present study describes a new culture protocol allowing the activation and proliferation of autologous tumor infiltrating T lymphocytes (TIL), and the generation of antitumor specific CTL in non-Hodgkin’s lymphoma (NHL). Cells from eight patients with indolent NHL were used. We performed 3-week co-cultures of TIL with irradiated autologous malignant B cells in the presence of low doses of IL-1β, IL-2 and IL-12. The proliferation, phenotype and cytotoxicity, and antitumor specificity of T cells recovered were studied. T-cell clonality was analyzed using TCRγ gene rearrangement amplification by a multiplex PCR. Under these culture conditions, TIL proliferated, and the CD8+ T lymphocytes that were in a minority at the beginning of the culture increased dramatically in 6 out of 8 cases. In two cases, CD4+ T lymphocytes expanded. We showed that an oligoclonal selection of reactive T cells occurred in culture. Specific cytotoxicity developed against autologous malignant B cells in the 6 cases where there was an expansion of CD8+ T lymphocytes. Inhibition experiments performed with mAb directed against HLA class I and II molecules, CD4, CD8 and TCRγδ showed that the cytotoxic effector cells were CD8+ T lymphocytes probably expressing TCRαβ+. Cytokine secretion was analyzed in culture medium, and we detected significant levels of IFN-γ, TNF-α, and IL-10 and no IL-4 (except in one case). Our results demonstrate that memory T cells from lymphoma patients can be amplified and differentiated into antitumor cytotoxic cells using a combination of the cytokines IL-1β, IL-2, and IL-12 in association with non modified tumor cells.