Generation of tumor-specific cytotoxic T-lymphocytes from peripheral blood of colorectal cancer patients for adoptive immunotherapy.

Abstract

3056 Background: Adoptive T-cell transfer (ACT) using autologous TIL, grown ex vivo and then infused into the cancer patient after lymphoablative chemotherapy, has emerged as an effective treatment for patients with metastatic melanoma. However, this approach has been hampered by the difficulty of isolating TILs from tumors other than melanoma, and of amplifying a sufficient quantity of autologous tumor-reactive T cells. So we decided to adopt a recently described procedure for generating in vitro large numbers of anti-tumor HLA-restricted CTLs, by stimulating patient’s CD8-enriched peripheral blood mononuclear cells (PBMCs) with DCs pulsed with apoptotic solid tumor cells (TCs) as a source of tumor antigens. Methods: 61 patients affected by CRC were enrolled. Tumor biopsies were obtained at surgery, together with 100 ml of heparinized peripheral blood. Tumors were dissociated to a single-cell suspension and cultured in order to obtain tumor cell line from each patient. Dendritic cells (DCs) were generated from previously separated PBMCs, using a magnetic positive selection of CD14+ monocytes, cultured in presence of Interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Anti-tumor cytotoxic T lymphocytes (CTLs) were elicited using DCs as antigen-presenting cells, autologous apoptotic tumor cells as source of antigens and T CD-8 lymphocytes enriched effectors, with weekly stimulation. To evaluate the cytotoxic activity of CTLs, interferon-γ (IFN-γ) secretion was assessed by ELISPOT. Results: Tumor cell lines and DCs were obtained from 19 out of 61 patients. ELISPOT was performed so far for 6 patients: strong IFN-γ secretion was detected at the third, fourth and fifth stimulations for one patient and at the second for another patient, whereas for three patients a weak secretion was detected during the second and the third stimulations. CTLs from one patient did not react to the stimulations. Conclusions: Generation of CTLs suitable for ACT immunotherapy is feasible from peripheral blood in patients with CRC.