Separation, phenotyping and limiting dilution analysis of T‐lymphocytes infiltrating human solid tumors

Tumor-infiltrating lymphocytes (TIL) were obtained by a combination of mechanical release and enzymatic disaggregation from 35 human solid tumors. The number of lymphocytes in TIL-enriched suspensions varied from 1 × 104 to 7.6 × 106 per wet gram of tumor. The TIL preparations separated by differential centrifugation on Ficoll-Hypaque gradients contained 10–95% of TII+ cells (mean 50%), and tumor cells accounted for the other major cellular component. Macrophages, NK cells, B cells and granulocytes were infrequently seen. Morphologically, TIL-T were small non-activated cells. They expressed the TII and T3 antigens but not the receptor for IL-2 (IL-2R) or HLA-DR antigens as determined by double immunofluorescence staining. Rare TII+/IL-2R+ cells were recovered only from colon and lung carcinomas. The mean T4/T8 ratio in 12 TIL preparations was 1.1 ± 0.8. Immunohistology with monoclonal antibodies (MAbs) performed in 31/35 tumors confirmed that the TII+ cells infiltrating solid tumors rarely expressed the IL-2R and that the cell content of suspensions enriched in TIL was comparable to that determined in situ. The recovered TIL were cloned in a microculture system that permits proliferation of nearly all normal peripheral blood T lymphocytes (PBL-T). Under these culture conditions, frequencies of the proliferating T lymphocyte precursors (PTL-P) were depressed in both the TIL preparations (< 0.01 to 0.39) and patients' PBL-T (0.05 to 0.5). These low frequencies of PTL-P were seen in patients with all tumor types, both primary and metastatic.