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

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Abstract
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 × 104to 7.6 × 106per 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 determinedin 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.

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