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Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and interleukin-2 (IL-2) is currently being investigated in the treatment of chemoresistant cancers. In protocols using high dosages of IL-2 and adoptive transfers of lymphokine-activated killer (LAK) cells, severe pulmonary toxicities, including pulmonary edema and respiratory failure, are regularly observed. Serial measurements of pulmonary function in patients receiving TIL and continuous infusions of moderate dosages of IL-2 showed the development of mild obstructive and restrictive defects. Partial reversibility of the FEV1 with bronchodilators was observed, suggesting the development of hyperreactive airways. Decreases in lung volumes, DLCO, and gas exchange occurred and may reflect pulmonary infiltration by inflammatory cells, increased lung water, or diminished chest wall function. We conclude that adoptive immunotherapy with TIL and IL-2 produces changes in systemic immune response that may contribute to abnormal pulmonary physiology.

Skin testing represents a direct method of assessing immune responses in vivo. Twenty-six patients with metastatic cancer of the lung, kidney, or melanoma were treated with adoptive transfers of autologous tumor-infiltrating or blood lymphocytes and continuous infusions of interleukin-2 (IL-2). Prior to therapy, cutaneous anergy to recall antigens was observed in 19 patients (73%), whereas 6 (27%) displayed normal delayed-type hypersensitivity (DTH) responses. When tested again at the end of therapy, DTH responses could not be elicited in any of the patients. Proliferative responses to skin test antigens, lectins, and IL-2 diminished progressively during therapy but returned to baseline values at 1 month. Unexpectedly, 14 of these patients (53%) developed immediate skin test responses to candida antigens and 5 (19%) to mumps antigens. These immediate responses were characterized by local erythema and induration that developed within minutes of injecting antigen. Biopsies displayed marked dermal edema and infiltration by eosinophils. Although serum IgE levels were not increased, immediate reactivity could be transferred by a heat-sensitive serum factor. The implications of this novel response are uncertain, and its development did not correlate directly with the anti-tumor effects of therapy. We conclude that adoptive immunotherapy with IL-2 produces a reduction in cutaneous DTH and diminished responses to mitogens while simultaneously promoting cutaneous allergy. We hypothesize that this may reflect diminished IL-2 production by antigen-specific helper T cells and that other lymphokines may promote these immediate hypersensitivity responses.

The culture of human T lymphocytes in interleukin-2 (IL-2) containing growth factor medium results in a significant shift in the T-lymphocytes subsets isolated from such cultures at weekly intervals. If normal peripheral blood mononuclear cells are stimulated with phytohemagglutinin (PHA) or in a mixed lymphocyte reaction (MLR), the resulting T lymphoblasts can be propagated in growth factor medium. Staining of the cultured cells with monoclonal antibodies was evaluated by indirect immunofluorescence on a laser-activated flow cytometer (Ortho Spectrum III). The antibodies used were: OKT3 (mature T lymphocytes), OKT4 (helper/inducer T lymphocytes), OKT8 (cytotoxic/suppressor T lymphocytes, OKT10 (immature and "activated" lymphocytes), OKT11a (cells which rosette with sheep erythrocytes), and OKIa-I (HLA-DR constant region). Both PHA and MLR activation resulted in initial preservation of the OKT4+ subset predominance over OKT8+ T lymphocytes noted on normal circulating blood lymphocytes. However, during culture in T-cell growth factor medium, there was a progressive increase in the percentage of OKT8+ cells, and a concomitant decrease in OKT4+ lymphoblasts. The increase in OKT8+ cells in the MLR-stimulated cultures was paralleled by an increase in specific cell-mediated cytotoxicity against the stimulating lymphocyte population. In addition to the shift in T-lymphocyte subset, there was virtual 100% staining with OKT3 and OKT11a, indicating the T-cell nature of the proliferating cells. OKT10 which was present on a small subset of fresh blood lymphocytes appeared rapidly in stimulated cultures, and was retained on virtually all lymphoblasts of either OKT4+ or OKT8+ subset. OKIa-1 cells increased slowly in PHA-stimulated cultures. HLA-DR+ T cells were detected earlier in MLR cultures. The activation of T lymphocytes results in a significant increase in the number of molecules of OKT11a bound per cell, in concert with the increased avidity of T lymphoblasts for sheep erythrocytes. The significant change in the phenotype and function of lymphoblasts isolated from long-term cultures demonstrates the importance of monitoring cultures, and the potential hazards in equating a cultured cell population with a freshly isolated one.

A trial of adoptive immunotherapy was performed in which long-term cultured, interleukin-2 (IL2)-dependent T-lymphocytes were administered to patients with metastatic adenocarcinoma of the lung. Lymphocytes were isolated from explants of cancer tissues that were cultured in medium with recombinant IL-2. These T-cells expressed surface markers of activation, and killed a broad panel of tumor targets. Intravenously injected 111indium-labeled T-cell blasts distributed primarily to lungs, liver, and spleen. Despite a paucity of infused lymphocytes detected by external imaging at sites of tumor, five of seven patients showed reduction of their cancers. However, in no case was greater than 50% reduction of total tumor burden achieved. Evidence of increased delayed cutaneous hypersensitivity to protein antigens was observed in three patients following therapy. We conclude that long-term cultured tumor-derived T-cells can be transferred safely into humans and that these cells may be capable of enhancing immune responses and mediating tumor reduction in vivo.

Human thymocytes normally proliferate poorly in PHA-stimulated cultures, as measured by thymidine uptake. Their response increases when supernatant factors from mitogen-stimulated human lymphocyte cultures (SUP) are added. The cells that proliferate in PHA + SUP-stimulated cultures continue to divide if they are resuspended with fresh SUP every 3 to 4 days. PHA-stimulated thymocytes are deficient in the production of the T cell growth factor interleukin 2 (IL 2). The phenotype of thymocytes prior to stimulation was 90% T6+, 80% T4+, 83% T8+, and 12% T3+, whereas that of the cells after 3 days in culture with PHA + SUP was 1% T6+, 18% T4+, 66% T8+, and 80% T3+. Lysis of T3+ cells from the thymocyte preparation before culture greatly reduced the proliferative response to PHA + SUP. This suggests that the PHA-induced proliferation of more mature thymus cells is restricted by a lack of essential growth factors (either IL 1 or IL 2) and that the co-stimulating activity of SUP on human cells is predominantly on thymus cells that already are T3+.

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.

We examined the activities of activated lymphocytes, interferon-gamma (IFN-gamma), and interleukin 2 (IL-2) in adoptive immunotherapy of pulmonary metastases. Pulmonary metastases produced in Balb/c mice by a single tail-vein injection of 5 X 10(5) murine sarcoma (MCB8) tumor cells on day 0 were treated with combinations of Con A-activated lymphocytes (CAL) (3 X 10(7) cells on days 3 and 7), IL-2 (5 X 10(4) U three times a day on days 3 to 8), and IFN-gamma (5 X 10(4) U/mouse on days 1 to 8). Treated tumors contained increased numbers of infiltrating Thy-1.2+ lymphocytes and a predominance of L3T4+(CD4+) lymphocytes. The level of expression of class I and class II MHC antigens by tumor cells in the lung was increased after treatment. Mice that received CAL + IL-2 + IFN-gamma showed approximately 80% reduction in tumor burden as compared to controls (P = 0.001). Mice treated with IL-2 + CAL, or IL-2 + IFN-gamma, displayed approximately 50% reduction (both P less than 0.02 as compared to triple therapy), whereas IL-2, IFN-gamma, or CAL administered as single agents had little effect on pulmonary metastases. We conclude that adoptive immunotherapy with activated lymphocytes and IL-2 is enhanced by IFN-gamma.

Tumour-infiltrating lymphocytes (TIL) were isolated and expanded from small tumour biopsy samples of twenty-eight patients (thirteen with malignant melanoma, seven with renal cell carcinoma, and eight with non-small-cell lung cancer). The patients were treated with autologous expanded TIL (about 10(10)) and continuous infusions of recombinant human interleukin-2(1-3 x 10(6) U/m2 per 24 h). 29% of the patients with renal cell cancer and 23% of those with melanoma achieved objective tumour responses lasting 3-14 months. Toxic side-effects were limited, and no patient required intensive-care monitoring. Adoptive immunotherapy with TIL and interleukin-2 may be an effective systemic approach to the treatment of some patients with malignant melanoma and renal cell carcinoma.