Cytokine regulation of tumor necrosis factor-alpha and -beta (lymphotoxin)-messenger RNA expression in human peripheral blood mononuclear cells.

The immune response at the molecular level is characterized by a carefully coordinated interplay of both cytokine production and receptor induction. The regulation of these molecules including the closely related tumor necrosis factors alpha (TNF) and beta (lymphotoxin, LT) is still incompletely understood. We have examined the effects of various cytokines on the expression of TNF and LT mRNA in human peripheral blood mononuclear cells (PBMC). Northern blot analysis with total cellular RNA from mixed populations of PBMC revealed that genes coding for TNF and LT were not spontaneously expressed. Treatment of PBMC with recombinant interleukin (IL)-2 resulted in a high level expression of TNF and LT mRNA. Whereas IL-1 beta was equally effective as IL-2 in inducing both TNF and LT mRNA, granulocyte-macrophage colony-stimulating factor selectively induced only TNF mRNA. Both TNF and LT mRNA were minimally induced by IL-1 alpha, IL-3, interferon (IFN)-alpha, or IFN-gamma. Similarly TNF alone had little effect on induction of TNF and LT mRNA. In conjunction with IL-2, cytokines such as IFN-alpha, IFN-gamma, or TNF did not interfere with IL-2 induction of TNF and LT mRNA. Interestingly, IL-4 in combination with IL-2 inhibited the IL-2-driven induction of TNF and LT mRNA. This inhibitory effect of IL-4 was also observed at the level of TNF and LT protein secretion. Furthermore, IL-4 was also inhibitory of IL-2-mediated induction of Tac mRNA in PBMC. These results extend the interrelationship of cytokine regulation of TNF and LT expression. In particular, they reveal the previously unrecognized function of IL-4 in antagonizing the IL-2 induction of TNF, LT, and Tac mRNA in PBMC.

Induction of endogenous cytokine-mRNA in circulating peripheral blood mononuclear cells by IL-2 administration to cancer patients.

The lymphokine IL-2 plays a central role in immune regulation. Recent clinical trials have shown that when administered systemically either alone, or in combination with lymphokine-activated killer cells, IL-2 can cause regression of metastatic tumors in some patients with a variety of otherwise refractory cancers. To evaluate the mechanism of in vivo action of IL-2, as well as the toxicity associated with its administration, we have studied the in vivo cytokine-mRNA expression of circulating PBMC in cancer patients undergoing treatment with high dose IL-2. Before IL-2 administration, we found low level or no evidence of cytokine-mRNA expression in PBMC. After IL-2 infusion, circulating PBMC showed enhanced proliferative activity and contained significant levels of mRNA for TNF-alpha and IL-6 as well as mRNA for the p55 IL-2R, Tac, but no mRNA coding for granulocyte-monocyte-CSF and TNF-beta (lymphotoxin). IL-1 beta mRNA was expressed at very low levels in circulating PBMC after IL-2 infusion. Each of these cytokine -mRNA was, however, inducible in vitro by stimulation of PBMC with IL-2 alone. The results of these in vivo studies suggest that IL-2 may be a physiologic inducer of TNF and IL-6 which, because of their pleiotropic effects, may be important endogenous signals in the body's immune response and account for some of the physiologic changes seen in patients receiving high dose IL-2.

Effects of transforming growth factor-beta on human lymphokine-activated killer cell precursors. Autocrine inhibition of cellular proliferation and differentiation to immune killer cells.

With subpopulations of human lymphoid cells that were enriched for lymphokine-activated killer (LAK) cell precursors, studies were performed to examine the effects of transforming growth factor-beta (TGF-beta) on their IL-2-dependent growth and differentiation to killer cells. The majority of the LAK precursor cells appeared to reside in nonadherent, non-T, and non-B lymphocyte populations that expressed CD11 and CD16 Ag. These cells were induced to proliferate and become LAK cells by high concentrations of rIL-2 alone in the apparent absence of any prior activation with mitogen or Ag. The partially purified lymphocyte subpopulations generated varying but several-fold greater levels of LAK killing on a per cell basis than did unfractionated lymphocytes. The exogenous addition of TGF-beta to the LAK precursor cultures, markedly inhibited IL-2-stimulated growth as well as the development of LAK activity in a dose-dependent manner. The antimitotic effect of TGF-beta was reversible; inhibition of proliferation could be largely restored by increasing the concentration of IL-2 in culture. In contrast, TGF-beta inhibition of cytotoxicity was relatively independent of the concentration of IL-2. Further, LAK precursors constitutively expressed TGF-beta mRNA and high affinity receptor for TGF-beta. Activation of LAK precursors with IL-2 alone, resulted in a three- to fivefold up-regulation of intracellular TGF-beta mRNA and TGF-beta biologic activity secreted in the culture media. Furthermore, Northern blotting revealed that the resting LAK precursors did not express the Tac-mRNA. Receptor binding studies with 125I-IL-2 suggested the presence of a single class of IL-2R with an apparent Kd of intermediate range (beta-chain of IL-2R) on the unstimulated cells. Stimulation with high concentrations of Il-2 induced Tac-mRNA (both the 3.5- and 1.5-kb transcripts) and resulted in the expression of high affinity IL-2R (Kd approximately 10(-11) M) on these cells. Suppression of IL-2-dependent responses by TGF-beta was accompanied by a selective down-regulation of the 1.5-kb Tac-mRNA as well as by reduction in high affinity IL-2R. The results suggest a negative autocrine control of TGF-beta on IL-2-dependent growth and differentiation of human LAK cells, possibly related to regulate the killer activation function.

Studies of serum-free culture medium in the generation of lymphokine activated killer cells.

Lymphokine activated killer (LAK) cells administered in conjunction with recombinant interleukin-2 can mediate the regression of metastatic tumor in some patients with advanced cancer. In these trials LAK cells were activated in medium containing 2% human type A or AB serum. We have found three commercially available, serum-free culture media which allow development of in vitro LAK activity by human peripheral blood lymphocytes. They are AIMV (Gibco), MASF-3 (Whitaker-MA Bioproducts) and HB-104 (Dupont). If 2-mercaptoethanol was added to these culture media they were also capable of generating murine LAK cells which were effective in reducing pulmonary metastases in the murine MCA-106 model. Although LAK cells generated in these media have not been tested in humans yet, potentially they could provide a safe, unlimited and less expensive source of culture fluid for generating the large numbers of LAK cells needed for human clinical trials.

Development of an automated closed system for generation of human lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) can mediate tumor regression in some patients with advanced cancer. The activation of large numbers of LAK cells was performed in roller bottles in a research laboratory setting and required meticulous aseptic technique, at least one skilled technician per patient and one laminar flow hood per patient. To reduce the complexity and expense of LAK cell generation for human immunotherapy trials we have developed a closed-system automated procedure using a continuous flow blood cell separator. PBL were obtained by standard apheresis techniques. Platelets and plasma were elutriated using countercentrifugal flow of saline in the cell separator machine. The washed PBL were underlaid with Ficoll-Hypaque (FH) in the original separation bag. Lymphocytes were then flushed into a collection bag where they were concentrated and washed with 2 liters of saline. Mean recovery from the automated FH technique was 54.6 +/- 4.3% compared to 62.3 +/- 4.0% using manual methods in 50 ml tubes (P greater than 0.05). Cells were diluted in the collection bag with RPMI 1640 +/- 2% human AB serum and could be dispensed in an automated fashion to polyolefin bags via a sample port with 1000-1500 U/ml IL-2. After 3-4 days of culture in 5% CO2 at 37 degrees C, activated cells from the bags were harvested and washed in a closed system using the continuous flow cell separator. Cell yield from the harvest was 79.2 +/- 5.4% in the automated system compared to 64.9 +/- 5.0% in the standard procedure using manual harvest of roller bottles (P less than 0.01). Lytic capacity of the cells against fresh human tumor in a 4 h 51Cr release assay was equivalent in cells processed either by the automated or the conventional manual method. The advantages of a closed system include decreased potential for microbial contamination and reduced labor and capital equipment costs. This technique may be easily adapted for use with other cell collection and culture systems.

Identification of specific cytolytic immune responses against autologous tumor in humans bearing malignant melanoma.

Tumor-infiltrating lymphocytes from six patients with metastatic malignant melanoma were expanded by culture in recombinant interleukin 2. Three of the preparations were highly cytotoxic against autologous fresh melanoma tumor cells, but not against autologous fresh normal cells or allogeneic fresh tumor targets. The other three were highly cytotoxic against autologous fresh melanoma tumor cells and also had a limited capacity to kill allogeneic fresh tumor targets. The tumor-associated specific killer cells could be expanded from threefold to 95,652-fold with maintenance of specific antitumor lysis. The expanded tumor-infiltrating cells were Leu-4+ T cells, and in five of six patients the majority were Leu-3+. These studies demonstrate that the melanoma-bearing patient raises an immune response against autologous tumor and presents a method for the generation of human lymphocytes with antitumor reactivity that may be useful in the adoptive immunotherapy of tumors.

Large scale production of human lymphokine activated killer cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine activated killer (LAK) cells in conjunction with recombinant interleukin 2 (RIL-2) is capable of reducing established metastatic cancer in a variety of animal tumor models. A major difficulty in the application of these efforts to the treatment of human cancer has been the activation in vitro of up to 2 X 10(11) human peripheral blood lymphocytes obtained by repeated leukaphereses. We have thus developed optimal and simplified techniques for the generation of human LAK cells for use in clinical trials. We have found that 1.5 X 10(9) lymphocytes separated on Ficoll-Hypaque gradients and incubated in 1000 ml of culture medium in a 2.3 liter roller bottle with 1000-1500 U of RIL-2 per ml, generated LAK cells capable of killing fresh human tumor cells in a 4 h chromium release assay. The culture medium used was RPMI 1640 with 2 mM glutamine, 2% heat-inactivated human AB serum, 50 micrograms/ml streptomycin and gentamicin and 50 U/ml penicillin. This technique allows activation of sufficient numbers of cells in a research laboratory setting to conduct human clinical trials. The administration of LAK cells generated in this fashion can mediate the regression of human tumors when administered in conjunction with IL-2.