Trafficking of syngeneic murine lymphokine activated killer T cells following intraperitoneal administration in normal and tumor bearing mice.

Nongenetically restricted T cells may be important host effector cells in women with ovarian cancer receiving intraperitoneal (ip) IL-2 therapy. We developed an in vitro technique to produce murine lymphokine-activated killer T cells. Murine splenocytes were cultured in the presence of 1000 U/ml IL-2 for 10 to 15 days. Phenotypical analysis showed 95% of total cells to express the pan T phenotype Thy 1.2 and no NK cell phenotypes by Day 7 in culture. These cells were labeled with 51Cr and their trafficking pattern after ip administration into normal and M5067 tumor bearing mice was examined. Various organs and tissues were collected at different timepoints and monitored for radioactivity. Within 4 hr., about 60% of the counts were associated with the bowel, peritoneum, and omentum of both normal and tumor bearing mice. About 15% of counts were associated with the blood, lung, kidney, spleen, and liver of both normal and tumor bearing mice.

Immunotherapy of ovarian cancer. II. In vitro generation and characterization of lymphokine-activated killer T cells from the peripheral blood of recurrent ovarian cancer patients.

We examined the in vitro sensitivity of continuous ovarian cancer cells to lymphokine-activated killer T cells (T-LAK) alone or in combination with cytokines. Lymphocyte viability in T-LAK cultures generated from normal donors and ovarian cancer patients declined in the first 2 to 4 days; however, the remaining cells in these cultures maintained a constant rate of proliferation for long periods in vitro. These cells became 90-95% CD3+ TCR+ -alpha/beta T-cells after 7-10 days in culture. The T-LAK cells from normal donors and cancer patients expressed an equal ability to induce lysis of a panel of human target cells (NK-sensitive K562, NK-insensitive RAJI, and two human ovarian tumor lines, SKOV-3 and OVCAR-3), demonstrating that they are nongenetically restricted killers. Preincubation of either the effector or target cells with tumor necrosis factor or interferon-gamma or addition of these cytokines directly to cytolytic assays did not alter the degree of cell lysis in vitro. This is a method for generating large numbers of autologous, cytolytically active T-LAK cells from the blood of ovarian cancer patients that could be employed in adoptive intraperitoneal immunotherapy.

Growth of the endometrial adenocarcinoma cell line AN3 CA is modulated by tumor necrosis factor and its receptor is up-regulated by estrogen in vitro.

We demonstrate that tumor necrosis factor (TNF) has a biphasic effect on the growth of the human endometrial adenocarcinoma cell line AN3 CA in vitro. Low levels (0.2-5 pg/ml) of TNF were moderately growth stimulatory (up to 20% enhancement), while levels over 100 pg were growth inhibitory (up to 45% inhibition). Northern blot analysis showed expression of the 75-kilodalton (kDa) TNF receptor mRNA, but no expression of the 55-kDa TNF receptor mRNA or TNF mRNA. The growth of these cells was not directly affected by physiological concentrations (10(-7)-10(-9) M) of 17 beta-estradiol (E2). However, [125I]TNF binding studies and Scatchard analysis showed that 18-h coculture with 10(-8) M E2 increased the number of TNF receptors expressed on these cells 3-fold. Quantitative mRNA analysis confirmed that 75-kDa TNF receptor mRNA expression increased within 4 h of incubation with E2. These observations suggest an interaction between the endocrine and the immune systems, with an important implication for the homeostasis of endometrial tissues.

Enhancement of lymphokine-activated T killer cell tumor necrosis factor receptor mRNA transcription, tumor necrosis factor receptor membrane expression, and tumor necrosis factor/lymphotoxin release by IL-1 beta, IL-4, and IL-6 in vitro.

Co-culture with IL-2 can induce human T lymphocytes to proliferate and become nongenetically restricted, lymphokine-activated killer (LAK) cells in vitro. Our studies were conducted with long term cultured, human T-LAK cells from peripheral blood, which are 95 to 99% CD3+. We found that proliferating 7 to 10-day human T-LAK cells express TNFR, by using a 125I-TNF binding assay. Additional treatment of these cells with the cytokines IL-1 beta, IL-4, or IL-6 rapidly up-regulated 55-kDa TNFR mRNA transcription and doubled TNFR membrane expression. Further studies revealed that these cytokines also increased the release of TNF and lymphotoxin (LT). Antibody neutralization studies indicated that IL-1 induces release of both TNF and LT; however, IL-4 and IL-6 induce primarily LT release. These results further support the concept that these cytokines are involved in the regulation of TNF/LT release, TNFR synthesis, and TNFR membrane expression. It is apparent that cytokines and their membrane receptors are involved in the autocrine/paracrine control of T cell proliferation, differentiation, and expression of functional activity after IL-2 stimulation in vitro.

The presence of antibodies to lymphotoxin and tumor necrosis factor in normal serum.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are cytokines that probably play a role in several autoimmune diseases. In this report, we describe the detection, in normal nonimmune serum, of naturally occurring antibodies that bind to recombinant human LT and TNF. The naturally occurring antibodies that bind to LT and TNF could explain why normal immunoglobulin is effective therapy in idiopathic thrombocytopenic purpura and Kawasaki syndrome.