Induction of interleukin-2 receptor by tumor necrosis factor α on cultured ovarian tumor-associated lymphocytes

Summary We have recently reported that autologous tumor-specific cytotoxic T lymphocyte (CTL) lines and clones can be developed from lymphocytes infiltrating ovarian malignant ascites (TAL). In this study, we investigated the biological effects of tumor necrosis factor (TNF) in the induction, expansion, long-term proliferation and lytic function of CD8⁺ TAL. TNF up-regulated the IL-2 receptor (IL-2R) chain (Tac antigen) on the surface of CD3⁺ CD8⁺ CD4^– TAL, enhanced the proliferation of autologous tumor-specific CTL, and potentiated their lytic function in long-term cultures. Furthermore, in the induction and expansion phase of CD8⁺ TAL, the presence of TNF was associated with a selective increase in CD8⁺ IL-2R⁺ (Tac⁺) cells, and subsequent decrease in CD4⁺ IL-2R⁺ (Tac⁺) cells. These results suggest that the observed facilitation of the outgrowth of CD8⁺ cells in TAL cultures may be due, at least in part, to the up-regulation of IL-2R, and indicate the usefulness of TNF in the analysis of signalling in autologous tumor-reactive CTL.     

Cellular and cytokine responses of the human central nervous system to intracranial administration of tumor necrosis factor α for the treatment of malignant gliomas

To elucidate the role of tumor necrosis factor (TNF) as a biological response modifier, we studied cellular and cytokine responses of the central nervous system to TNF administered intracranially in a phase I clinical trial for patients with malignant gliomas. Six patients received injections of TNF (1.25×10³–10×10³ U/injection) into the tumor cavities, and regional fluids (RF) and lumbar cerebrospinal fluids (CF) were serially sampled before and after the injections. Recruitment of neutrophils occurred, mostly peaking 8 h after TNF injection, and fewer numbers of CD4⁺ T cells and monocytes/macrophages migrated, subsequently peaking at 24 h. The CF leukocytosis persisted for 48 h and was associated with an increased level of neutrophil chemotactic activity in the CF. This neutrophil chemotactic activity was attributed to interleukin-8 (IL-8) by HPLC. The level of IL-6 activity in the CF and RF consistently increased; beginning 2 h after TNF injection and reaching the maximum between 8 h and 12 h. It returned to the basal level within 48 h. IL-1 was detected in the CF of three patients, its level peaking at 8 h. Prostaglandin E₂ also increased after injection of TNF, peaking between 4 h and 12 h and then gradually decreasing. Transforming growth factor was found in all cases tested and one patient showed a significant change after TNF injection. IL-2 activity, interferon (INF) activity, IFN, and granulocyte/macrophage-colony-stimulating factor were not detected in the CF or RF. In conclusion, TNF is biologically effective in inducing migration of immune cells and generating multiple cytokine responses in the human central nervous system.     

Steady state changes in mitochondrial electrical potential and proton gradient in perfused liver from rats fed a high fat diet

In this work the protonmotive force (p), as well as the subcellular distribution of malate, ATP, and ADP were determined in perfused liver from rats fed a low fat or high fat diet, using density gradient fractionation in non acqueous solvents.Rats fed a high fat diet, despite an enhanced hepatic oxygen consumption, exhibit similar p to that found in rats fed a low fat diet, but when we consider the two components of p, we find a significant decrease in mitochondrial/cytosolic pH difference (pH_m) and a significant increase in mitochondrial membrane potential (_m) in rats fed a high fat diet compared to rats fed a low fat diet, which tend to compensate each other. In rats fed a high fat diet the concentration ratio of malate and ATP/ADP does not reflect the changes in pH_m and _m, which represent the respective driving force for their transport.The findings are in line with an increase in substrate supply to the respiratory chain which is, however, accompanied by a higher energy turnover in livers from HFD rats. By this way the liver could contribute to the lack of weight gain from the high caloric intake in HFD rats.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Cytokines in Brain Ischemia—The Role of TNFα

1. The role of cytokines and other inflammatory mediators in the progression of ischemic brain injury is a new and exciting era of research. Evidence in support for a role for TNF in this respect is emerging as evidence on de novo upregulation of TNF following ischemia is now well established.2. TNF administered directly to the brain parenchyma elicits local microvascular injury in the form of pericapillary edema and leukocyte adhesion to cerebral capillaries.3. TNF administered into the cerebroventricular space prior to ischemia augment the extent of tissue damage and neurological deficits.4. Specific and potent inhibitors of TNF synthesis or TNF receptors must be developed and tried to prove firmly a role for TNF in ischemic brain injury.     

Tumor necrosis factor α modifies resistance to interferon α in vivo: First clinical data

Summary Patients with Philadelphia-positive chronic-phase chronic myelogenous leukemia (CML) resistant to interferon (IFN) were treated in a phase I/II study with recombinant human tumor necrosis factor to overcome IFN resistance. Doses of 40, 80, 120 or 160 µg/m² TNF were given as 2-h infusions on 5 consecutive days every 3 weeks. IFN (4 × 10⁶ IU/m² s.c., daily) treatment was continued. Six patients were treated, completing 1–24 (median, 12) treatment cycles. Five of the six patients achieved partial hematological remission, while the remaining patient had to stop treatment because of WHO grade 4 thrombocytopenia following the first TNF cycle. No complete hematologic remission or cytogenetic improvement was seen. Side-effects were similar to those described for both substances alone. Maximum tolerable TNF doses usually varied between 80 µg/m² and 160 µg/m². To examine possible pathways of TNF activity in these patients, interferon receptor status and (2–5)-oligoadenylate synthetase levels were examined in peripheral blood mononuclear cells. Both parameters remained unchanged during TNF treatment. These preliminary data point to significant clinical efficacy of additionally applyed TNF in IFN-resistant CML patients.     

The extrathyroidal conversion of T4 to T3 in the striped racer snake,Elaphe taeniura

The extrathyroidal conversion of thyroxine to triiodothyronine in the snake, Elaphe taeniura, has been determined in vitro. The liver, kidney and pancreas are important organs showing significant 5-deiodinase activity. The pancreas has a higher conversion rate (18.5±3.58 pmol·min^-1·mg protein^-1) than other vertebrate tissues that have been studied. The 5-deiodinase activity is dependent on substrate (thyroxine) concentration, cofactor, i.e. dithioerythritol concentration, temperature, duration of incubation and pH. It is sensitive to iopanoic acid, propylthiouracil, salicylate and propranolol. It is also indicative that the 5-deiodinase activity increased and decreased, respectively, in snakes with experimentally induced hyper- and hypo-thyroidism. These characteristics suggest that snake 5-deiodinase is similar to that of mammals, probably of type I category.Abbreviations ANOVA analysis of variance - BSA bovine serum albumin - BW body weight - cpm counts per minute - 5D 5-deiodinase - DTE dithioerythritol - EDTA ethylenediamine tetraacetate - IOP iopanoic acid - K _m Michaelis-Menten constant - L/D Light/Dark - MW molecular weight - NRS normal rabbit serum - PEG polyethylene glycol - %B percentage of added label found in the pellet - PTU propylthiouracil - RIA radioimmunoassay - rT₃ 3,5,5-triiodothyronine - SPSS Statistical Package for the Social Sciences - T3 3,5,3-triiodothyronine - T4 thyroxine - TRIS Tris (hydroxymethyl) aminomethane - Tx thyroidectomized - V _max maximum velocity of enzyme reaction     

The prebiotic chemistry of nucleotides

Diiminosuccinonitrile (DISN), formed by the oxidation of diaminomaleonitrile (DAMN), has been investigated as a potential prebiotic phosphorylating agent. DISN effects the cyclization of 3-adenosine monophosphate to adenosine 2, 3-cyclic phosphate in up to 39% yield. The mechanism of this reaction was investigated. The DISN-mediated phosphorylation of uridine to uridine monophosphate does not proceed efficiently in aqueous solution. The reaction of DISN with uridine-5-phosphate and uridine results in the formation of 2,2-anhydronucleotides and 2,2-anhydronucleosides respectively, and other reaction products resulting from an initial reaction at the 2- and 3-hydroxyl groups. The clay mineral catalysis of the cyclization of adenosine-3-phosphate was investigated using homoionic montmorillonites.     

Calpeptin suppresses tumor necrosis factor-α-induced death and accumulation of p53 in L929 mouse sarcoma cells

The cytokine tumor necrosis factor (TNF) induces caspase-dependent cell death in a subset of tumor cells. In this report, we show a novel suppressive effect of calpeptin, a calpain inhibitor, on TNF-induced cell death and accumulation of p53 in L929 mouse fibrosarcoma. Exposure to 10 ng/ml TNF induced cell death in >50% of L929 cells within 12 h and stimulated accumulation of p53 (8-fold). Preincubation of cells with calpeptin blocked both TNF-induced cell death and accumulation of p53 as examined with Western blot. TNF-induced accumulation of p53 was in part contributed by increase of p53 mRNA level (2.2-fold) in a calpeptin-insensitive manner. Interestingly, other calpain inhibitors tested did not show these effects like calpeptin and TNF treatment did not increase apparent calpain activity in L929 cells, suggesting that calpeptin may have another function besides targeting calpain. Expression of dominant negative mutant p53Val¹³⁵ reduced the incidence of TNF-mediated cell death. Taken together, our findings suggest that TNF induces calpeptin-dependent, but calpain-independent accumulation of p53 protein as a necessary step leading to death in L929 cells.     

Effects of hypoxia and intracellular iron chelation on hypoxia-inducible factor-1α and -1β in the rat carotid body and glomus cells

The present investigation provides for the first time, unambiguous information on the occurrence of hypoxia-inducible factors (HIF-1 and HIF-1 proteins) in normoxia (Nx) and their interaction with hypoxia (Hx) and intracellular Fe²⁺ chelation in the rat carotid body (CB) glomus cells. HIF-1 bound to HIF-1 translocated into the nucleus is identified on the basis of immunohistochemistry and immunofluorescence. In Nx, a weak expression of HIF-1 was observed in CB glomus cells. However, exposure of CB and glomus cells to Hx (Po₂7 Torr) and Nx with ciclopirox olamine (CPX, 5 M) for 1 h showed a significant (P<0.001) increase in HIF-1 protein. The CBs and glomus cells exposed to Nx, Hx, and Nx with CPX showed a constant level of HIF-1 protein expression. HIF-1 subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. Hydroxylation of HIF-1 by prolyl hydroxylase for proteasomal degradation was dependent on iron, 2-oxoglutarate, and oxygen concentration. The intracellular iron that acts as a cofactor for prolyl hydroxylase activity belongs to the labile iron pool and can be easily chelated. Thus, chelation of intracellular labile iron by CPX in Nx significantly increased HIF-1 in CB glomus cells. Thus, the results are consistent with the hypothesis that HIF-1 which is present in the glomus cells translocates to the nucleus during exposure to Hx and to CPX in Nx.     

Structure of F1-ATPases

F₁-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F₁-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type ₃₃ and ₂₂₂₂₂ were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F₁-ATPases.     

Bacillus stearothermophilus KP 1064 pullulan hydrolase

Summary A pullulan hydrolase of Bacillus stearothermophilus KP 1064 was purified homogeneously. The molecular weight, Stokes radius, sedimentation coefficient (s₂₀, w), extinction coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 115,000, 4.16 nm, 5.5 S, 1.92 cm²·mg^-1 and 4.4, respectively. The enzyme consisted of two identical subunits each comprising a methionine residue at the NH₂-terminus. The enzyme hydrolysed pullulan, amylopectin, soluble starch, amylose, -and -limit dextrins, - and -cyclodextrins, phenyl-d-maltoside, maltotriose, and maltopentaose. The main products from amylose and pullulan were maltose and panose, respectively. The substrate specificity, along with the pattern of products, suggested the assignment of the enzyme to a unique type of maltogenic -amylase (1,4-d-glucan glucanohydrolase, EC. 3.2.1.1).Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Sendai, 30 March 1983.     

Fibroblast growth factor increases TNFα-mediated prostaglandin E2 production and TNFα receptor expression in human fibroblasts

To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF, we tested the effect of FGF on TNF-mediated PGE₂ production and TNF receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE₂ production, it enhanced the amount of PGE₂ produced in response to TNF between 3 and 11-fold. FGF stimulated TNF-induced PGE₂ production independent of potential TNF-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF induced-PGE₂ production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF-induced PGE₂ production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF receptor expression. Untreated fibroblasts expressed 3,900 receptors/cell, while cells treated with FGF for 6h expressed 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF (K_d 3.8×10^–11 M). The FGF-mediated increase in TNF receptor expression and TNF-mediated PGE₂ production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF-mediated PGE₂ production in human fibroblasts.     

Deuterium isotope effects and fractionation factors of hydrogen-bonded A:T base pairs of DNA

Deuterium isotope effects and fractionation factors of N1...H3–N3 hydrogen bonded Watson–Crick A:T base pairs of two DNA dodecamers are presented here. Specifically, two-bond deuterium isotope effects on the chemical shifts of ¹³C2 and ¹³C4, ²¹³C2 and ²¹³C4, and equilibrium deuterium/protium fractionation factors of H3, , were measured and seen to correlate with the chemical shift of the corresponding imino proton, _H3. Downfield-shifted imino protons associated with larger values of ²¹³C2 and ²¹³C4 and smaller values, which together suggested that the effective H3–N3 vibrational potentials were more anharmonic in the stronger hydrogen bonds of these DNA molecules. We anticipate that ²¹³C2, ²¹³C4 and values can be useful gauges of hydrogen bond strength of A:T base pairs.     

Differential accumulation of mRNAs encoding extracellular and intracellular PR proteins in tomato induced by virulent and avirulent races of Cladosporium fulvum

Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a -1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa -1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic -1,3-glucanase and a basic 35 kDa -1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic -1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa -1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.     

Indirect induction of a Staphylococcus aureus prophage by P11de a plasmid phage hybrid.

Summary Studies on the rate of synthesis of the and subunits of RNA polymerase in haploid strains of Escherichia coli K12 containing poorly-suppressed rif _am ^o mutations provide conclusive evidence that synthesis of at least these two subunits is regulated.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Production of tumor necrosis factor α and interferon γ in interleukin-2-treated melanoma patients: Correlation with clinical toxicity

Summary Interleukin-2 (IL-2)-based immunotherapy regimens are accompanied by dose-limiting toxicity consisting of fever, tachycardia, chills and capillary leak syndrome. We hypothesized that the toxicity was caused by the induction and release of endogenous cytokines such as tumor necrosis factor (TNF) and interferon (IFN). We measured the serum levels of TNF and IFN in IL-2-treated melanoma patients and attempted a correlation with clinical toxicity. A total of 23 patients received either 6 × 10⁶ IU or 12 × 10⁶ IU Cetus IL-2/m² by i. v. bolus daily for 5 consecutive days on weeks 1, 3 and 5. Serum TNF and IFN levels were measured by enzyme-linked immunosorbent assay. Clinical toxicity was scored each day by objective measurements of hypotension, tachycardia, fever and chills/rigors. Clinical toxicity and IFN levels correlated nicely, peaking on the 5th day of each treatment cycle. The kinetics and magnitude of TNF production, however, were not predictable and did not correlate with either IFN or toxicity. Some patients had modest increases in TNF production while others had markedly increased levels during the second and third treatment weeks. Remarkably, these high levels persisted during nontreatment weeks and after completion of therapy. This clinical study demonstrates novel kinetics for immunoreactive TNF in IL-2 cancer patients, which do not correlate well with toxicity.This work was supported by NIH Grants CA 50 780 (J. E.) and CA 29 605, CA 12 582 (D. L. M.) and the U. C. Tobacco-Related Disease Research Program RT-62 (J. E.). J. E. is the recipient of an NCI Clinical Investigator Award (KO8-01360) and is a Dorothy and Leonard Straus Scholar at UCLA     

Phenotypic analyses of lymphokine-activated killer cells that release interferon γ and tumor necrosis factor α

Summary We recently reported that interleukin-2(IL-2)-activated peripheral blood lymphocytes and CD3⁺, lymphokine-activated killer (LAK) cell clones release tumor necrosis factor (TNF) and interferon (IFN) when stimulated with K562 erythroleukemia cells. We examined the phenotype of IL-2-activated peripheral blood leukocytes that secrete TNF and IFN when stimulated with K562 cells and demonstrated that TNF secretion is not due to the presence of contaminating mononuclear phagocytes. Further, we demonstrate that IL-2-activated natural killer (NK) cells release only IFN when stimulated with K562 cells while T lymphocytes exposed to monoclonal anti-CD3 and K562 cells secrete both TNF and IFN. However, T cells stimulated only with K562 cells did not release IFN or TNF while the admixture of these T cells with NK cells, when stimulated with K562 cells, released levels of TNF comparable to those produced by the unseparated cells. At present it is unclear whether only one or both effector cell types respond to K562 by releasing TNF or why the presence both cell types is needed.This work was supported by grants from the national Institutes of Health (CA 23074 and CA 17094) and the Arizona Disease Commission (8277-000000-1-0-YR-9301)