Atacicept in relapsed/refractory multiple myeloma or active Waldenström's macroglobulinemia: a phase I study.

BACKGROUND: Advanced multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are incurable B-cell malignancies. This is the first full clinical report of atacicept, a fusion protein that binds to and neutralises the B-cell survival factors, B-lymphocyte stimulator (BLyS) and A proliferation-inducing ligand (APRIL), in MM and WM. METHODS: In this open-label phase-I study, 16 patients with advanced disease (12 MM, 4 WM) received one cycle of five once-weekly subcutaneous injections of atacicept (2, 4, 7 or 10 mg kg(-1)). Patients with stable disease after cycle 1 entered an extension study (either two additional cycles (2, 4 and 7 mg kg(-1) cohorts) or 15 consecutive weekly injections of atacicept 10 mg kg(-1)). RESULTS: Atacicept was well tolerated, systemically and locally; the maximum tolerated dose was not identified. Of 11 patients with MM who completed initial treatment, five patients were progression-free after cycle 1 and four patients were progression-free after extended therapy. Of four patients with WM, three patients were progression-free after cycle 1. Consistent with atacicept's mechanism of action, polyclonal immunoglobulin isotypes and total B cells were reduced. Bone-marrow density, myeloma cell numbers and plasma concentrations of soluble CD138 also decreased. CONCLUSION: Atacicept is well tolerated in patients with MM and WM, and shows clinical and biological activity consistent with its mechanism of action.

Safety, pharmacokinetics and pharmacodynamics of recombinant human tumour necrosis factor-binding protein-1 (Onercept) injected by intravenous, intramuscular and subcutaneous routes into healthy volunteers.

The safety, pharmacokinetics and pharmacodynamics of recombinant human tumour necrosis factor-binding protein-1 (r-hTBP-1, Onercept) were investigated after intravascular and extravascular injection, in three studies in healthy volunteers. Subjects received Onercept as single intravenous doses of 5, 15, 50 and 150 mg, or single IV, IM, SC injection of 50 mg, or six repeated SC injections of 50 mg. Based on vital signs, hematology and blood chemistry, antibodies to study drug and local tolerability, r-hTBP-1 exhibited a remarkably safe profile. There was no evidence of alteration of hepatic oxidative metabolism. Recombinant-hTBP-1 showed linear pharmacokinetics that could be described by a triexponential model, and exhibited an initial half-life of 30 min, an intermediate half-life of 4 hours and a terminal elimination half-life of about 15 hours, although it was prolonged to 21 hours after repeated SC injections. The total clearance was estimated at 4 l/h. The initial (Vc) and steady state (Vss) volumes of distribution were approximately 4 l and 10 l, respectively. Renal clearance was minimal, representing around 2.5% of the total clearance, and remained constant after increasing doses of r-hTBP-1. The absorption was slow and biphasic. The immunoactivity of r-hTBP-1 was closely related to its biological activity, although the assessment was limited to only some of the samples. As anticipated in normal healthy volunteers, the pharmacodynamic response was generally not different from placebo. Total TNF-alpha serum levels increased slightly, 1 hour following IV administration of 50 mg and 150 mg r-hTBP-1. However, no major increase in the active entity levels (free TNF-alpha) was observed. In addition, no TNF-alpha-driven biological response was observed, i.e. C-reactive protein, IL-6 and fibrinogen remained almost constant, as did transferrin and albumin. Its safety profile and pharmacokinetic characteristics make Onercept a candidate drug suitable for antagonising pathologically high levels of TNF-alpha as reported in inflammatory, immune and cardiovascular diseases.

Influence of interferon beta-1a dose frequency on PBMC cytokine secretion and biological effect markers.

Interferon-beta regimens for immune-mediated diseases, such as multiple sclerosis (MS), have not been compared regarding their biological effects. In this randomized, parallel-group, placebo-controlled study, cytokine secretion by mitogen-stimulated PBMCs and serum response markers were assessed in volunteers receiving subcutaneous recombinant IFN beta-1a (Rebif, Ares-Serono) 22 microg once a week (QW), 22 microg three times a week, 66 microg QW, or placebo. The production of IL-1beta, IL-6, IFN-gamma, TNF-alpha and TNF-beta markedly decreased during 24-48 h after each injection, with limited dose-dependency and no evidence of tolerance or effect augmentation over 1 month. IL-10 secretion remained unchanged. The increase in serum beta2-microglobulin, neopterin and 2-5A-synthetase was more sustained. Thus, IFN-beta-induced immunomodulation in vivo strongly depends on the administration schedule, the time-integrated effect being 2-3 times greater when a same weekly dose is divided in three injections.

Interferon-beta inhibits activated leukocyte migration through human brain microvascular endothelial cell monolayer.

Perivascular leukocyte infiltration into the central nervous system is characteristic of multiple sclerosis (MS) pathology. Interferon-beta (IFN-beta) has shown efficacy in the treatment of patients with MS, but the relevant mechanisms remain incompletely understood. In this study the effects of IFN-beta on leukocyte transendothelial migration were investigated using cells relevant to MS pathogenesis, namely human brain microvascular endothelial cells (HB-MVEC). Activated, but not resting leukocytes exhibited a high transendothelial migration capacity. HB-MVEC prestimulated with tumor necrosis factor (TNF) and IFN-gamma significantly promoted leukocyte transendothelial migration. IFN-beta inhibited the activated leukocyte transendothelial migration on TNF/IFN-gamma-activated HB-MVEC in a dose-dependent manner. A matrix metalloproteinase (MMP) inhibitor and monoclonal antibodies to lymphocyte function antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1), but not to very late antigen-4 or to vascular cell adhesion molecule-1 significantly inhibited the transendothelial migration of stimulated leukocytes, suggesting that this phenomenon involves the LFA-1/ICAM-1 interaction and MMP. However IFN-beta did not interfere with the binding of leukocytes to HB-MVEC unless IFN-beta was preincubated with leukocytes or added to HB-MVEC at the time of stimulation. Furthermore IFN-beta did not modulate the expression of adhesion molecules on either stimulated leukocytes or activated HB-MVEC, but partially reduced TNF and interleukin-1 production from stimulated leukocytes during coculture with HB-MVEC. Interestingly, in the presence of IFN-beta, a significant down-regulation of MMP-9 release from stimulated leukocytes was found, especially for the activated form of MMP-9. These results indicate that inhibition of leukocyte transendothelial migration is an important mechanism accounting for the beneficial effects of IFN-beta in the treatment MS patients.

Effects of urinary gonadotrophin preparations on human in-vitro immune function.

Among commercially available urinary human menopausal gonadotrophin (HMG) material, gonadotrophins comprise <5% of the total protein content. Thus, during a typical ovarian stimulation cycle with HMG, several milligrams of non-relevant proteins are administered that may lead to unwanted side effects, including allergic or other hypersensitivity reactions. The effects of two recombinant and four urinary gonadotrophin preparations of different purity upon the function of T cells from healthy blood donors were studied. Only one of the HMG preparations significantly enhanced the spontaneous proliferation of peripheral blood mononuclear cells. Phytohaemagglutinin-induced proliferation was not modified by any preparation, while two preparations significantly increased proliferation in the mixed lymphocyte reaction. Three of the HMG preparations induced the release of interleukin (IL)-1. Highly purified FSH, either urinary or recombinant, showed no effect. None of the preparations induced detectable IL-2 production, whereas only one HMG preparation tended to decrease IL-2 secretion. No major changes in CD25 expression were induced by any of the gonadotrophins. Cytokine measurement by immunoassays detected only IL-1beta in two commercially available preparations. The various effects exhibited by the crude urinary preparations were not a result of the gonadotrophin content and differed from product to product, suggesting that the contaminants present in these preparations are not identical. This could contribute to unpredictable clinical manifestations of allergic or other immune reactions.

Modulation of soluble and membrane-bound TNF-induced phenotypic and functional changes of human brain microvascular endothelial cells by recombinant TNF binding protein I.

In this study, the effects of TNF binding protein I (TBP I) on TNF-induced changes of human brain microvascular endothelial cells (MVEC) were investigated. TBP I completely abolished TNF-induced IL-6 production and E-selectin induction, while it partially inhibited TNF-induced IL-8 production and up-regulation of ICAM-1 and VCAM-1. Moreover, TBP I significantly inhibited TNF-induced cytotoxicity and leukocyte adherence on human brain MVEC. The inhibitory activity of TBP I for TNF was dose-dependent and related to the time of administration after TNF stimulation. In addition, TBP I inhibited membrane-bound TNF induced activation of human brain MVEC, but the concentration required was about 10-fold higher than that for soluble TNF. These results indicate a therapeutic potential for TBP I in diseases of the central nervous system associated with TNF overproduction.

The coagulation and fibrinolytic responses of baboons after in vivo thrombin generation--effect of interleukin 6.

Disseminated intravascular coagulation (DIC) may lead to severe thrombotic or hemorrhagic complications. The present work was undertaken to study the effect of interleukin 6 (IL-6) on variations of key coagulation and fibrinolytic parameters in plasma in a baboon model of experimental DIC induced by injection of factor Xa and phospholipids at dosages leading to partial (48%) or complete fibrinogen depletion. Transient increases of D-dimer, fibrinopeptide A, thrombin-antithrombin and the activated partial thromboplastin time were observed. Each parameter had a particular (time and Xa/phospholipid dose dependent) pattern of changes. The principal effect of IL-6 was a more rapid restoration of fibrinogen concentrations and of overall coagulation tests. Injection of factor Xa/phospholipids led also to a rapid increase of tissue-type plasminogen activator (t-PA) the extent of which was dependent on Xa/phospholipid dose. Pretreatment with IL-6 induced a threefold increase of basal t-PA and a corresponding increase of the t-PA response. Plasminogen activator inhibitor type 1 (PAI-1) concentrations did not change after low dose Xa/phospholipids, but increased eightfold after high dose Xa/phospholipids, IL-6 pretreatment induced within 8 h a twentyfold increase of PAI-1 but no further increase was observed after injection of factor Xa/phospholipids. Thus, in vivo thrombin generation leads to dynamic modifications of the coagulation and fibrinolytic systems. The principal effect of IL-6 is a more rapid normalization of overall coagulation tests, due to normalization of fibrinogen, and an increased t-PA release response which is partially counteracted by increased PAI-1 concentrations.

The effect of factor Xa/phospholipid infusion on the acute phase response in baboons.

Disseminated intravascular coagulation (DIC) is a frequent complication of septicemia or tissue injury and may be accompanied by elevations of interleukin-6, a mediator of the acute phase response. It is not known whether thrombin or fibrin deposition may directly induce an acute phase response. To study this, we employed a baboon model of in vivo thrombin generation, induced by the administration of purified bovine Factor Xa and phospholipid vesicles. Two Xa/phospholipid dosages were used, a low dosage (2 animals) leading to a rapid 49% decrease in fibrinogen and a high dosage (two injections at 5h interval; 3 animals) leading to complete fibrinogen depletion. Thereafter, fibrinogen levels increased in both treatment groups, reached a maximum of 2.52 +/- 0.23 g/l (mean +/- SE, n = 5; p < 0.01 with respect to basal levels) at day 2, and returned to normal by day seven. In five control (injection of 0.15% NaCl) baboons no significant changes of fibrinogen were observed (maximal values: 1.88 +/- 0.12 g/l). Serum concentrations of C-reactive protein, an acute phase protein, increased from 3.7 +/- 0.4 mg/l to a maximum of 33.0 +/- 7.3 at day one, which was five-fold higher (p < 0.01) than in control animals at day one (6.2 +/- 0.5 mg/l). Transient increases were observed within 6h for interleukin-6 from basal values of 6.2 +/- 1.7 ng/l to peak plasma levels of 42.9 +/- 21.4 ng/l, a value three-fold higher (p = 0.07) than in control animals (14.8 +/- 4.0 ng/l). The preliminary results of this observational study suggest that factor Xa/phospholipid infusion is followed by an acute phase response, leading after one day to significant increases of fibrinogen and of C-reactive protein.

Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions.

BACKGROUND: Glycoprotein hormones influence the development and function of the ovary, testis and thyroid by binding to specific high-affinity receptors. The extracellular domains of these receptors are members of the leucine-rich repeat (LRR) protein superfamily and are responsible for the high-affinity binding. The crystal structure of a glycoprotein hormone, namely human choriogonadotropin (hCG), is known, but neither the receptor structure, mode of hormone binding, nor mechanism for activation, have been established. RESULTS: Despite very low sequence similarity between exon-demarcated LRRs in the receptors and the LRRs of porcine ribonuclease inhibitor (RI), the secondary structures for the two repeat sets are found to be alike Constraints on curvature and beta-barrel geometry from the sequence pattern for repeated beta alpha units suggest that the receptors contain three-dimensional structures similar to that of RI. With the RI crystal structure as a template, models were constructed for exons 2-8 of the receptors. The model for this portion of the choriogonadotropin receptor is complementary in shape and electrostatic characteristics to the surface of hCG at an identified focus of hormone-receptor interaction. CONCLUSIONS: The predicted models for the structures and mode of hormone binding of the glycoprotein hormone receptors are to a large extent consistent with currently available biochemical and mutational data. Repeated sequences in beta-barrel proteins are shown to have general implications for constraints on structure. Averaging techniques used here to recognize the structural motif in these receptors should also apply to other proteins with repeated sequences.

Glycosylated recombinant human tumor necrosis factor binding protein-1 reduces mortality, shock, and production of tumor necrosis factor in rabbit Escherichia coli sepsis.

OBJECTIVE: To examine the effect of glycosylated recombinant human tumor necrosis factor binding protein-1 (r-hTNF binding protein-1), the extracellular domain of the tumor necrosis factor receptor p55 produced in mammalian cells, in a rabbit model of circulatory shock due to Escherichia coli. DESIGN: Prospective, randomized, controlled trial. SETTING: University hospital research laboratory. SUBJECTS: Eighteen female, New Zealand white rabbits. INTERVENTIONS: Anesthetized rabbits, infused with E. coli (10(9) organisms/kg), were pretreated with either r-hTNF binding protein-1 or saline. Mean arterial pressure, central venous pressure, cardiac output, and heart rate were recorded every 20 mins for 1 hr before, and for 4 hrs after, the infusion of E. coli. Blood samples were obtained at 1-hr intervals for platelet count and white blood cell count, r-hTNF binding protein-1, and tumor necrosis factor (TNF) measurements. MEASUREMENTS AND MAIN RESULTS: Administration of r-hTNF binding protein-1 resulted in improvement of mean arterial pressure, cardiac output, and systemic vascular resistance, as compared with the vehicle-treated group (p < .05). Treatment with r-hTNF binding protein-1 was associated with 100% survival, as compared with 55.6% of the saline-treated rabbits (p < .05). Approximately 85% of r-hTNF binding protein-1 was cleared from the circulation 1 hr after the bolus injection (from 171 +/- 27 micrograms/mL at time = 0, to 27 +/- 4 micrograms/mL at 60 mins, decreasing to 6 +/- 2 micrograms/mL for the next 3 hrs). The r-hTNF binding protein-1-treated rabbits had lower serum TNF bioactivity during the first 2 hrs (p < .01). The decreased bioactivity of TNF was confirmed by a specific radioimmunoassay for rabbit TNF. However, at 4 hrs, the vehicle-treated rabbits had lower serum bioactive TNF concentrations (p < .05). The decrease in TNF concentrations in the r-hTNF binding protein-1-treated rabbits resulted from decreased production and, in part, from carry-over of r-hTNF binding protein-1 into the bioassay. CONCLUSIONS: Treatment with r-hTNF binding protein-1 improved hemodynamic variables and survival of E. coli-challenged rabbits. Administration of r-hTNF binding protein-1 suppressed bioactivity of TNF in the circulation of these rabbits, and the production of TNF as well.

In vivo modulation of coagulation and fibrinolysis by recombinant glycosylated human interleukin-6 in baboons.

To assess the effect of interleukin-6 (IL-6) on the coagulation and the fibrinolytic systems, we administered a single subcutaneous injection of recombinant glycosylated human interleukin-6 (r-hIL-6) 100 micrograms per kg body weight) to four baboons (Papio ursinus). Four saline injected baboons served as controls. In serial plasma or serum samples collected over a period of seven days we measured several key parameters of the coagulation and the fibrinolytic systems, IL-6 and a set of acute phase proteins. Three hours after the injection, the serum IL-6 levels peaked at 50 ng/ml and then gradually declined with a terminal half-life of around 4 hours. The biological efficacy was demonstrated by the significant increases of several acute phase proteins, circulating platelets and the decrease of prealbumin and fibronectin. Between days 1 and 3, marked effects on the coagulation system were observed with a prolongation of the activated partial thromboplastin time, prothrombin time and thrombin time. Plasma concentrations of fibrinopeptide A and D-dimer increased. The antithrombin III antigen and activity levels decreased, but the thrombin-antithrombin III complex concentrations did not change. The fibrinolytic system rapidly showed striking modifications after 6-8 hours, the concentrations of tissue-type plasminogen activator and of plasminogen activator inhibitor type 1 peaked at respectively four and thirty times the basal concentrations. No changes were seen in the control group. We conclude that besides its well-known acute phase inducing and hematopoietic activities, subcutaneous rhIL-6 also modulates several parameters of the coagulation and the fibrinolytic systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of haptoglobin expression in IL-6 treated HepG2 cells by ELISA and by RNA hybridization--evaluation of a quantitative method to measure IL-6.

Interleukin-6 (IL-6) is known to be an important modulator of acute phase (AP) protein expression in hepatocytes both in vivo and in vitro. In the present study the inducing activity of IL-6 on the expression of the AP protein haptoglobin (HP) by the human hepatoma cell line HepG2, has been evaluated. HP mRNA inducibility was analysed by Northern and slot-blot hybridization, while HP protein was detected by means of an ELISA procedure. A dose-response relationship from 0.3 to 4.8 ng/ml of a human recombinant IL-6 preparation derived from a Chinese hamster ovary (CHO) cell line was observed after 48 h of treatment. Comparable results were obtained by analysing both HP mRNA expression and HP protein secretion. Detectable induction of HP protein secretion was observed with as little as 25 pg/ml of IL-6. The effect of IL-6 was potentiated by dexamethasone, while an inhibition on HP mRNA inducibility could be prevented by lowering the foetal calf serum (FCS) concentration to 1%. Preliminary data indicate that neither IL-1 beta nor TNF-alpha were able to induce significantly HP mRNA expression and protein secretion. The activity ratio between two IL-6 preparations (from CHO and E. coli cells) obtained with a conventional IL-6 bioassay (i.e., T1165 cell growth assay) was comparable to that obtained in the induction of HP expression. The nominal specific activity of the CHO-derived IL-6 was two to three times higher with both responses.

Protective effect of natural TNF-binding protein on human TNF-induced toxicity in mice.

The in vivo protective effect of urinary TNF-binding protein (uTBP) on acute TNF-induced lesions and lethality was assessed in BALB/c mice. Two animal models, the local Shwartzman reaction and galactosamine (GaLN) induced TNF sensitization, were used. In the former, local cutaneous haemorrhagic necrosis induced by 10 micrograms of recombinant human TNF alpha (r-hTNF) was prevented with iv doses of uTPB as low as 1 microgram when administered concomitantly or 10 micrograms when injected intravenously 60 min before or 30 min after the lesion eliciting-dose of r-hTNF. In the latter model, injection of 1 microgram or r-hTNF caused the death of all mice within 36 h. Either 100 or 250 micrograms of uTBP given intravenously simultaneously with r-hTNF/GaLN totally prevented this mortality. In contrast to anti-human TNF monoclonal antibodies, these very same doses of uTBP significantly protected mice even when injected after the lethal r-hTNF dose. These data confirm in relevant in vivo pathological models the TNF inhibiting capacity of the natural soluble TNF receptor I.

Urinary TNF-binding protein (TNF soluble receptor) protects mice against the lethal effect of TNF and endotoxic shock.

We tested the effect of urinary TNF-binding protein (uTBP) on the toxic effect of TNF (0.5 micrograms/mouse, i.v.) in adrenalectomized mice sensitized with IL-1 to increase susceptibility to TNF. In this experimental model, mortality was 67%, but decreased to 13% when uTBP (250 micrograms/mouse, i.v.) was administered simultaneously with TNF. The protective effect of uTBP was dose-dependent, and time course experiments indicated a protective effect when uTBP was administered before or up to one hour after TNF. Some protection was also obtained when uTBP was given three hours after TNF. Urinary TBP improved the survival of mice after a lethal dose of LPS (1.2 mg/mouse, i.p.), suggesting its possible efficacy in the therapy of septic shock or other TNF-mediated pathologies.

Recombinant glycosylated human interleukin-6 accelerates peripheral blood platelet count recovery in radiation-induced bone marrow depression in baboons.

This report was aimed at confirming the potential clinical use for a genetically engineered glycosylated human interleukin-6 (rhIL-6) in hematopoiesis. Its tolerance and efficacy were assessed on hematopoietic restoration after neutron radiation-induced bone marrow injury on baboons, which represent an adequate model of parallelism for studying hematology in the human. The particular neutron radiation absorption pattern in the body allows the preservation of underexposed bone marrow areas that mimics an autotransplantation-like situation. An initial dose finding study (1 microgram up to 20 micrograms/kg/d for 8 consecutive days) in normal baboons established a dose-dependent response regarding the peripheral platelet count (range of increase, 1.5- to 4-fold). A significant elevation in white blood cell (WBC) count, as well as a substantial reversible normochromic normocytic anemia, were observed for the highest doses only (10 and 20 micrograms/kg/d). All rhIL-6 administered doses were clinically well tolerated. In myelosuppressed baboons, a selected dose of 10 micrograms/kg/d of rhIL-6 for 13 consecutive days significantly lessened the degree of induced thrombocytopenia as compared with the control group (P = .01) and shortened the time to occurrence of the nadir, showing that the onset of recovery occurs much earlier, ie, an average of 5 days (P = .003), in the treated group. Moreover, this accelerated platelet recovery is evidenced by an 8-day shorter mean time back to baseline values (P = .03) in the rhIL-6--treated animals. At this dose no effect was observed on the WBC recovery pattern. Importantly rhIL-6 did not accentuate the radiation-induced anemia and was clinically well tolerated. All tested monkeys recovered from their induced pancytopenia and no animal loss was recorded. IL-6, tumor necrosis factor, and IL-1 blood measurements are reported. In conclusion, rhIL-6 is a potent thrombopoietic factor for the treatment of induced thrombocytopenia in nonhuman primates at a clinically well-tolerated dose.

Pharmacokinetics and tissue distribution of human urinary tumor necrosis factor binding protein in mice.

Iodinated natural human urinary tumor necrosis factor binding protein I (125I-uTBP) was iv injected into BALB/c mice, and its pharmacokinetics and tissue distribution were assessed during a short-term (0-1 hr) and for a long-term (0-24 hr) period. The blood 125I-uTBP concentration displayed a biphasic pattern that was adequately described by a biexponential function with estimated half-lives of 0.1 and 3.8 hr. The apparent volume of distribution (Vc) of the central compartment was 3 ml, which approximated the mouse blood volume. The clearance (CL) derived either from a model-dependent or a model-independent method of analysis was 2.5 and 2.9 ml/hr, respectively. One hr after the iv administration of 125I-uTBP, the radioactivity accumulated in the major organs and tissues. The highest concentrations in terms of pg per organ were seen in the skin and in the liver. When expressed as pg 125I-uTBP per mg organ, the distribution was the highest in the gallbladder, bladder, kidneys, and lungs. At 24 hr, the distribution of 125I-uTBP represented about 10% of the amount measured at 1 hr. The rank order of accumulation of the radiolabeled uTBP in the major organs, expressed as pg per organ at 24 hr was skin greater than liver greater than kidneys greater than lungs greater than gut greater than spleen greater than gallbladder.

Differential effect of gamma-irradiated and heat-treated lymphocytes on T cell activation, and interleukin-2 and interleukin-3 release in the human mixed lymphocyte reaction.

Heat-inactivated (45 degrees C/1 hr) lymphocytes selectively activate suppressor T cells in the mixed lymphocyte reaction (MLR), while no significant proliferation and cytotoxic T lymphocyte activation can be detected. It is not well understood why hyperthermic treatment abolishes the stimulatory capacity of lymphocytes since HLA-DR molecules remain detectable immediately following heat exposure. In order to further characterize the requirements for Ts activation we studied the effects of hyperthermic treatment on cellular protein and DNA synthesis and cell surface protein expression in proliferating T and B cells; interleukin (IL)-1, IL-2, and IL-3 release following allogeneic stimulation with heat treated cells (HMLR); and IL-2 receptor expression as an indicator of T cell activation in the HMLR. Hyperthermic treatment reduced cellular protein synthesis as estimated by 14C-leucine uptake to about 15%, and DNA synthesis (3H-thymidine incorporation) to about 5% of untreated control cells. In contrast to y-irradiated cells, viability of heated cells rapidly declined within the first 24 hr. Hyperthermic treatment doubled binding of mouse immunoglobulin paralleled by an increased expression of IL-2 and transferrin receptors, while expression of HLA-DR and 4F2 proteins appeared unchanged. Stimulation with heated cells triggered the release of IL-1- and an IL-3-like bioactivity but did not induce IL-2 synthesis and/or release, thus explaining the lack of proliferation in the HMLR. Addition of exogenous IL-2 but not IL-1 restored HMLR proliferation. A comparison of allostimulation with y-irradiated and heat-treated cells revealed that significantly fewer T cells were induced to express IL-2 receptors at day 3 (14% vs. 8%, P less than 0.001) and at day 6 (42% vs. 21%, P less than 0.05) with heat-inactivated stimulators. We conclude that metabolically compromised lymphocytes activate Ts and are sufficient to stimulate IL-1 and IL-3 synthesis but do not transmit an unknown signal required for the activation of IL-2 synthesis and IL-2 receptor expression on a yet-to-be-defined T cell subset.

A unique T-cell receptor complex expressed on human fetal lymphocytes displaying natural-killer-like activity.

We have recently derived a series of cloned cell lines displaying natural killer (NK) cell-like activity from normal human fetal blood (25 weeks). The lines were obtained after repeated stimulation of mononuclear cells with allogeneic Epstein-Barr virus (EBV)-transformed B lymphocytes and are interleukin-2 (IL-2) dependent. Initial characterization of the clones has been reported previously. Certain of these clones have been found to have unusual surface characteristics, namely, they are recognized by several well-defined anti-T3 antibodies, but do not react with WT31, which is thought to recognise an invariant epitope of the human (Ti-alpha beta) structure. Transcription of the genes encoding the alpha- and beta-chains of the T-cell receptor was assessed in two of these clones (F6A4 and F6C7). Ti-beta genes were found to be expressed, whereas alpha messenger RNA was not detected in Northern blot analysis. These data strongly suggest that these cells do not produce a stoichiometric T3/Ti-alpha beta receptor complex. However, experiments performed with a monoclonal antibody (anti-NKFi) developed against F6C7 cells demonstrated the existence of a unique clonotypic structure [relative molecular mass (Mr) 85,000 (85K)] which is surface-associated with T3 proteins. Furthermore, both anti-T3 and anti-NKFi were found to block cytotoxic effector function. Together, the results support the view that T3 proteins are involved in non-major histocompatibility complex (MHC)-restricted cytotoxic reactions mediated by certain circulating fetal lymphocytes which are likely to use a clonotypic structure distinct from both the 'first' (alpha beta) and the putative 'second' (gamma delta) T-cell receptor to recognize their target. The present studies were designed to characterize this structure.

Allogeneic responses of human fetal (22- to 25-week) peripheral blood lymphocytes: preferential recruitment of cytotoxic effector cells with both CTL activity and NK-like function.

In the present study, we have characterized human cytotoxic effector lymphocytes generated following in vitro immunization of normal fetal (22- to 25-week) peripheral blood mononuclear cells (FPBMC) by an allogeneic Epstein-Barr virus-transformed B-cell line termed LAZ388. Primary stimulations led to strong FPBMC proliferation. However, subsequent addition of LAZ388 cells to the cultures on Day 8 did not trigger conventional secondary responses. In fact, further proliferation of activated FPBMC required the addition of exogeneous interleukin 2. Cytotoxic activity generated in the mixed-lymphocyte reactions was assayed against LAZ388 immunizing cells as well as against the highly susceptible natural killer (NK) target cell line K562. Eight days after stimulation by LAZ388, there was no specific lysis and a moderate NK-like activity. However, following second and subsequent stimulations a strong killing was measured against both LAZ388 and K562 cells. Blocking experiments performed with relevant monoclonal antibodies suggested that cytotoxicity against immunizing cells was conventionally directed at MHC gene products. Effector cells were further studied using cloning procedures; it was found that all cloned cell lines able to kill LAZ388 cells were also strongly active against K562. Both types of cytotoxic function appeared to be mediated via surface receptors physically or at least functionally associated with T3 proteins.

Characterization of antileukemia cells' cytotoxic effector function. Implications for monitoring natural killer responses following allogeneic bone marrow transplantation.

We have studied here cytotoxic function of three cloned cell lines--TC12, 48, and 50--derived from circulating lymphocytes that were potentially able to eliminate residual tumor cells in a patient transplanted for treatment of acute lymphocytic leukemia. These cloned cells, which have both phenotypic and functional characteristics of natural killer lymphocytes, were tested in chromium release assays against a panel of 16 uncultured populations of leukemia cells. In addition, their activity was compared with that of cloned and uncloned NK cells from normal individuals. It was found that TC clones induced a much weaker degree of killing against fresh tumor cells compared with conventional NK target cell lines such as K562 or MOLT 4. In addition, there was great heterogeneity in their individual lytic capacity against the various leukemia blasts (TC12, 48, and 50 cells killed in a significant fashion, respectively 7, 1, and 4 of the 16 leukemias), reflecting the functional diversity of normal NK cell populations. Thus, for a fraction of leukemias, there was no correlation between lytic ability of TC cells and that of uncloned lymphokine-activated large granular lymphocytes from normal peripheral blood. Together, these results support the view that direct identification of patients' cytotoxic lymphocytes screened against in vivo relevant tumor cells is necessary to evaluate potentially beneficial immunologic responses in the context of bone marrow transplantation.