The neutralization of type I IFN biologic actions by anti-IFNAR-2 monoclonal antibodies is not entirely due to inhibition of Jak-Stat tyrosine phosphorylation.

A panel of monoclonal antibodies (mAb) derived against human interferon-alpha/beta receptor-2 (IFNAR-2) was evaluated for their ability to antagonize the biologic effects of type 1 interferons (IFN-alpha1, IFN-alpha2a, and IFN-beta). Anti-IFNAR-2 mAb 117.7, 35.9, 53.2, and 51.44 neutralized type I IFN-mediated antiviral, antiproliferative, and major histocompatibility complex (MHC) class I upregulation functions. However, only mAb 51.44 neutralized IFN-alpha2a and IFN-beta-mediated natural killer (NK) cell cytotoxicity. In BIAcore and cell binding studies, only mAb 51.44 and 234.28 inhibited IFN-alpha2a and IFN-beta binding to its receptor. The receptor blockade by mAb 51.44 and 234.28 resulted in the inhibition of IFN-alpha2a and IFN-beta-induced tyrosine phosphorylation of Jak1, Tyk2, Stat1/2/3, and IFNAR-1/2 and inhibition of IFN-stimulated gene factor 3 (ISGF3) formation. mAb 117.7, 35.9, and 53.2, although antagonists of IFN's biologic activities, did not block the binding of IFN-alpha/beta to its receptor. The 117.7 mAb, representative of this class of receptor nonblocking mAb, induced hyper-tyrosine phosphorylation of IFNAR-2 in the presence of IFN-alpha/beta but did not inhibit IFN-alpha/beta-induced Jak-Stat tyrosine phosphorylation and ISGF3 complex formation. These results show that the neutralization of type I IFN biologic actions by anti-IFNAR-2 mAb cannot be entirely explained by inhibition of Jak-Stat tyrosine phosphorylation.

Soluble human lymphocyte activation gene-3 modulates allospecific T cell responses.

Lymphocyte activation gene (LAG)-3, a member of the Ig superfamily, has been characterized as an activation antigen of T cells and NK cells. LAG-3 has been proposed as an alternate ligand for HLA class II due to some sequence homology and similarities in exon-intron organization with CD4. Here, we report the functional evaluation of a soluble Ig fusion molecule of human LAG-3 (LAG-3-Ig) in T cell activation assays. Cytofluorimetry studies revealed LAG-3-Ig binding predominantly to class II-expressing cells. In functional assays, inhibition of primary allogeneic mixed lymphocyte response (MLR) and murine-human xenogeneic MLR was observed in the presence of LAG-3-Ig. Effects of LAG-3-Ig addition were not observed on mitogen-, recall antigen- or superantigen-mediated stimulation. Cytotoxic T lymphocyte effector functions were also not affected by LAG-3-Ig. Inhibition of alloresponses by LAG-3-Ig occurred within the first 24 h of activation, resulting in a strong inhibition of IL-2 production. Unlike blockade of the CD28 receptor, however, LAG-3-Ig-mediated inhibition could not be reversed by exogenous IL-2 supplementation. Cytofluorimetric analysis of the phenotype of cells exposed to LAG-3-Ig in MLR cultures revealed a decrease in IL-2 receptor expression (CD25) on CD4+ cells in all donors tested. Based on the results from these studies, we conclude that LAG-3-Ig inhibits alloresponses of naive peripheral blood lymphocytes, by blocking the activation of a subpopulation of allo reactive cells.

Interleukin-12-mediated killer activity in lung cancer patients.

The authors investigated the interleukin (IL)-12-inducible killer activity of blood mononuclear cells (MNC) from 30 untreated primary lung cancer patients and 24 control subjects. Cytotoxicity was assayed as 4-h 51Cr release from Daudi lymphoma cells or lung cancer cells (H-69, N-291 and PC-9). MNC from lung cancer patients exhibited similar killer activity to those from control subjects after in vitro incubation with IL-12 for 4 days. Effective killer induction by IL-12 was observed even in MNC from advanced lung cancer patients and patients with small cell lung cancer. IL-12 and a suboptimal dose of IL-2 had additive effects in inducing killer activity in MNC from both lung cancer patients and control subjects. On the other hand, with an optimal dose of IL-2, IL-12 suppressed killer induction. Addition of IL-12 alone or in combination with IL-2 resulted in interferon (IFN)-gamma production by MNC from lung cancer patients as well as control subjects. These observations suggest that IL-12 could be useful for immunotherapy of lung cancer in humans.

Effects of the callipyge phenotype on serum creatinine, total cholesterol, low-density lipoproteins, very-low-density lipoproteins, high-density lipoproteins, and triacylglycerol in growing lambs.

The goals of this study were to investigate the effects of the callipyge (CLPG) phenotype on serum creatinine and lipid profiles of growing lambs. Preliminary studies in our laboratories indicated that creatinine may have utility in distinguishing the CLPG phenotype and that expression of the CLPG gene altered concentrations of serum total cholesterol (TC). As a result, in this study, we examined the influence of the CLPG gene on concentrations of creatinine, TC, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), and triacylglycerol (TG) at varying stages of maturity in lambs. Ten homozygous (c/c) Polypay ewes were crossed with Dorset rams heterozygous for the CLPG gene (C/c). From this cross, 20 lambs (13 females and 7 males) were born, of which 11 were homozygotic (c/c) and 9 were heterozygotic (C/c; CLPG) based on muscle weights and longissimus dorsi (LD) area at slaughter. Blood samples were taken at monthly intervals and serum lipid constituents were assayed. At 1 mo of age, no differences (P > .05) in plasma lipids were detectable between phenotypes. However, at 2 mo age, CLPG lambs had higher (P < .01) concentration of TG, TC, HDL, and VLDL compared to homozygotic (c/c) lambs. Triglycerides and VLDL were elevated (P < .05) in CLPG lambs at 3 mo of age. By slaughter, no differences (P > .05) in serum lipid constituents were detectable between genotypes. Hence, the increase in serum TC is due to elevated levels of HDL and VLDL. These observations indicate that creatinine may be used to distinguish CLPG lambs and that the CLPG gene alters serum lipid profiles during the postnatal period.

Influence of systemic chemotherapy on anti-P-glycoprotein antibody-dependent cell-mediated cytotoxicity in patients with small cell lung cancer.

Anti-P-glycoprotein antibody (MRK-16)-dependent cell-mediated cytotoxicity (ADCC) by blood mononuclear cells (MNC) was examined in patients with small cell lung cancer (SCLC) before and after systemic chemotherapy. The effect of in vitro treatment of MNC with interleukin (IL)-2 and macrophage-colony-stimulating factor (M-CSF) was also examined. The ADCC reaction was assessed by a 6 h 51Cr-release assay using a multidrug-resistant (MDR) SCLC cell line (H69/VP cells). The MRK-16 monoclonal antibody was able to augment spontaneous cytotoxicity by MNC, even in SCLC patients. Pretreatment of MNC with IL-2 significantly augmented their ADCC ability in SCLC patients, while M-CSF had no effect on ADCC activity. After the first cycle of systemic chemotherapy, the ADCC activity tended to decline, but ADCC of MNC pretreated with IL-2 was not affected. The results suggest that anti-P-glycoprotein antibody, in combination with a cytokine such as IL-2, may be therapeutically useful against human SCLC resistant to chemotherapeutic drugs.

Human alveolar macrophages augment natural killer cell stimulatory factor (interleukin-12)-inducible killer activity from autologous blood lymphocytes.

Interleukin-12 (IL-12), also known as natural killer cell stimulatory factor (NKSF), was found to induce cytotoxic activity from human blood T cells and NK cells. The present study was undertaken to examine the effect of human alveolar macrophages (AM) on induction by IL-12 cytotoxic cells from blood lymphocytes. AM were obtained by bronchoalveolar lavage from healthy donors. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were also isolated by centrifugal elutriation from peripheral blood of the same donors. Cytotoxicity of lymphocytes was measured by 4-h 51Cr release assay. IL-12 stimulated blood lymphocytes to produce interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), and this effect was augmented by co-cultivation with monocytes or AM. AM-upregulated induction of cytotoxic lymphocytes was stimulated with IL-12, and this effect was significantly abrogated by addition of antibodies against IFN gamma and TNF alpha. Induction by IL-12 of IFN gamma production and cytotoxic activity of CD8+ cells was also augmented by co-cultivation with monocytes or AM. AM were more effective than monocytes in augmenting the cytotoxic activity of IL-12-stimulated lymphocytes and CD8+ cells. These observations suggest that in situ induction of IL-12-stimulated cytotoxic cells in the lung may be regulated by complex cytokine networks, depending on participation of monocytes and alveolar macrophages.

Induction mechanism of human blood CD8+ T cell proliferation and cytotoxicity by natural killer cell stimulatory factor (interleukin-12).

Natural killer cell stimulatory factor (NKSF/IL-12) has been found to induce cytotoxic activity of human blood T cells. In the present study, the effect of NKSF on induction of cytotoxic CD8+ T cells in the presence or absence of monocytes was examined. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were isolated by centrifugal elutriation from peripheral blood of normal donors. Then, CD8+ cells were isolated with antibody-bound magnetic beads from purified lymphocytes. The cytotoxicity of CD8+ cells was measured by 51Cr release assay for 4 h. NKSF enhanced the proliferative response of CD8+ cells stimulated with suboptimal concentrations of interleukin-2 (IL-2), but rather inhibited their proliferative and cytotoxic responses on stimulation with an optimal concentration of IL-2. NKSF stimulated CD8+ cells to produce interferon gamma (IFN gamma) irrespective of the presence of added IL-2, and this effect was augmented by co-cultivation with monocytes. Blood monocytes upregulated induction of cytotoxic CD8+ cells stimulated with NKSF alone, and this effect was abolished by addition of antibody against IFN gamma, but not of antibody against tumor necrosis factor alpha. Induction of NKSF-inducible cytotoxic CD8+ cells was inhibited by addition of transforming growth factor beta, but not of IL-4. These observations suggest that in situ induction of NKSF-stimulated cytotoxic CD8+ cells may be regulated by complex cytokine networks, depending on the participation of monocytes.

Interleukin-10 is a potent inhibitor of tumor cytotoxicity by human monocytes and alveolar macrophages.

The effects of purified human interleukin-10 (IL-10) on the expression of antitumor activity of human monocytes and alveolar macrophages (AMs) obtained by centrifugal elutriation and bronchoalveolar lavage, respectively, from the same healthy donors were examined. Monocytes and AMs were incubated for 16 h in medium with lipopolysaccharide (LPS) in the presence or absence of IL-10 or IL-4, and then their tumoricidal activity was assayed by measuring 125I-IUdR release from human melanoma (A375) cells. Addition of IL-10 to cultures of monocytes or AMs with LPS resulted in dose-dependent suppression of their cytotoxicity against A375 cells, the suppression of the activity of monocytes being the higher. IL-10 also suppressed the synergistic effects of interferon-gamma and desmethyl muramyldipeptide in activation of monocytes. IL-10 inhibited the early induction phase of monocyte activation but not the effector phase (monocyte-mediated cytotoxicity). IL-10 plus IL-4 inhibited the antitumor activities of AMs and monocytes much more than either IL-10 or IL-4 alone. IL-10 and IL-4 at suboptimal concentrations also showed synergistic inhibitory effects. These findings suggest that IL-10 may be important in vivo in down-regulating the antitumor activities of monocytes and AMs in the lung by inhibiting their productions of antitumor effector molecules.

Augmentation by transferrin of IL-2-inducible killer activity and perforin production of human CD8+ T cells.

The effects of human transferrin (Tf) on lymphokine (IL-2)-activated killer (LAK) induction from blood lymphocytes of healthy donors was examined. LAK cells were induced by 6-day incubation in medium with recombinant human IL-2 of lymphocytes, and their cytotoxic activity was assessed by measuring 51Cr release from NK-resistant Daudi cells. Tf alone did not induce any LAK activity, but in combination with IL-2, it augmented LAK induction dose- and time-dependently. This augmenting effect was completely abolished by pretreatment with anti-Tf antiserum. Tf augmented the proliferative response of lymphocytes to IL-2 and their expressions of receptors for IL-2 and Tf. CD8+ T cells were isolated from purified blood lymphocytes using antibody-bound magnetic beads. Addition of Tf to cultures of CD8+ cells resulted in significant augmentation of killer cell induction and perforin (PFP) production after 4 days stimulation with IL-2. These results indicate that Tf is important in generation of IL-2-inducible killer properties and PFP activity of human CD8+ T cells.