Search

Mature natural killer (NK) cells use Ca2+-dependent granule exocytosis and release of cytotoxic proteins, Fas ligand (FasL), and membrane-bound or secreted cytokines (tumor necrosis factor [TNF]-alpha) to induce target cell death. Fas belongs to the TNF receptor family of molecules, containing a conserved intracytoplasmic "death domain" that indirectly activates the caspase enzymatic cascade and ultimately apoptotic mechanisms in numerous cell types. Two additional members of this family, DR4 and DR5, transduce apoptotic signals upon binding soluble TNF-related apoptosis-inducing ligand (TRAIL) that, like FasL, belongs to the growing TNF family of molecules. Here, we report that TRAIL produced or expressed by different populations of primary human NK cells is functional, and represents a marker of differentiation or activation of these, and possibly other, cytotoxic leukocytes. During differentiation NK cells, sequentially and differentially, use distinct members of the TNF family or granule exocytosis to mediate target cell death. Phenotypically immature CD161(+)/CD56(-) NK cells mediate TRAIL-dependent but not FasL- or granule release-dependent cytotoxicity, whereas mature CD56(+) NK cells mediate the latter two.

Extracellular signal-regulated kinases (ERK, also known as mitogen-activated protein kinases) are serine-threonine kinases transducing signals elicited upon ligand binding to several tyrosine kinase-associated receptors. We have reported that ERK2 phosphorylation and activation follows engagement of the low affinity receptor for the Fc portion of IgG (CD16) on NK cells, and is necessary for CD16-induced TNF-alpha mRNA expression. Here, we analyzed the involvement of ERK in NK cell-mediated cytotoxicity and IFN-gamma expression induced upon stimulation with targets cells, coated or not with Abs. Our data indicate that, as with immune complexes, ERK2 phosphorylation occurs in human primary NK cells upon interaction with target cells sensitive to granule exocytosis-mediated spontaneous cytotoxicity, and that this regulates both target cell- and immune complex-induced cytotoxicity and IFN-gamma mRNA expression. A specific inhibitor of mitogen-activated protein kinase kinase reduced both spontaneous and Ab-dependent cytotoxicity in a dose-dependent manner involving, at least in part, inhibition of granule exocytosis without affecting effector/target cell interaction and rearrangement of the cytoskeleton proteins actin and tubulin. Involvement of ERK in the regulation of Ca2+-dependent cell-mediated cytotoxicity was confirmed, using a genetic approach, in primary NK cells infected with a recombinant vaccinia virus encoding an ERK inactive mutant. These data indicate that the biochemical pathways elicited in NK cells upon engagement of receptors responsible for either spontaneous or Ab-dependent recognition of target cells, although distinct, utilize ERK as one of their downstream molecules to regulate effector functions.

Cytokine-mediated enhancement of spontaneous cytotoxicity depends, at least in part, on modulation of the expression of surface molecules responsible for recognition of target cell structures and triggering or inhibition of the cytotoxic machinery. We previously demonstrated that expression of transcription factors (e.g., Egr-1, JunB, and c-Fos) is differentially regulated by IL-2 and IL-12. Here we show that expression of CD161/NKR-P1A, a molecule involved in triggering cytotoxicity, is specifically upregulated by IL-12. CD161 transcription, mRNA accumulation, and surface expression are increased by IL-12. Other cytokines sharing the IL-2R beta- and/or common gamma-chains (i.e., IL-15, IL-4, and IL-7) do not mediate these effects. In an effort to analyze the mechanisms by which IL-2, IL-12, and IL-15 differentially regulate gene transcription, we have isolated a novel gene, 197/15a, the expression of which in NK and T cells is down-regulated by IL-2 and IL-15, up-regulated by IL-12, and not affected by IL-4 and IL-7. IL-2 and IL-15 act, at least in part, repressing 197/15a transcription; their effect on 197/15a mRNA accumulation is partially independent of novel protein synthesis, likely not mediated by JunB, Bcl-2, or Bax, and requires the activity of rapamycin-sensitive molecule(s). The observation that IL-2 and IL-12 differentially modulate CD161 expression suggests the existence of cytokine-specific mechanisms of modulation of spontaneous cytotoxicity based on the regulation of expression of surface molecules involved in target cell recognition and/or triggering of the cytolytic machinery.

We used a monoclonal antibody (mAb) (B203.13, IgM) generated from a mouse immunized with the human B/myeloid bi-phenotypic B1b cell line, to analyse haemopoietic cells. The antigen recognized by this mAb is expressed on most adult and umbilical cord blood CD21+ B cells, at minimal density on mature monocytes, and is undetectable on granulocytes, T, natural killer (NK) cells, and erythrocytes. Within umbilical cord blood and adult bone marrow haemopoietic progenitor cells, the B203.13 mAb recognized a surface marker, present on progenitor cells of several haemopoietic lineages, that was transiently expressed on early erythroid and T/NK progenitors, and was preferentially maintained on cells of the B and myeloid lineages. Within the CD34+ cells, B203.13 was expressed on early committed myeloid (CD33+) and erythroid (CD71dim) progenitor cells, as confirmed in colony formation assays. The mAb also reacted with cells of B and myeloid chronic leukaemias and cell lines. These data define B203.13 mAb as a novel reagent useful for the characterization of haemopoietic progenitors and leukaemias.

To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (delta SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. delta SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of delta SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing delta SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of delta SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of alpha 2 integrin by delta SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/membrane fraction, delta SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.

The evidence accumulated in the past few years has indicated that natural killer (NK) cells, originally identified on the basis of their ability to mediate spontaneous cytotoxicity, play a primary role in regulating immune responses and homeostasis via release of cytokines. Polyclonal populations of NK cells can be induced to produce an overlapping set of soluble factors. Some of these, represented primarily by IFN-gamma, TNF-alpha, and several growth factors for hematopoietic cells, have been clearly identified; others may represent novel cytokines. Production of specific cytokine subsets by distinct NK-cell subpopulations has not been documented, and the same cytokines are produced, in response to distinct stimuli, by both resting and activated NK cells, with the differences being for the most part quantitative, rather than qualitative. No cytokines capable of regulating NK-cell proliferation in an autocrine fashion have been described, and, with the exception of IL-5, cytokine production is not correlated with the proliferative capability of the cells. In this review, the stimuli leading to cytokine production are discussed in light of the knowledge of the molecular mechanisms by which distinct ligands (target cells, ligands for specific receptors, antibodies to triggering molecules, cytokines) regulate expression of cytokine-encoding genes and variably modulate each others' effects. The biological consequences of the production of specific cytokines are related to their effects on the NK cells themselves and on other hematopoietic and nonhematopoietic cell types. These are discussed, in an attempt to provide a picture that helps in understanding how NK cells may participate in regulating inflammatory and immune responses independently, at least in part, of their cytotoxic functions.

The ability to perform biological studies on Natural Killer (NK) cells requires effective methods for their isolation from hematopoietic cells of other lineages. NK cells are a discrete lymphocyte subset distinguishable from B- and T-lymphocytes on the basis of both physical and phenotypic characteristics that can be exploited for then purification. Techniques based on differential cell buoyancy (centrifugation on discontinuous density gradients, such as Percoll [1]) have been used to enrich NK cells from mixed lymphocyte populations, but do not allow purification of these cells to homogeneity. The mononuclear cell suspensions obtained, although enriched in NK cells, also contain variable proportions of other cell types (notably monocytes and/or activated T- and B-lymphocytes) (2) and subsets of NK cells of higher density are lost in these preparations.

BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukeic phenotype of Philadelphia chromosome-positive cells. c-RAF-1 serine/threonine kinase is known to be activated by receptor and nonreceptor tyrosine kinases. To determine whether c-RAF-1 plays a role in the growth of BCR/ABL-dependent cells, we examined whether c-RAF-1 associates with and/or is regulated by BCR/ABL and, if so, whether this interaction is functionally significant for BCR/ABL-dependent growth of chronic myelogenous leukemia cells and for growth factor-dependent proliferation of normal bone marrow cells. We show that c-RAF-1 enzymatic activity is regulated by BCR/ABL, although the protein does not associate with BCR/ABL. Downregulation of c-RAF-1 expression with antisense oligodeoxynucleotides or cDNA constructs, and inhibition of c-RAF-1 activity by its dominant negative mutants, inhibited both BCR/ABL-dependent growth of chronic myelogenous leukemia cells and growth factor-dependent proliferation of normal hematopoietic progenitors and the MO7 cell line without affecting the BCR/ABL-and growth factor-independent proliferation of HL-60 cells. These results indicate that c-RAF-1 plays an important role in Philadelphia chromosome-positive and normal hematopoiesis.

Problem: Giemsa stained cell isolates prepared from chorionic villus samples (CVS) contain granulated cells morphologically similar to large granular lymphocytes., Method: Phenotypic characterization of these cellular isolates by two-color immunofluorescence and subsequent in vitro culture in the presence of recombinant interleukin-2 (rIL-2) were done in order to determine whether CVS could serve as a source of decidual lymphocytes., Results: A major fraction of the CVS-derived lymphocytes were characterized as decidual NK cells, exhibiting high levels of CD56 expression (CD56+bright), without concomitant expression of CD16. The T cell population present in CVS-derived lymphocytes contained both CD4+ and CD8+ cells in a ratio somewhat reduced compared to that found in peripheral blood. While both T cells and CD56+bright cells from CVS proliferate in vitro in response to rIL-2 alone, preferential growth of CD56+bright cells was accomplished using a selective culture technique wherein co-culture with an irradiated, B lymphoblastoid cell line promoted the growth of CD56+ cells., Conclusion: CVS contains decidual NK cells and T cells that proliferate in response to rIL-2 and/or third party stimulator cells. These culture techniques will allow investigations into the maturation and/or activation of decidual NK cells and T cells.

An in vivo model for human melanoma was established with the growth of CR3 and DE5 human melanoma tumor cells following i.v. injection into C.B.-17 severe combined immunodeficient mice depleted of murine natural killer (NK) cells. The ability of human NK cells to mediate antitumor activity in vivo was investigated by evaluating the number of lung nodules and survival of mice given injections of human NK cells i.v. early after injection of CR3 tumor cells. Under these conditions, human NK cells effectively reduced lung nodule counts and prolonged survival when coinjected with interleukin 2 (IL-2). Multiple injections of IL-2 given during the first 16 h post-NK injection did not further enhance the tumor reduction. Significantly increased antitumor activity against CR3 tumor cells in vivo was observed in mice receiving NK cells coinjected with IL-2 and interleukin 12 (IL-12) in comparison to NK cells and IL-2 only. However, coinjection of IL-12 with human NK cells alone did not reduce the tumor burden. These results demonstrate the antitumor activity of human NK cells against human melanoma in severe combined immunodeficient mice and its augmentation by IL-2, alone or in combination with IL-12, suggesting that this model can be used to further investigate the interaction between human NK cells and human tumors.

Natural killer cell stimulatory factor (NKSF) or interleukin-12 (IL-12) is a heterodimeric cytokine with pleiomorphic effects on T and NK cells, including induction of lymphokine production, mitogenesis, and enhancement of spontaneous cytotoxic activity. Similarly to IL-2, NKSF/IL-12 enhances NK cell-mediated cytotoxicity within a few hours and independently from induced proliferation. This effect is independent from other induced cytokines, because it is not prevented by antibodies neutralizing interferon (IFN)-alpha, IFN-beta, IFN-gamma, IL-2 or tumor necrosis factor (TNF)-alpha and, unlike the induction of IFN-gamma production by peripheral blood lymphocytes, it does not require HLA class II-positive accessory cells. Enhanced cytotoxicity is accompanied by morphologic changes in NK cells, including a significant increase in the number of cytoplasmic granules. In addition to the previously described ability to enhance the cytotoxic activity of NK cells against tumor-derived target cells, NKSF/IL-12 is also a potent stimulator of cytotoxicity against virus-infected cells, either fibroblasts acutely infected with herpes viruses or T cell lines chronically infected with human immunodeficiency virus-1. NK cell-mediated antibody-dependent cytotoxicity or anti-CD16 antibody-redirected lysis is not significantly enhanced by NKSF/IL-12. However, the ability of resting peripheral blood T cells to mediate anti-CD3 antibody-redirected lysis is enhanced by 18-h incubation with NKSF/IL-12, indicating that this lymphokine can modulate the cytotoxic capability of both NK and T cells.

In this study we identify and characterize a subset of human peripheral blood T cells, present in all individuals, that has features previously described for T cells either separately or in special circumstances. These cells are found in purified suspensions of resting peripheral blood lymphocytes within the CD8+ T lymphocytes, express alpha beta T cell receptor (TCR), and can be identified and isolated because of high-density expression of surface CD11b (TCR alpha beta +/CD3+/CD8+/CD11b+ cells). They coexpress constitutively the IL-2 receptor beta chain, Fc gamma RIIIA, and CD56. Although they do not mediate spontaneous cytotoxicity, CD3+/CD8+/CD11b+ cells have cytotoxic potential, demonstrated in redirected cytotoxicity assays with P815 target cells in the presence of anti-Fc gamma RIII (CD16) or anti-CD3 monoclonal antibodies. Stimulation of CD3+/CD8+/CD11b+ cells with rIL-2 induces proliferation, cytotoxicity against NK-sensitive and NK-resistant target cells, and expression of surface activation antigens, including IL-2 receptor alpha chain (CD25). CD3+/CD8+/CD16+/CD56+ cell clones with cytotoxic functions including those mediated by engagement of surface CD16 were obtained by limiting-dilution cloning of purified CD3+/CD8+/CD11b+ cells in the presence of rIL-2 and autologous feeder cells. Our data support the hypothesis that the CD3+/CD8+/CD11b+/CD16+ cells represent a discrete peripheral blood lymphocyte subset that could be the physiological counterpart of that expanded in several pathological conditions and in large granular lymphocyte lymphocytosis.

Receptors for the Fc region of the immunoglobulin G (IgG) (Fc gamma R) have been recognized as a link between humoral and cellular immune responses. A soluble form of Fc gamma RIII (CD16) has been found in seminal plasma (SP), which may modulate immunosuppression of antisperm immune responses in the male and female reproductive tracts. SP from some individuals apparently have lower levels of Fc gamma RIII, but it is not known whether the diminished activities are due to low receptor concentration or steric interference from IgG. To test the hypothesis that different levels are due to steric interference, relative levels of Fc gamma RIII were measured in SP using monoclonal antibody 3G8 in an amplified enzyme-linked immunosorbent assay (ELISA) system. Men who were positive for antisperm antibodies (ASA) by Sperm Mar and direct immunobead assay (N = 26) and negative for ASA (N = 26) were tested. Individuals who were ASA positive had lower detectable levels than those who were ASA negative (t = 1.99, P = 0.05). Therefore, variation in Fc gamma RIII levels may be due to steric interference from IgG. IgG subclass concentrations in SP of both groups were determined using an ELISA method and compared to Fc gamma RIII levels. Slight correlations were seen for IgG1 (r2 = 0.237, P < 0.001), IgG2 (r2 = 0.204, P < 0.001), and total IgG (r2 = 0.299, P < 0.001) in relation to Fc gamma RIII levels in ASA-negative SP specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

We analyzed the phenotypic changes associated with monocyte activation and differentiation using a newly developed monoclonal antibody (B148.4). Among peripheral blood leukocytes, the antigen recognized by this antibody is expressed on monocytes and granulocytes at high and low density, respectively. Antigen expression is lost during in vitro differentiation of monocytes and is absent on tissue macrophages, indicating that expression of this antigen is related to monocyte differentiation. Only 1 alpha,25-dihydroxyvitamin D3 and phorbol diesters, of several inducers tested, up-regulate B148.4 antigen expression on cells (monocytes and certain myeloid cell lines) that constitutively bear the antigen, without, however, allowing its maintenance during monocytic differentiation or inducing it on negative cells. By immunoprecipitation from B148.4+ U937 cells, the antigen is a complex of a major 116-kd and two minor 38- and 46-kd molecules. Analysis of eight different tissues reveals that the antigen is shared with endothelial cells. Biochemical characteristics, cellular distribution, and expression pattern on monocytes, myeloid cell lines, and AML cells upon culture with different stimuli indicate that B148.4 is a novel monocyte differentiation antigen.

The ability of human B cells to produce granulocyte-macrophage (GM)-CSF and IL-3 was investigated. B cells, isolated from tonsils or from the peripheral blood of patients with B cell chronic lymphocytic leukemia using mAb and immune rosettes, were cultured with or without Staphylococcus aureus Cowan strain I. GM-CSF and IL-3 were measured in the culture supernatants using a bioassay based on the selective proliferative response of the MO7e megakaryoblastic cell line to IL-3 or GM-CSF. S. aureus Cowan I-stimulated normal B cells released measurable amounts of GM-CSF but not of IL-3 as determined in neutralization assays with specific mAb in the MO7e cell line test. Some of the unstimulated normal B suspensions also produced GM-CSF, albeit in lower quantities. When normal B cells were fractionated into small (resting) and large (activated) B cells by Percoll density gradients, spontaneous GM-CSF production was detected only in the large cell fractions, but small cells were induced to produce GM-CSF upon S. aureus Cowan I stimulation. On a per cell basis, tonsillar B cells were found capable of releasing more GM-CSF than activated peripheral blood monocytes. The amount of GM-CSF produced by B cells was always inferior to that released by stimulated peripheral blood T cells or NK cells. The purified B cell suspensions from all 14 B cell chronic lymphocytic leukemia patients studied released GM-CSF but not IL-3 in the culture supernatants, sometimes even in the absence of stimuli. Northern blot analysis on total or poly(A)+ RNA confirmed the presence of GM-CSF, but not of IL-3, mRNA transcripts in both normal and malignant B cells. The results of these studies support the notion that activated human B lymphocytes release sufficient GM-CSF to play a role in the control of both hematopoiesis and the inflammatory process.

In the past year, natural killer cells have been the subject of much active investigation. The analysis of the effect of cytokines on the generation, proliferation and function of natural killer cells, and the definition of the lymphokines that they produce, have been particularly important areas of research in view of their possible application in adaptive immunotherapy, combined with biological response modifiers.

The low-affinity Fc receptor for immune-complexed IgG (Fc gamma RIII; CD16) present on in vitro cultured human monocytes are encoded by an Fc gamma RIII-2 gene that, by cDNA sequence analysis, is identical to that expressed on tissue macrophages and on natural killer cells. In macrophages, Fc gamma RIII-2 encodes a glycoprotein of 52-62 kDa, with a peptide backbone of 33 kDa identical to that of the homologous receptor on natural killer cells. Like this and unlike in polymorphonuclear neutrophils, Fc gamma RIII (CD16) on cultured monocytes is insensitive to phosphatidylinositol-specific phospholipase C, is not allelic for the neutrophil NA alloantigens NA-1/NA-2, is not recognized by a monoclonal antibody (1D3) detecting an epitope present only on neutrophil Fc gamma RIII (CD16) and functions to trigger cytotoxicity upon ligand binding.

Human interferons (IFs) can induce a several-fold increase in the natural cytotoxicity of human lymphocytes on target cell lines. IFs increase the efficiency of cytotoxicity and the number of natural killer (NK) cells. In mixed cultures of lymphocytes and other tumor-derived or virus-infected cells, endogenous IF is produced, which mediates 70-90% of the observed cytotoxicity. The effect of IF on target cells is antagonistic to its effect on lymphocytes: the susceptibility to lysis of cells treated with IF decreases. Whereas normal fibroblasts are protected by IF, virus-infected cells and most tumor-derived cells are not. The protective effect is specific for NK cells cytotoxicity: IF-treated target cells are lysed to the same extent as the untreated controls by antibody-dependent killer cells, by phytohemagglutinin-stimulated lymphocytes, by cytotoxic T lymphocytes, and by antibodies and complement. NK cells bind to IF-treated fibroblasts, but are unable to lyse them. The cytotoxic ability of NK cells is inactivated after interaction with normal fibroblasts, but not with IF-treated fibroblasts. Unlabeled, normal fibroblasts but not IF-treated fibroblasts compete for the cytotoxicity mediated by NK cells in 51Cr-labeled target fibroblasts. IF, by stimulating very efficient, nonspecific cytotoxic cells, and by protecting normal cells from lysis, might render the NK cell system an inducible defense mechanism against virus-infected and tumor cells.

Immune (gamma) interferon (IFN-γ) is produced during an immune response by antigen-specific T cells and probably also by natural killer (NK) cells recruited by the T cell-product interleukin 2 (IL-2). IFN-γ was discovered and originally measured on the basis of its anti-viral activity, but its complex anti-cellular activities probably reflect its biological role more clearly. In this review, Giorgio Trinchieri and Bice Perussia discuss some aspects of the biology of IFN-γ, its pleiomorphic anti-cellular effects and its ability to modulate cellular responses to other regulatory factors., (Copyright © 1985. Published by Elsevier B.V.)

At least two distinct HLA-DR+ cell subsets lacking surface markers specific for B cells, monocytes or other known lineages are present in human peripheral blood. One subset is non-adherent to plastic, produces interferon-alpha (IFN-alpha) when incubated with cytomegalovirus-infected target cells and provides an accessory function required for natural killer (NK) cell-mediated lysis of such cells. These non-adherent HLA-DR+ cells express the surface antigen recognized by antibody anti-D44 and do not stimulate mixed leucocyte reaction (MLR). The other HLA-DR+ cell subset is loosely adherent to plastic, produces only minimal levels of IFN-alpha when incubated with cytomegalovirus-infected target cells and does not provide the accessory function required for NK cell-mediated lysis of such cells. These HLA-DR+ cells stimulate a strong MLR, do not express D44 antigen and meet the criteria of dendritic cells (DC) morphologically and functionally.

We analyzed the combined effect of tumor necrosis factor and 1 alpha,25-dihydroxyvitamin D3 on the differentiation of human myeloid cell lines HL-60, ML3, and U937. The two compounds synergize in inducing the morphological, phenotypic, enzymatic, and functional characteristics of cells of the monocytic lineage. Immune gamma-interferon synergizes with each compound to induce differentiation. However, recombinant tumor necrosis factor is much more effective than recombinant gamma-interferon in potentiating the effect of 1 alpha,25-dihydroxyvitamin D3 and, alone, is also more effective than recombinant gamma-interferon in inducing expression of the high-affinity Fc receptor on ML3 cells. The possible physiological or pathological relevance of the synergistic effect of tumor necrosis factor and 1 alpha,25-dihydroxyvitamin D3 on monocytic differentiation is discussed.

T lymphocytes and natural killer (NK) cells exert both stimulatory and suppressive effects that regulate growth and differentiation of hematopoietic cells. Activated T and NK cells have been demonstrated in different pathological states of bone marrow failure and are proposed to play a role in the pathogenesis of the disease. T and NK cells have also been shown to be responsible for bone marrow graft rejection in both allogeneic and syngeneic donor/recipient combinations. Lymphocytes can regulate hematopoietic cell growth by direct cellular contact or by releasing soluble factors, such as colony-stimulating factors, immune interferon, lymphotoxin, and tumor necrosis factor, active on hematopoietic precursor cells.

We compared the effects of recombinant (r) tumor necrosis factor (TNF) and lymphotoxin (rLT) on in vitro colony formation by erythroid, multipotential and granulocyte, monocyte (CFU-GM) precursor cells. Recombinant granulocyte, macrophage-colony stimulating factor and bone marrow preparations enriched for hematopoietic precursors were used in order to examine the direct effects of the cytokines on precursors in the absence of other soluble factors or mature cells that may influence colony formation. Both rTNF and rLT inhibited colony formation by erythroid and multipotential precursor cells. rTNF and rLT did not inhibit the more mature (day-7) CFU-GM, although a synergistic inhibition was observed when the cytokines were added to cultures together with recombinant interferon gamma (rIFN gamma). However, rTNF did inhibit day-7 CFU-GM when conditioned medium from the human bladder carcinoma cell line 5637 was used as a source of colony-stimulating activity. rTNF inhibited the more immature (day-14) CFU-GM regardless of the source of colony-stimulating activity used, and was a stronger inhibitor of day-14 CFU-GM than rLT, suggesting a difference in the biological effects of the two cytokines. Noninhibitory concentrations of rTNF or rIFN increased the number of monocyte colonies formed, identified by cytochemical staining. These data suggest a mechanism for TNF and IFN gamma-induced inhibition of CFU-GM through induction of monocyte differentiation rather than through a direct toxic effect.

Inhibitors of glycosylation and carbohydrate processing were used to probe the functional consequences of specific, differential alterations in glycosylation of monoclonal IgG secreted by hybridoma clones. Neither the absence of glycosylation nor the presence of atypical oligosaccharides significantly influenced binding of the monoclonal antibody to the cell surface antigen recognized. However, lymphocyte-mediated antibody-dependent cytotoxicity was enhanced significantly, as compared to native (unmodified) IgG-sensitized target cells, when target cells were sensitized with IgG bearing the atypical oligosaccharides induced metabolically by castanospermine, N-methyldeoxynojirimycin, deoxymannojirimycin or monesin, but not by swainsonine. The enhanced cytotoxicity was mediated by natural killer cells but not by monocytes or interferon-activated polymorphonuclear leukocytes. By contrast, antibody-dependent cytotoxicity mediated by activated polymorphonuclear leukocytes against target cells sensitized with the IgG glycosylation phenotypes induced by swainsonine and tunicamycin, but not by castanospermine, was decreased in comparison to cytotoxicity against target cells sensitized with native IgG. The enhanced lymphocyte-mediated cytotoxicity was Fc receptor-dependent. A panel of monoclonal antibodies directed against different human tumor target cells was used to demonstrate that the castanospermine-induced IgG phenotype generally enhanced antibody-dependent tumoricidal activity mediated by natural killer cells. However, differences in lymphocyte response to an alteration in IgG glycosylation were observed.

A study of T and B cell surface markers was carried out on peripheral blood lymphocytes from diabetic patients, employing immunofluorescent staining for membrane Ig, and the E rosette test. According to the immunofluorescent studies the % of sIg bearing lymphocytes decreases with age; this trend is more evident in healthy individuals. Lymphocytes from diabetic patients posses a higher amount of membrane IgG than age-matched controls. It was only in the younger diabetic patients that a definite decrease was observed in the % of IgM bearing lymphocytes. The evaluation of E rosette-forming lymphocytes revealed a similar age-associated reduction both in diabetics and in controls. Some common features of the pattern of T and B cell markers on lymphocytes from younger diabetic patients and older controls suggest that the former undergo, at least from the immunological standpoint, a more precocious ageing process. It is possible that some metabolic disorders in lymphocyte activity may play a role in this, resulting in a deficiency in the control of immunological mechanisms.

Two monoclonal antibodies directed against distinct epitopes of recombinant human immune interferon (rIFN-gamma) were used to investigate the relationship between the molecular organization of IFN-gamma and its various biological activities on cultured human melanoma cells. Both monoclonal antibodies inhibited the increase in the expression of cell surface human lymphocyte antigens Class I and II antigens and the antiproliferative and antiviral actions of rIFN-gamma. On the other hand neither monoclonal antibody affected the binding of rIFN-gamma to melanoma cells and its ability to reduce the expression of a high molecular weight-melanoma associated antigen. These data indicate that the functional domains of IFN-gamma responsible for antiviral activity, increased human lymphocyte antigen expression and antiproliferative effects on human melanoma cells may be distinct from that (those) involved in reduced expression of the high molecular weight-melanoma associated antigen and in IFN-gamma binding to cell receptors.

Hybrid cell lines secreting antibodies specific for human gammaglobulin (HGG) were prepared by cell fusion and cloning techniques. The monoclonal antibodies were tested for their antibody reacts with a different antigenic determinant of HGG. One reacts with isolated kappa (kappa) light chains, one with isolated lambda (lambda) light chains, and one with the Fc fragment of IgG1 molecules. The reactivity patterns of two additional monoclonal antibodies are more complex. One reacts with a determinant present on the Fc of all IgG subclasses and the other binds to a determinant on the Fab of IgG molecules. The two monoclonal antibodies reactive with light chains also bind to surface components of human B cells. The murine immunoglobulin (Ig) class of each clone product was identified.

When a group of 104 aged subjects was screened for autoimmune reactions, positive reactions for the rheumatoid factor and/or autoantibodies (ANA, anti-thyroid, PCA, anti-smooth muscle, anti-mitochondria) were recorded in 40.4%. Immunological functions were studied in 32 positive aged subjects, 32 age- and sex-matched negative controls, and 32 young subjects. Some differences attributable to the process of aging were quite evident, such as a depression in the percentage of E rosette forming peripheral lymphocytes and in their response to PHA, and an increase in the percentage of IgG-bearing peripheral lymphocytes and in the serum levels of IgA and three complement fractions (C'3, C'4, and C'3-PA). No clear-cut picture was noted when autoimmunity-positive and -negative, aged subjects were compared. However, some differences between sexes suggest that autoimmune reactions are linked to a depressed T cell function mainly in males, whereas the reverse is true for females.

In this paper, we characterize the antigen recognized by the monoclonal antibody B73.1 and the modification occurring at the membrane of the positive cells after interaction with the antibody. The B73.1-defined antigen is a protein of 50,000 to 72,000 daltons that is sensitive to pronase but not to trypsin treatment. B73.1 antibody, and its F(ab')2 fragment, directly block, at high concentrations, the binding of IgG antibody-sensitized erythrocytes to the Fc receptors (FcR) of a subpopulation of lymphocytes and neutrophils. B73.1 antibody dissociates rapidly from the positive cells, but concomitant modulation of both B73.1 antigen and FcR is induced when cells are incubated in the continuous presence of antibody or when B73.1 antibody is cross-linked at the cell membrane with an anti-mouse immunoglobulin antiserum. Reaction of lymphocytes with immune complexes also induces modulation of both FcR and B73.1 antigen, without affecting the expression of other antigens on the positive cells. The possibility that the antigen is internalized and digested by the cell after reaction with the antibody is discussed. B73.1 antibody inhibits antibody-dependent cytotoxicity mediated by lymphocytes (K cells) and neutrophils, whereas it does not affect spontaneous cytotoxicity of NK cells. These results suggest the B73.1-defined antigen might be the FcR or a structure closely related to it on K/NK cells.

In this report, we characterize the major human peripheral blood nonadherent mononuclear cell subset that is responsible for the production in vitro of alpha-interferon (alpha IFN) in response to influenza, Sendai, and Newcastle disease viruses. Using a panel of anti-monocyte, anti-B, anti-T and anti-natural killer cell monoclonal antibodies to purify and recover both positive and negative cell populations, we show that the major alpha IFN-producing cells are HLA-DR(+) cells with no other surface markers characteristic of either lymphocytes or myelomonocytic cells. These cells copurify, on Percoll density gradients, with cells mediating NK activity, but the cell population responsible for alpha IFN production can be distinguished unambiguously from NK cells based on the former's reactivity with an anti-HLA-DR monoclonal antibody, the lack of reactivity with antibodies that detect the low-affinity receptor for IgG on human natural killer cells and granulocytes, and the inability to mediate spontaneous cytotoxicity. Double immunofluorescence assays indicate that cells of this subset, the lineage of which is as yet undetermined but which might be related to dendritic cells, constitute a minor proportion (approximately 1-1.5%) of the nonadherent mononuclear cells from healthy donors.

Lymphocytes bearing complement receptors were separated from lymphocyte suspensions using C3-reacted Sepharose columns as solid phase immunoabsorbents. The retained CR+ lymphocytes were recovered by elution of the column with IgG fraction purified from a rabbit antiserum to human C3. This paper describes the method, its specificity and the yields of cells obtained.

An increase in the percentage of IgG bearing peripheral blood lymphocytes is observed in aged subjects as compared with young ones. Such a finding is probably due to the presence, in 'aged' sera, of a higher concentration of immune complexes, bound to lymphocytes through their Fc or Complement receptors.

Several monoclonal antibodies specific for human cells of the myelomonocytic lineage at discrete stages of differentiation have been described. In this review I briefly summarize the results obtained with these reagents and discuss their possible use for the study of cell lineage and functional activities of normal peripheral blood leukocytes, for the analysis of myelomonocytic cell differentiation in vivo and in vitro and for the antigenic phenotyping of leukemia cells.

In human one-way mixed lymphocyte reactions we have observed an increase in the percentage of sIgD bearing lymphocytes, detected by direct membrane immunofluorescent staining. This increase does not seem to be attributable either to the synthesis of sIgD receptor molecules by previously negative T or null cells or to a non specific stimulation of B cells by a factor released in the course of the MLR.

A serious and often ignored source of artifacts when testing the specificity of antibodies is the contamination of leukocyte preparations with platelets which subsequently adhere to monocytes. The presence of Ca2+ chelating agents or acetylsalicylic acid in the washing buffers inhibits adhesion of platelets to monocytes, thus permitting an accurate distinction among antibodies that are specific for monocytes, platelets or both. The analysis of the specificity of various new or recently described monoclonal antibodies reactive with these cell types is reported here.

We describe five monoclonal antibodies that react with four discrete antigens present on human platelets. Antibodies B2.12 and B59.2 precipitate the glycoprotein IIb-IIIa complex from radiolabeled platelet membrane extracts and inhibit platelet aggregation induced by adenosine diphosphate (ADP), collagen, or epinephrine. The antigen recognized by the two antibodies is present on megakaryocytes but either absent entirely or expressed in small amounts on platelets from Glanzmann's thrombasthenic patients. The antigen recognized by antibody B37.3 is absent from thrombasthenic platelets. Antibody B1.12 reacts with an antigen shared by platelets and 20% of peripheral blood lymphocytes and is a potent inducer of platelet aggregation. Antibody B2.10 reacts specifically with platelets and megakaryocytes but does not affect platelet functions. Thus, these reagents are useful tools in diagnostic and functional studies of both normal and abnormal platelets.