OKM1 Monoclonal Antibody Research Articles

Protein kinase C-beta is required for macrophage differentiation of human HL-60 leukemia cells.

The requirement for protein kinase C (PKC)-beta in phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of human HL-60 promyelocytic leukemia cells was studied by using the variant HL-525, which is deficient in PKC-beta and is resistant to PMA-induced differentiation. Transfecting these resistant HL-525 cells with expression vectors containing either PKC-beta I or PKC-beta II cDNA resulted in clones that displayed PKC-beta transcript levels similar to or higher than those of the parental HL-60 cells or cells from a PMA-susceptible HL-60 clone, HL-205. These productive transfectants also exhibited PMA-induced cell attachment and spreading, inhibition of cell replication, reactivity to the OKM1 monoclonal antibody, and the ability to phagocytize opsonized beads, which are all characteristic macrophage markers. No PMA-induced differentiation markers were observed in any of the PKC-beta I or PKC-beta II transfectants that did not exhibit an increased PKC-beta RNA level or in cells transfected with control plasmids. These results indicate that restoration of the PKC-beta isozyme deficiency by productive gene transfection causes HL-525 cells to revert to a phenotype like that of the parental HL-60 cells, which is characterized by susceptibility to PMA-induced macrophage differentiation. Therefore, we can conclude that PKC-beta is one of the essential elements in the PMA-induced signal transduction pathway which leads to macrophage differentiation in HL-60 cells and perhaps in other related cell types.

T Cells and Trachoma: Their Role in Cicatricial Disease

Frozen sections of tarsoconjunctival biopsies with trachomatous scarring from 14 black adults undergoing corrective surgery for trichiasis, and "normal" tissue from three postmortem controls, were immunohistochemically stained for the major T- and B-cell subsets, and for macrophages and monocytes. T cells outnumbered B cells by 2 to 17 times, and macrophages and monocytes by approximately 20 times in all specimens. Biopsies were categorized as “inflamed” if a cumulative inflammatory score of cellular staining in the substantia propria with CD4, CD8, and OKM1 monoclonal antibodies was greater than that of control tissues. CD4+ lymphocytes predominated over CD8+ lymphocytes in 5 of 7 inflamed biopsies, whereas CD8+ lymphocytes predominated over CD4+ lymphocytes in 5 of 7 noninflamed biopsies. Lymphoid aggregates were present in five inflamed biopsies, but lacked germinal centers, centrally located B cells, or parafollicular T cells typical of the acute stage of trachoma. CD4+ and CD8+ lymphocytes also were observed in the epithelium and lumen of Meibomian glands. These observations indicate that the inflammatory infiltrate of the tarsoconjunctiva in the cicatricial stage of trachoma is comprised predominantly of T cells, and suggests that T cells may be involved in the genesis of tarsal thickening and conjunctival scarring seen in the later stages of trachoma.

Cell surface markers, inositol phosphate levels and membrane potential of lymphocytes from young and old human patients

It is well known that most physiological functions change with aging, including the immune response. Data concerning the aging of lymphocyte subpopulations are conflicting. The antigen density of peripheral blood lymphocytes has been determined by fluorescently tagged OKT-3, OKT-4, OKT-8, OKT-11 and OKM1 monoclonal antibodies in a carefully selected aged (over 87 years) population, and compared to that of young subjects. A substantial difference was found in the percentage distribution of OKT8 and OKM1 subsets. The volume of lymphocytes of the elderly population was significantly less than that of the young. The effect of various monoclonal antibodies on phosphatidylinositol breakdown has also been studied. It was found that only OKT3, acting through the CD3 antigen receptor, was able to induce inositol phosphate formation in both young and elderly, although in the latter population this occurred at a lower level. Because the plasma membrane plays a regulatory role in this process, an important and sensitive functional parameter, the membrane potential, was also monitored and influenced by changing the extracellular K+ concentration. The lymphocytes of the elderly population responded less sensitively to changes in extracellular potassium concentration.

Deficiency of leukocyte adhesion molecules

Study of patients congenitally deficient in expression of the CD11/CD18 dimer has clarified the fundamental role of these transmembrane glycoproteins of the integrin family in multiple functions of polymorphonuclear leukocytes (PMNL). Deficient expression is associated with severe deficiency of PMNL aggregation, adhesion to protein-coated surfaces and endothelial monolayers, locomotion, chemotaxis, phagocytosis and bacterial cell killing. Severely affected patients develop leukocytosis and die in infancy of overwhelming infection. A patient we studied at autopsy died from septicemia and pneumonia. There was striking lack of any acute inflammatory response near sites of necrosis and infection. In the lungs, in contrast, a distinct but deficient acute focal inflammatory response was observed.We used colloidal gold immunolabeling to study the subcellular localization of the CD11/CD18 adhesion molecules in human PMNL and their expression and movement on cell surfaces after stimulation with the chemotactic peptide formyl-met-leu-phe (fMLP). Human PMNL in pyrogen-free PBS were fixed for lh in 1% glutaraldehyde, dehydrated to 70% ethanol and embedded in LR White resin at 55°C. A polyclonal rabbit antiserum recognizing CD18 and absorbed with CD18-deficient PMNL was used to label sites of storage in thin sections, using 5-nm goat-anti-rabbit-IgG gold. We compared sites labeling for CD18, lactoferrin and gelatinase in adjacent thin sections. For surface labeling, cells were fixed in 1% glutaraldehyde for lh, exposed to OKM1 monoclonal antibody, washed and labeled using 36-nm rabbit-anti-mouse-IgG-gold. Controls for both studies included substitution of other or nonspecific primary and secondary antibody. Cells were attached to polylysine-coated coverslips, critical-point dried in CO2, carbon coated and examined by scanning EM using backscattered electron imaging. Other cells were exposed to f(ab’)2 fragments of OKMl and to f (ab’)2-rabbit-anti-mouse prior to fixation, followed by labeling with 36-nm goat-anti-rabbit-gold, or exposed to f(ab’)2-OKMl conjugated to 36-nm gold particles prior to fixation.

Leukaemic B cells from patients with chronic lymphocytic leukaemia suppress immunoglobulin production by lymphocytes from normal donors.

We observed that highly purified E-rosette-negative largely leukaemic B cells from 9 out of 15 patients with chronic lymphocytic leukaemia (CLL) significantly suppressed immunoglobulin production by mixtures of T4 and B cells from normal donors in the presence of pokeweed mitogen (PWM). This suppression by leukaemic B cells was concentration-dependent. Addition of equal numbers of B cells from normal donors to the mixtures of normal T4 and B cells increased, or had no effect on the production of IgM, IgA, and IgG. Treatment of purified largely leukaemic B cells from patients with CLL with either the anti-B1 or anti-Leu 1 monoclonal antibody plus complement abolished their ability to suppress immunoglobulin production. In contrast, treatment with either the anti-Leu 5 or the OKM1 monoclonal antibody plus complement had no effect on the suppression. These results suggest that leukaemic B cells from certain patients with CLL may exhibit, or can be induced to exhibit, immunosuppressive properties.

Cannabinoids induce incomplete maturation of cultured human leukemia cells.

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 microM delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody or the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 microM THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. Pronounced among these changes was an increase in the synthesis of at least 10 proteins that are found abundantly in monocytes. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype; the THC-treated cells failed to exhibit other monocyte markers such as attachment to the surface of tissue culture dishes or morphological maturation beyond the promonocyte stage. However, treatment of these "incompletely" matured cells with either phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. Two other cannabinoids, cannabidiol and cannabinol, which were more cytotoxic than THC at comparable doses, also caused an increase in the expression of maturation markers, but at doses higher than those required for THC. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced "incomplete" cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

Studies on human blood lymphocytes with iC3b (type 3) complement receptors. I. Granular, Fc-IgG receptor positive and negative subsets in healthy subjects and patients with systemic lupus erythematosus.

By using the OKM1 monoclonal antibody and the fluorescence-activated cell sorter to identify lymphocytes bearing iC3b (type 3) complement receptors, two principal populations of OKM1+ lymphocytes have been identified in human peripheral blood. One subset exhibited azurophilic granules and Fc receptors for IgG stained by Leu-11. The other population did not display FcR, but was enriched in cells reacting with OKT3 and OKT8 (low intensity). In healthy subjects, approximately 60% of CR3+ lymphocytes were granular FcR-bearing cells and only 18% co-expressed OKT3 determinants. In patients with systemic lupus erythematosus (SLE), CR3+ lymphocytes were predominantly FcR negative cells and 71% lacked granules. Only 33% reacted with Leu-11, but 50% co-expressed OKT3, 44% reacted with OKT8+, and 15% were OKT4+. We tested the hypothesis that agranular OKT3+ Leu-11- lymphocytes, such as those found in SLE patients, contained the precursors of natural killer (NK) cells. Leu-11+ cells were removed from normal lymphocytes by complement lysis, and the remaining cells were treated with recombinant IFN-alpha, IFN-gamma, or IL 2. These procedures were ineffective in generating typical NK effector cells. Our studies do not support the hypothesis that CR3+ Leu-11- lymphocytes are the precursors of granular Leu-11+ NK cells.

Ligand binding by the p150,95 antigen of U937 monocytic cells: properties in common with complement receptor type 3 (CR3).

U937 cells (a monocytic cell line) grown in the presence of phorbol myristate acetate were surface labeled with 125I and the iC3b-binding proteins isolated by affinity chromatography on iC3b-Sepharose in the presence of divalent cations. Three polypeptides of 170, 150 and 95 kDa were found to bind specifically to iC3b-Sepharose. The polypeptides of 170 and 95 kDa were identified as the alpha and the beta subunits of CR3 by immunoprecipitation with OKM1 monoclonal antibody. The 150-kDa polypeptide was not immunoprecipitated by antibodies to the alpha subunit of CR3 or LFA-1. However, the 150-kDa polypeptide, together with the 95-kDa polypeptide, was immunoprecipitated with an anti-beta subunit-specific antibody IB4, which immunoprecipitates LFA-1, CR3 and p150,95. These results indicated that the 150-kDa polypeptide is the alpha subunit of the p150,95 antigen. The binding of p150,95 and CR3 to iC3b-Sepharose is specific as neither binds to C3u-Sepharose. A monoclonal antibody, 3.9, which immunoprecipitated the 150 and a 95-kDa polypeptide from U937 cells was characterized as being directed against the alpha-subunit of the p150,95 antigen. Phorbol myristate acetate-stimulated U937 cells from rosettes with EAC3b and EAiC3b but not with EAC3d cells. Monoclonal antibody 3.9 does not inhibit either type of rosetting, but we were unable to exclude a role for p150,95 in adherence of iC3b-coated particles. Since there is no rosetting with C3d-bearing particles it is unlikely that p150,95 is a receptor for C3d, a role for p150,95 which has been suggested by others (Wright, S.D., Licht, M.R. and Silverstein, S.C., Fed. Proc. 1984. 43: 413).

Similar effects of treatment with alpha interferon on the protein synthesis of human large granular lymphocytes, T cells, and monocytes.

Preparations of human large granular lymphocytes (LGL), T cells, and monocytes (MC) were obtained through centrifugation on Percoll gradients and preparative E-rosetting. The different preparations contained more than 80% of the appropriate cell type, as judged by their ability to lyse 51Cr-labelled K562 cells, cell morphology, and the presence of cell surface structures recognized by the OKT3, OKT10, Leu 7 and OKM1 monoclonal antibodies. The protein synthesis is unstimulated and alpha interferon (IFN-alpha)-treated cells of the different types was studied by subjecting 35S-methionine-labelled cell extracts to two-dimensional gel electrophoresis. The general pattern of protein synthesis in LGL and T cells was virtually identical, whereas at least 7 major proteins were synthesized at a higher rate in monocytes. The effects of IFN-alpha on the protein synthesis of LGL and T cells were identical, IFN-alpha increasing the rate of synthesis of 9 proteins. These proteins were also expressed, but not always IFN-augmentable, in monocytes. No additional, cell-type associated, IFN-inducible proteins were found. This suggests that the augmenting effect of IFN-alpha on the cytotoxic capacity of LGL, T cells, and monocytes may be to affect common steps in their lytic machineries.

Defective natural killer cytotoxicity and polymorphonuclear leukocyte antibody-dependent cellular cytotoxicity in patients with LFA-1/OKM-1 deficiency.

Four children with an immunodeficiency involving the absence of leukocyte membrane glycoproteins reacting with anti-LFA-1 and OKM-1 monoclonal antibodies were unable to mediate adherence-dependent leukocyte functions. Even with normal Fc receptor function, their PMN-ADCC and MC-NKC were markedly deficient. Single cell analysis demonstrated deficient antibody-mediated PMN-target cell adherence. Monoclonal antibodies against LFA-1 and OKM-1 reproduced this immunodeficiency in leukocytes from normal adults. LFA-1/OKM-1 mediates a PMN-target cell adhesive step.

T-lymphocyte subsets in human lymph nodes: Relative increase of OKT-8 + cells in neoplastic and reactive B-cell proliferation

Cell suspensions obtained from 54 human lymph nodes involved by different pathological conditions were characterized by conventional markers and by the OKT-3, OKT-4, OKT-8, OKIa-1, and OKM-1 monoclonal antibodies. In 18 cases of reactive lymphoid hyperplasia, the majority of lymph node cells were mature T lymphocytes (E-RFC = 56 ± 9%; OKT-3 + = 63 ± 10%); among T-cell subsets, OKT-4 + cells were 49 ± 8% whereas OKT-8 − cells were 21 ± 8% ( T4 T8 = 2.7 ± 1.1 ). This distribution of T-cell phenotypes was not similar in the different histological types of reactive lymphoid hyperplasia. In fact, an increase in the percentage of OKT-8 + cells (25 ± 9%; P < 0.05) and a decrease in the values of the T4 T8 ratio (2.1 ± 1.0; P < 0.05) were observed in 9 cases of reactive lymphoid hyperplasia of follicular type when they were compared to the mixed and sinus types. In 13 lymph nodes involved by B-cell lymphoma, the percentage of T lymphocytes was markedly reduced (E-RFC = 21 ± 12%; OKT-3 + = 27 ± 18%) and the percentage of OKIa-1 + cells (51 ± 15%) was significantly ( P < 0.01) increased as compared to reactive nodes; in addition, in these cell suspensions, an increase in the relative proportion of OKT-8 + cells ( T4 T8 = 1.4 ± 0.7; P < 0.01 ) could also be demonstrated. Finally, a clear prevalence of OKT-4 + cells on OKT-8 + cells was demonstrated in 5 cases of tuberculous lymphadenitis ( T4 T8 = 3.9 ± 1.5 ; NS) and in 18 cases of Hodgkin's disease ( T4 T8 = 4.2 ± 2.0; P < 0.01 ). In tuberculous lymphadenitis, a significant increase ( P < 0.01) in the percentage of OKM-1 + cells could also be demonstrated.

Natural cytotoxicity of human blood monocytes: Production of monocyte cytotoxic factors (MCF) during interaction with tumor cells

Human blood monocytes obtained by EDTA-reversible adherence to autologous serum-coated plastic dishes expressed natural cytotoxicity against NK-sensitive K562 cells in a 4-h 51Cr release assay. These monocytes released soluble cytotoxic factors, termed monocyte cytotoxic factors (MCF), when cultured with target cells. In contrast, blood monocytes obtained by adherence to fetal calf serum-coated plastic surfaces failed to kill K562 cells and to produce MCF. Although some lysis could be detected at 18 h, optimal lysis of K562 cells by MCF was observed after 48 h incubation in a microcytotoxicity assay using trypan blue dye exclusion. The addition of actinomycin D to the cytotoxicity assay enhanced the sensitivity and then NCF activity was detectable in a 18-h Cr release assay. Neither supernatants produced by culture of monocytes alone nor lysates of monocytes were cytotoxic. In addition, cytochalasin A inhibited both direct cell-mediated lysis and generation of MCF. Optimal production of MCF occurred after 6–24 h of interaction with K562 cells, although significant activity was already present by 3 h. Treatment of monocytes with OKM1 monoclonal antibody plus complement abrogated both cell-mediated lysis and MCF generation, whereas Leu-11b plus complement were ineffective. These results indicate that human blood monocytes can release MCF during interaction with tumor cells and that this may be involved in the lytic mechanism of monocyte-mediated natural cytotoxicity.

Demonstration of specific receptors for fluoresceinated casein on human neutrophils and monocytes using flow cytometry.

Casein is chemotactic for human neutrophils (PMNs) and monocytes. The binding of fluorescein (FITC)-conjugated casein (mixture of alpha, beta, and kappa-casein) and purified alpha-casein to PMNs, monocytes, and lymphocytes was analyzed using flow cytometry. These studies demonstrate that 75-95% of PMNs and 46-85% of monocytes have membrane receptors for casein while lymphocytes lack these receptors. The binding of FITC-casein and FITC-alpha-casein was specific and was blocked only by unlabeled casein and alpha-casein, but not by ovalbumin, bovine or human serum albumin, beta-casomorphin, C5a, or formyl-methionyl-leucylphenylalanine (fMLP). The binding of FITC-casein was reversible when PMNs were stained with this fluorescent agent and subsequently incubated with unlabeled casein. Double-labeling studies of mononuclear cells using FITC-casein and the OKM1 monoclonal antibody in conjunction with a rhodamine conjugated anti-Ig second antibody demonstrate that mononuclear cells binding FITC-casein also stain with the OKM1 monoclonal antibody, indicating a specificity for monocytes.

Characterization of human T lymphocytes that express the C3b receptor.

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.

Suppressor cells of the human NK activity: Characterization of the cells and mechanism of action

The surface marker characteristics and mechanism of action of small- to medium-sized NK suppressor lymphocytes, which can be found in both umbilical cord blood and adult peripheral blood, have been studied. Evidence suggestive of T-cell origin of the lymphocytes consisted of E-rosette formation, reactivity with OKT3 monoclonal antibody, and dot-like acid α-naphthyl acetate esterase (ANAE) staining pattern typical of T cells. Furthermore, no reactivity was seen with OKT6 and OKM1 monoclonal antibodies and the presence of intracytoplasmic immunoglobulin was excluded by indirect immunofluorescence microscopy, making the involvement of monocytes, B cells, and thymocytes less likely. As regards the mechanism of action, the role of prostaglandins was unlikely since indomethacin had no effect on the level of suppression. The role of soluble mediators was further examined by blocking cell secretion with monensin. In these experiments monensin treatment of the suppressor cells did not unwind suppression, suggesting that mechanisms other than secretion of suppressive factors were operative. The importance of cell-to-cell contact was demonstrated by the following observations: (i) A short contact of effector lymphocytes with suppressor lymphocytes, followed by their physical separation, resulted in decreased cytotoxic activity of the effector cells, (ii) Suppression could be mediated through Nuclepore filters, which allowed cell processes to pass through the filter, but not through filters which did not allow cell-to-cell contact. The suppressor cells were resistant to irradiation (2500 rad) and treatment with dexamethasone and puromycin. Viable cells were not needed, since paraformaldehyde-fixed suppressor cells could also mediate inhibition of K562 killing.

Cytochemical profile of immunoregulatory T-lymphocyte subsets defined by monoclonal antibodies.

Immunogold staining in combination with enzyme cytochemistry was used to determine the cytochemical profile of the immunoregulatory T-lymphocyte subpopulations defined by the monoclonal antibodies OKT3, OKT4, OKT8, and OKM1 in normal peripheral blood. Leukocyte suspensions were first incubated with the monoclonal mouse antibodies and then with colloidal gold-labeled goat antimouse antibodies. The cells were fixed and cytocentrifuge preparations were made. Cytochemical reactions for the detection of peroxidase, acid alpha-naphthyl acetate esterase, acid phosphatase, and beta-glucuronidase were performed on these preparations. Under light microscopy, lymphocytes reacting with the monoclonal antibodies had numerous dark granules around their surface membrane. In the cytoplasm the intracellular enzymatic activities were stained. The T-lymphocytes were characterized by a dot-like activity for the three enzymes. No significant difference could be found between the cytochemical profile of the T-helper (OKT4 positive) and T-cytotoxic suppressor cell populations (OKT8 positive). A few cells with lymphocyte morphology reacted with the OKM1 monoclonal antibody. Their cytochemical characteristics were different from those of mature T-cells (OKT3 population) or mature monocytes. From the comparison of their cytochemical characteristics, we can conclude that there is little correlation between the immunoregulatory T-lymphocyte subsets defined by these monoclonal antibodies and those defined by Fc receptors.

Phenotypic heterogeneity of the OKM1-positive lymphocyte population: Reactivity of OKM1 monoclonal antibody with a subset of the suppressor/cytotoxic T-cell population

Upon analysis on a fluorescence-activated cell sorter, about 20% of E-rosette-positive cells from normal donors were found to stain with OKM1, a monoclonal antibody reacting primarily with cells of myelomonocytic origin. These OKM1(+) cells are not monocytes since they have small light scatter and are peroxidase negative. They also stain with OKT11 and antibody 9.6, two monoclonal antibodies against an antigen identical to or related to the E receptor. In two-color immunofluorescence experiments, the existence of an OKM1(+) OKT8(+) cell was demonstrated. This was also confirmed with additive staining experiments. This cell type constitutes ~10% of the E-rosette(+) cell population. OKM1 reacts with approximately 33% of OKT8(+) cells. Conversely, 48% of OKM1(+) E-rosette(+) cells are also OKT8(+). The same overlap was found between Leu 2a and OKM1 while no overlap between OKT4 and OKT8 staining cells, or between OKT4 and OKM1 staining cells was detected. The OKM1(+) lymphocyte population was isolated by removing OKT3(+) cells from nylon wool-nonadherent cells: 43 ± 13% of these cells also reacted with Ab 9.6 and 33 ± 10% with OKT8. At least three subpopulations of OKM1(+) lymphoid cells can therefore be identified: one without any T cell marker, one that is E-receptor positive, and one that has, in addition to the E receptor, a definite T-cell marker (OKT8). In addition, OKT8(+) cells can be separated into OKT8(+) OKT3(+) and OKT8(+) OKT3(−) populations. Only the OKT8(+) OKT3(+) population suppresses Ig synthesis in pokeweed mitogen-stimulated cultures.

Characterization of E-rosette-positive Fc-receptor-bearing cells by flow cytometry

A subpopulation of human peripheral blood mononuclear cells which have the capacity to form spontaneous rosettes with sheep erythrocytes (E +) have been observed to also express a receptor for the Fc portion of immune complexed IgG. In this study quantitative flow cytometry is used to investigate the binding of human IgG to human E + cells and to determine the relationship of these IgG-binding cells to the subsets of cells defined by OKT3 and OKM1 monoclonal antibodies. It was observed that a number of E + cells bear an Fc receptor for monomeric human IgG; that all subclasses of IgG can bind to this subpopulation; that this subset overlaps with the T cell identified with rosetting techniques; and that virtually all of these E + Fc receptor-positive cells binding monomeric IgG are phenotypically OKM1+ and OKT3−.

Monoclonal antibodies which distinguish between human NK cells and cytotoxic T lymphocytes.

Although it is widely accepted that T cells have a major role in specific tumour immunity, there is now much evidence that natural killer (NK) cells, which exist in many species and spontaneously lyse certain tumour cells in vitro, provide early resistance against tumour growth. Human NK-cell activity can be augmented in vitro by interferon and its inducers, including polyinosinic:polycytidylic acid (poly I:C); furthermore, NK-like activity is generated in mixed leukocyte cultures (MLCs) as is specific cytotoxic T-cell (Tc) activity. When effector cells generated in human MLCs lyse allogeneic or autologous virus-transformed or tumour cells, it has been difficult to evaluate the relative contributions of Tc and NK-like cells to the lysis because the latter, like T cells, can form rosettes with sheep erythrocytes and react with xenogeneic anti-human thymocyte serum. We report here that monoclonal antibodies against human mononuclear cell subpopulations can distinguish Tc from NK and NK-like cells. OKT3 or OKT8 monoclonal antibodies (reactive with virtually all or a subset of T cells, respectively) with complement (C') ablate MLC-generated Tc activity against allogeneic normal cells but do not decrease lysis of HLA-negative, NK-sensitive K562 leukaemia cells by NK, poly I:C-activated NK or MLC-activated NK-like cells. In contrast, OKM1 monoclonal antibody (reactive with a low proportion of non-adherent mononuclear cells as well as macrophages) with C' causes marked diminution of NK and poly I:C-activated NK-cell activity.