Characterization with monoclonal antibodies of T lymphocytes bearing Fc receptors for IgE (T epsilon cells) and IgG (T gamma cells) in atopic patients

Peripheral blood T lymphocytes from nonatopic control donors, asymptomatic atopic donors, and patients with moderate to severe atopic dermatitis were analyzed for Fc receptors for IgE (T epsilon cells) and IgG (T gamma cells) by rosette assays and were characterized with monoclonal antibodies. The T cells were reacted first with monoclonal antibodies, followed by fluoresceinated F(ab')2 goat antimouse Ig; they were then rosetted, and subsequently the rosetting cells were examined for immunofluorescence. Seven nonatopic control donors had less than 0.1% T epsilon cells and a mean +/- SD of 10.5% +/- 4.1 T gamma cells. Seven asymptomatic atopic donors with low IgE levels (2 to 233 IU/ml) varied from less than 0.1 to 1.3% T epsilon cells and 7.2% +/- 3.7 T gamma cells. Six of seven patients with moderate to severe atopic dermatitis and IgE levels of 1339 to 24,261 IU/ml had less than 0.1% T epsilon cells and significantly fewer T gamma cells (3.1% +/- 2.7, p less than 0.01) than the nonatopic control donors and the atopic donors in remission. Both T epsilon and T gamma cells reacted with the pan-T cell antibody Lyt-3 (anti-sheep red cell receptor) but not with antibodies OKT3, OKT4, or OKT6. Subpopulations of both T epsilon and T gamma cells reacted with antibodies OKT8 and the antimonocyte antibody OKM1. The OKM1+ cells did not appear to be monocytes, however, because the T cells did not react with another antimonocyte antibody, BRL.2, and were negative for nonspecific esterase activity. (ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

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.     

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.     

Isolation of human mononuclear cell subsets by counterflow centrifugal elutriation (CCE). I. Characterization of B-lymphocyte-, T-lymphocyte-, and monocyte-enriched fractions by flow cytometric analysis

Rapid separation of large numbers of human peripheral blood mononuclear cells into fractions enriched for B lymphocytes, T lymphocytes, or monocytes was accomplished by counterflow centrifugal elutriation (CCE). The first fraction contained 98% of the platelets. Ten additional fractions containing subpopulations of mononuclear cells were collected by sequential increases in the flow rate while maintaining a constant centrifuge speed. Analysis of the fractions using monoclonal antibodies revealed that fraction 2, which was free of esterase-positive monocytes, was highly enriched for B cells. T lymphocytes (OKT3+) were the predominant cell type found in fraction 4. No enrichment for T-lymphocyte-helper (OKT4+) or -suppressor (OKT8+) subpopulations was observed in the lymphocyte containing fractions. Three fractions (7-9), highly enriched for esterase-positive cells, were predominantly OKM1+ monocytes with no evidence of selective separation of monocyte subpopulations. Thus, cell fractions enriched for B cells, T cells, and monocytes could be obtained, by utilizing CCE, in large enough quantities to enable analysis of their functional properties. Of particular interest was the ability to separate small, resting B lymphocytes from monocytes.     

Induction of monocyte procoagulant activity with OKT3 antibody

OKT3 monoclonal antibody (MoAb), a mouse MoAb against cluster of differentiation 3 (CD3) molecule, induced a large amount of procoagulant activity (PCA) in human peripheral blood mononuclear cells (PBM). The PCA-inducing capability in OKT3 MoAb was abolished by absorption with T lymphocytes or Sepharose-conjugated antibody to mouse IgG. Most of the PCA in PBM was associated with monocytes. There was a dose-dependent increase in PCA when increasing numbers of T cells were added to the monocytes in the presence of OKT3 MoAb. OKT3 MoAb did not induce PCA in either T cells or monocytes alone. T cells pulsed with OKT3 MoAb only in the presence of monocytes could induce PCA in monocytes. Culture supernatants (CS) from PBM stimulated with OKT3 MoAb did not enhance PCA in monocytes; however, it did induce PCA in the human monocyte-like cell line (U937) which differs in some properties from monocytes; this activity could be abolished by the MoAb against human interferon-gamma (IFN-gamma). Nevertheless, neither human IFN-gamma nor interleukin 1 or 2 had significant direct effect in inducing PCA in U937 cells; CS from either monocytes or T cells alone stimulated with OKT3 MoAb did not induce PCA in U937 cells. This apparent discrepancy suggests that there may be factors in CS that induce PCA in U937 cells only in the presence of IFN-gamma. The PCA induced in monocytes or U937 cells was tissue factor-like because of the dependence on coagulation factors V, VII, and X. These observations suggest that OKT3 MoAb is a potent T cell-dependent monocyte PCA inducer and stimulates T cells only in the presence of monocytes. The direct cellular interaction between monocytes and stimulated T cells appears to be necessary to elicit monocyte PCA with OKT3 MoAb stimulation. Thus, monocytes may play a dual role, not only as effector cells, but also as cells that collaborate with T cells after OKT3 MoAb stimulation so as to produce PCA.     

Depletion of human NK cells with monoclonal antibodies allows the generation of cytotoxic T lymphocytes without NK-like cells in mixed cultures

In vitro stimulation of human mononuclear cells with x-irradiated autologous lymphoblastoid cell line (LCL) or allogeneic normal cells in mixed leukocyte cultures (MLC) was previously shown to result in the generation of OKT3+ OKT8+ cytotoxic T lymphocytes (CTL) lytic for allogeneic and autologous LCLs and also of natural killer- (NK) like cells that are OKT3- and primarily OKT8- and are lytic for HLA- NK-sensitive K562 cells. The origin of the NK-like cells was not previously known because, although the majority of fresh human NK cells react with monoclonal antibodies OKM1 and B73.1, lymphocytes bearing these markers are not detected several days after the onset of MLC, when NK-like cells are present. In this study, experiments were undertaken to determine whether NK-like cells generated after stimulation with x-irradiated pooled allogeneic normal cells (poolx) or with autologous LCL are derived from cells expressing antigens reactive with monoclonal antibodies OKM1 or B73.1, which react with fresh NK cells. Mononuclear cells, depleted of monocytes, were stained with OKM1 or B73.1 and fluorescein-labeled goat anti-mouse IgG. Lymphocytes depleted of OKM1+ or B73.1+ cells, by fluorescence-activated cell sorting, and lymphocytes that were stained but not sorted were stimulated for 7 days with either poolx or autologous LCL. The generation of NK-like activity was decreased at least 90% after depletion of cells reactive with OKM1 or B73.1, whereas the generation of CTL against autologous and allogeneic LCL was minimally affected. These findings show that NK-like cells generated in MLC are derived from cells that express the phenotype of fresh NK cells (OKM1+ or B73.1+) and that CTL can be generated in cultures in which relatively little NK-like activity is concomitantly detected, by depleting NK cells with monoclonal antibodies before stimulation.     

Identification of the T cell subset that produces human gamma interferon

Positive and negative selection procedures combined with cytofluorographic analysis and lysis with monoclonal antibodies were utilized to identify the T lymphocyte subset that produces human gamma interferon (gamma-IFN) (formerly referred to as "immune" or "type II" interferon) in response to mitogen stimulation. Lymphocytes were separated on the basis of their Fc receptors for IgG or IgM, their nonreactivity with IgM or IgG antibodies, and their reactivity with the monoclonal antibodies OKT4, OKT8, OKT11a, and OKM1. Isolated T cell subsets were incubated with the gamma-IFN inducer, phytohemagglutinin. Three days after induction, the cell supernatants were harvested and assayed for interferon. The T cell subset that produces gamma-IFN was identified as E rosette positive with the phenotype: T gamma, T non-micro, OKM1+, OKT4-, OKT8- and OKT11a+. gamma-IFN production by cells was resistant to doses of x-irradiation that abrogate mitogen-induced T suppressor function but was highly sensitive to low doses of 4-hydroperoxycyclophosphamide. These data demonstrate that gamma-IFN is produced by the T gamma, OKM1+ lymphocyte subset, but these cells may also require the presence of accessory monocytes for elaboration of gamma-IFN. The anti-proliferative activity of gamma-IFN may be responsible for the previously described suppressor function of this subset, and gamma-IFN production by T gamma cells may distinguish this subset from the suppressor/cytotoxic functions of the OKT8+ subset or the mitogen-induced OKT4+ suppressor.     

Treatment with recombinant IFN-gamma decreases cell surface CD4 levels on peripheral blood monocytes and on myelomonocyte cell lines

The human cell surface protein CD4 is not only an important accessory molecule in the activation of MHC class-II-restricted T cells, but has also been implicated to be a receptor for the human immunodeficiency virus HIV-I on lymphoid and monocytic cells. We have found that a 24-h treatment of the promonocytic leukemia cell line U937 with rIFN-gamma decreases the expression of the CD4 Ag by 50% as measured by cytofluorographic analysis. The decrease in CD4 expression was dependent on the concentration of rIFN-gamma, with maximal effects occurring at 20 to 200 U/ml. The decrease appeared to be due to actual loss of the CD4 molecule from the cell surface rather than masking of a particular epitope, inasmuch as similar results were obtained with the OKT4 and OKT4A antibodies. The effect of rIFN-gamma to decrease CD4 expression was not due to a general loss of cell surface Ag, because the binding of OKM1 and anti-HLe-1 increased after rIFN-gamma treatment. Treatment of rIFN-gamma also decreased cell surface CD4 expression on the promyelocytic leukemia cell line HL-60, and on the monocytic cell line THP-1, although the extent of the decrease was less than on U937 cells. Freshly isolated normal peripheral blood monocytes treated for 48 h with rIFN-gamma bound much less OKT4 or OKT4A antibody than cells incubated in the absence of rIFN-gamma. Moreover, treatment with rIFN-gamma reduced the percentage of peripheral blood monocytes that were positive for the CD4 Ag. In contrast with the decrease in CD4 levels on rIFN-gamma-treated monocytes, treatment with rIFN-gamma had no effect on CD4 levels on peripheral blood T lymphocytes or T cell lines.     

Distinct classes of human T-cell activation antigens

The characterization of three groups of antigens expressed by activated human T lymphocytes and detected by monoclonal antibodies is reported. Antigens defined by OKT19, OKT21, and OKT22 do not appear on in vitro activated T cells until increases in DNA synthesis become apparent and are not detected on most Interleukin 2 (IL-2)-independent cell lines and normal peripheral blood lymphocytes, monocytes, and granulocytes. Cell surface molecules reactive with the monoclonal antibodies OKT23 and OKT24 are displayed prior to any notable increase in DNA synthesis and are present on IL-2 independent cell lines, irrespective of lineage. T23 and T24 do not appear on peripheral blood cells and their distribution more closely resembles that of the T9 antigen (the receptor for transferrin) than antigens of the other groups. The third group of antigens, T14 and T20, have been classified as "early" antigens relative to DNA synthesis. They are expressed by distinct populations of normal lymphoid cells as well as by some IL-2-independent cell lines. Display of each group of activation antigens on T lymphocytes can be induced by either phytohemagglutinin, purified protein derivative from tuberculin, or allogeneic non-T cells, is not restricted to the OKT4+ or OKT8+ subsets, and is predominant on cells exhibiting the light-scattering properties of blast cells. The relative lack of expression of these antigens among normal peripheral blood cells make them attractive candidates for identifying changes in the status of immune activation.     

Human splenic sinusoidal lining cells express antigens associated with monocytes, macrophages, endothelial cells, and T lymphocytes

The antigenic and functional properties of splenic sinusoidal lining cells (SLC) have not been studied extensively. Some investigators have suggested that SLC are actively phagocytic, and thus part of the mononuclear phagocyte system. Others dispute this and assign the functions of endothelial cells to the SLC. During studies in situ of phenotypic subpopulations of human splenic macrophages (M phi), we found that SLC share membrane antigens (HLA-DR and OKM5), an enzyme (lysozyme), and histochemical properties (nonspecific esterases) with monocytes and M phi. In addition, we showed that LSC, like endothelial cells, synthesize factor VIII of the clotting system and also bear the receptor for transferrin. Our previous studies found that SLC express the antigens found on helper/inducer (OKT4, Leu-3a,b) and suppressor/cytotoxic (OKT8, Leu-2a) T lymphocyte subsets. We have confirmed these observations, and have shown by means of preincubation with soluble complexes of anti-human IgG-human IgG that the detection of T cell and other antigens on SLC is not due to nonspecific binding of antibodies by Fc receptors. By using techniques designed to isolate and purify splenic M phi, we were able to obtain SLC in suspension and to demonstrate that they retain the antigens detected in situ. Thus, the human splenic SLC expresses a unique combination of antigens, histochemical properties, and cell products in common with monocytes, M phi, and T lymphocytes.     

Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies

A series of seven monoclonal antibodies directed at determinants on human peripheral blood monocytes were produced and characterized. The antibodies were separated into three groups based on cell distribution and percentages of monocytes bearing antigen. Hybridoma antibodies, termed OKM1, OKM9, and OKM10, recognized antigen(s) expressed on the majority of adherent monocytes, null cells, and granulocytes. The second group, comprising OKM5 and OKM8, reacted with most adherent monocytes and platelets. OKM3 and OKM6, comprising a third group of antibodies, reacted with a subpopulation of adherent monocytes and platelets. OKM antibodies were not expressed on lymphocytes, thymocytes, and lymphoblastoid cells, with the exception of OKM3 which reacted with three B-cell lines. SDS gels of immunoprecipitates formed with OKM antibodies yielded the following tentative molecular weight results: OKM1 and OKM9 antigens appeared to be 160,000 (nonreduced) and 170,000 (reduced); OKM10 precipitated two polypeptides of 170,000 and 115,000 (reduced); OKM5 and OKM8 precipitated a single polypeptide of 88,000 (reduced, nonreduced); OKM6 antigen appeared to be 116,000 (nonreduced) and 130,000 (reduced).     

Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production.

We have investigated the roles of cytokines in the modulation of human immunodeficiency virus (HIV) production in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3. HIV-infected U937 cells exhibited markedly lower levels of CD4 and HLA-DR antigens than uninfected cells did. Vitamin D3 induced a time-dependent macrophagelike differentiation, as determined by monitoring the expression of some surface antigens by means of the monoclonal antibodies OKM1, OKM5, OKM13, OKM14, OKT4, anti-HLA-DR, TecMG2, TecMG3, LeuM3, LeuM1, anti-HLA-DP, and anti-HLA-DQ. Treatment with hydroxyvitamin D3 resulted in a marked increase in HIV production compared with control cultures. Interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were detected in the culture media, whereas interferon (IFN) was not generally found. Using the polymerase chain reaction technique, we found HIV-infected U937 cells to express detectable levels of mRNAs for alpha interferon (IFN-alpha), IFN-beta, TNF-alpha, and IL-1 beta. The addition of TNF resulted in a marked increase of HIV production, whereas IL-1 beta was ineffective. In contrast, both IFN-alpha and IFN-beta exerted some inhibitory effect on HIV production, which was more marked in vitamin D3-treated cultures than in untreated cultures. HIV production was significantly increased by antibodies to IFN-alpha in both untreated and vitamin D3-treated cultures. Anti-IFN-beta antibody increased HIV production only in vitamin D3-treated cells. In contrast, anti-TNF-alpha antibodies markedly decreased HIV production in both control and differentiating U937 cells. Vitamin D3 treatment resulted in a higher expression of TNF receptors in differentiating cells than in control HIV-infected cells. These data demonstrate a strong correlation between HIV production and macrophagelike differentiation in chronically infected U937 cells and suggest that endogenous IFN and TNF exert opposite effects in the regulation of virus production in both undifferentiated and vitamin D3-treated cell cultures.     

T cell unresponsiveness to the mitogenic activity of OKT3 antibody results from a deficiency of monocyte Fc gamma receptors for murine IgG2a and inability to cross-link the T3-Ti complex

We recently identified defective monocyte accessory function as the cause of T cell unresponsiveness to the mitogenic activity of OKT3 antibody in cultures of peripheral blood mononuclear cells (PBMC) from five healthy subjects, members of one family. We now report that the underlying abnormality in nonresponders is at the level of monocyte Fc gamma receptors for murine IgG2a. T cell unresponsiveness was not restricted to the signal provided by OKT3 but occurred also for two other anti-T3 antibodies of the IgG2a subclass, in contrast to a normal proliferative response to IgG1 anti-T3 antibodies in one of the OKT3 nonresponders. By using cytofluorography, we found that monocytes from responders but not from nonresponders bound OKT3-FITC to their membrane. The binding could be blocked by mouse IgG2a and by human IgG, but not by mouse IgG1 nor by serum albumin. The data suggest that, through specific Fc gamma receptors for murine IgG2a, monocytes bind the Fc portion of OKT3 during T cell activation. The function of this Fc gamma receptor binding was further studied by culturing PBMC from nonresponders on plates coated with affinity-purified goat anti-mouse IgG antibodies as a substitute for monocyte Fc gamma receptors. The addition of OKT3 to nonresponder PBMC, cultured on such plates, resulted in T cell activation, as evidenced by thymidine incorporation, IL 2 production, and expression of IL 2 receptors. Soluble anti-mouse IgG was not able to substitute for monocyte Fc gamma receptors. The results demonstrate the existence of polymorphism in monocyte Fc gamma receptors for murine IgG2a. They also substantiate that an essential helper function of monocytes in T cell activation by anti-T3 is to provide a matrix for multimeric binding of the Fc portion of the anti-T3 antibodies in order to cross-link T3 molecules.     

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.     

Human mononuclear phagocyte-associated antigens: I. Identification and characterization

Rabbit antisera were produced against a lymphokine-activated human macrophage cell line, U937 (αU937), and human peritoneal macrophages (αPEMØ). After absorption with AB erythrocytes, pooled platelets, and B-lymphoblastoid cell lines, both antisera reacted by microcytotoxicity, indirect immunofluorescence (IF), and radioimmunoassay (RIA) with adherence-purified human peripheral blood monocytes, splenic and peritoneal macrophages, and leukemic myelomonoblasts. A panel of normal human T lymphocytes, B lymphocytes, and erythroid-myeloid or lymphoblastoid cell lines failed to react with both αU937 and αPEMØ. Although both heteroantisera reacted against polymorphonuclear leukocytes (PMNs), after absorption with PMNs specific reactivity against mononuclear phagocytes remained. Absorption of αU937 and αPEMØ with myelomonoblastic leukemia cells (AMML) removed IF and RIA activity against both PMNs and monocytes but not against splenic and peritoneal macrophages. In contrast, absorptions of both heteroantisera preparations with splenic macrophages abolished their IF and RIA reactivity not only to splenic and peritoneal macrophages but also to peripheral blood monocytes and leukemic myelomonoblasts. These results are consistent with (1) both antisera defining specific monocyte/macrophage-associated antigens(s) which are distinct from MHC-coded HLA-A,B,C, and DR antigens, and (2) expression of common monocyte/macrophage-associated antigen(s) and uniquely associated antigen(s) selectively expressed on tissue macrophages. These reagents will be useful in delineating human monocyte/macrophage differentiation as well as the immunological functions of mononuclear phagocytes.     

Modulation induction of the T3 antigen by OKT3 antibody is monocyte dependent

We investigated the influence of monocytes on the susceptibility of the T3 antigen on human T cells to modulation induction by OKT3 antibody. In the absence of monocytes, the T3 antigen was only minimally susceptible to modulation. After the addition of 20% monocytes to the culture, however, complete modulation was readily observed. Furthermore, we found that even in the absence of OKT3 antibody, monocytes were able to down-regulate the expression of the T3 antigen, although to a lesser extent. The ability of monocytes to enhance antigenic modulation proved to be a more general phenomenon. Each individual T cell antigen, however, differed in its susceptibility to modulation by antibody, monocytes, or both, thereby establishing its own characteristic pattern. In addition, after complete modulation of the T3 antigen, the addition of monocytes to the culture thereafter had a distinct inhibitory effect on the reexpression of the T3 antigen. Monocyte enhancement of T3 modulation is significantly reduced when using the OKT3 F(ab')2 fragment, as is OKT3 mitogenesis. After pulsing the monocytes with OKT3 antibody before adding them to the culture, T3 modulation became nearly complete even in the absence of added OKT3 antibody. Monocyte-induced modulation proved not to be MHC restricted, thus allowing for comparative analysis of this effect between monocytes and other cell types. A moderate, however, incomplete modulation enhancement was observed with the human monocyte cell line U937 and with Daudi cells. This finding proved to coincide with the distinct ability of these cell lines to bind OKT3 antibody by their Fc receptors, as was the case with monocytes. In contrast, neither Fc receptor binding nor T3 modulation enhancement was observed with the cell lines Cess and G7. In addition, no effective T3 modulation was observed with glutaraldehyde-fixed monocytes. The overall results seem to indicate that effective modulation of the T3 antigen by OKT3 antibody requires the active participation of Fc receptors on monocytes.     

Cellular origin and interactions involved in gamma-interferon production induced by OKt3 monoclonal antibody

OKT3 monoclonal antibody, a human T cell mitogen, induced interferon production by cultured mononuclear cells at 10(-11) M concentrations. Interferon was secreted only under conditions wherein OKT3 was mitogenic, and production was correlated with cell proliferation. Thus, like mitogenesis, interferon secretion reached a peak 3 days after OKT3 stimulation, was inhibited by a factor(s) in human serum, and required 1000 times higher concentrations of Fab and F(ab')2 fragments of OKT3 for induction. The interferon was most likely of "gamma" (immune) type, because pH 2 and 56 degrees C treatments denatured it, whereas anti-alpha or -beta interferon antibodies did not. Mononuclear cells were fractionated into subpopulations that contained OKT4+ cells (helper/inducer T cells), OKT8+ cells (cytotoxic/suppressor T cells), and OKM1+ cells (monocytes) by combining sheep red blood cell rosetting and complement-mediated lysis using monoclonal antibodies against specific cell types. Both OKT4+ and OKT8+ cells proliferated upon OKT3 stimulation with the absolute requirement of OKM1+ cells. However, OKT4+ cells plus OKM1+ cells were necessary for the secretion of interferon. Studies with selective pretreatments with mitomycin C suggested that gamma-interferon was secreted by the OKT4+ cells and that the OKM1+ population subserved an accessory function.     

Phenotypic Analysis of New World Primate Mononuclear Cell Surface Antigens

We have applied a panel of monoclonal antibodies against antigens present on the surface of human mononuclear cells to the study of mononuclear cell surface antigens expressed by seven species of New World primates. Antibodies to the sheep erythrocyte receptor (OKT11a) to a thymocyte antigen (OKT10), to the I region of the major histocompatibility locus (OKIa), and to an antigen found on the surface of human monocytes (OKM1) cross-reacted with mononuclear cell surface antigens of most of the species studied. Antibodies to antigens which have been correlated with functional capabilities in the human system (OKT4, OKT5, OKT8, 3A1) were much less reactive with platyrrhine mononuclear cells. These reagents may be quite useful in studies of primate phylogeny and immunology.     

Activation of resting T lymphocytes by anti-CD3 (T3) antibodies in the absence of monocytes

The antigen receptor molecules on human T lymphocytes are noncovalently associated on the cell surface with the CD3 (T3) molecular complex. Perturbation of this complex with anti-CD3 monoclonal antibodies induces T cell activation. Previous studies have demonstrated that this process requires the participation of monocytes. In the present report, we demonstrate that purified, resting (G0 phase) T cells incubated with monoclonal anti-CD3 antibodies proliferate in response to purified interleukin 2 (IL 2), in a lymphokine dose-dependent fashion. Anti-CD3 antibody or IL 2 alone did not trigger cell division. The effect was specific for anti-CD3 antibodies because monoclonal antibodies reactive with other surface molecules (OKT4, OKT8, L368) were inactive. Furthermore, the same phenomenon was observed when anti-CD3 antibody Leu-4 (IgG1) was incubated with cells of individuals whose monocytes cannot process antibodies of the IgG1 subclass (Leu-4 nonresponders). In addition, both F(ab')2 and Fab fragments of anti-CD3 antibody OKT3 were also capable of rendering T cells receptive to the IL 2 growth signal. These data indicate that neither monocytes nor CD3 receptor cross-linking are required absolutely for resting T cell activation, provided that IL 2 is supplied exogenously. T lymphocytes treated with anti-CD3 antibodies proliferated in response to both purified mitogen-induced and recombinant IL 2. Antibodies to the IL 2 receptor (anti-Tac) inhibited the proliferation. Thus, the most likely mechanism for anti-CD3 antibody-mediated triggering is induction of IL 2 receptors.