Enhancement of the functional activities of human T cells after their interaction with SRBC

We have examined the functional consequences of the exposure of human lymphocytes to sheep red blood cells (SRBC). Peripheral blood lymphocytes (PBL) were incubated wih SRBC under optimal conditions for their interaction and, after lysis of the erythrocytes, the unfractionated PBL were examined for several T cell functions. Only after exposure to SRBC, but not after incubation with mouse, ox, chicken, or human erythrocytes, the unfractionated PBL showed an increased reactivity in the following functions: 1) Production of T cell growth factor, after PHA stimulation; 2) mitogenic response to suboptimal doses of PHA and Con A, and 3) response in mixed lymphocyte culture. Other functional activities, such as natural cytotoxicity (NK) and antibody-dependent cellular cytotoxicity (ADCC) were also enhanced by the interaction of PBL with SRBC, but the increases in cytotoxic activities were not consistently detected. Taken together, these results indicate that the interaction of PBL with SRBC has functional consequences in the reactivity of T cells producing an enhancement of several in vitro T cell functions.     

Mitogenic responsiveness and monocyte-lymphocyte interaction of early and late rosette-forming cell populations of human peripheral blood lymphocytes.

Adherent cells in human peripheral blood mononuclear cells were removed by the attachment to the plastic surface of tissue culture dishes. After removal of adherent cells, early rosette-forming cells (early RFC), which were characterized by early (5 min) rosette formation with sheep blood cells (SRBC) at an SRBC to lymphocyte ratio of 8:1, were separated from nonrosetting cells by sedimentation on Ficoll-Hypaque gradient. Total (60 min) rosette formation was carried out with the early RFC-depleted cell population on the gradient interface by the use of neuraminidase-treated SRBC at an SRBC to lymphocyte ratio of 20:1 and the resulting rosette-forming cells (late RFC) were sedimented by gradient centrifugation. These T cell subpopulations, early RFC-enriched and late RFC-enriched, were reasonably pure with respect to the ability to bind SRBC and contained less than 0.5% monocytes. Monocyte preparations, which were obtained after vigorous washing of the adherent cell layers on tissue culture dishes, responded to phytohemagglutinin P (PHA-P) or concanavalin A (Con A) with negligible incorporation of 3H-thymidine. There was no significance difference in the responsiveness to PHA-P between early RFC-enriched and late RFC-enriched populations either in the absence or in the presence of graded numbers of additional autologous monocytes. However, the response of early RFC-enriched population to Con A was significantly poor as compared with that of late RFC-enriched one unless additional monocytes were added. In the presence of 20% autologous monocytes in the culture, the Con A-induced response of early RFC-enriched population was markedly enhanced to reach close to that of late RFC-enriched population. These results suggest that early RFC and late RFC might be different from each other in their responsiveness and in their need for monocytes on the stimulation with Con A.     

Failure of OKT3 monoclonal antibody to induce lymphocyte mitogenesis: a familial defect in monocyte helper function

Monoclonal antibodies to the T3 molecule on human T cells have mitogenic activity. Although anti-T3 antibodies of the IgG1 subclass (e.g., UCHT1) induce mitogenesis in lymphocyte cultures from only 60 to 70% of normal donors, antibodies of Ig2a subclass (e.g., OKT3) invariably have been found to be mitogenic in all subjects tested up to the present. This paper describes a family (a mother, six daughters, and one son) in which five members failed to respond mitogenically to OKT3 although the proportion of OKT3-reactive cells in their peripheral blood was normal. Mitogenic responses to PHA, Con A, and PWM were normal. Five members comprising four OKT3 nonresponders were also unresponsive to UCHT1. Unresponsiveness to OKT3 and unresponsiveness to UCHT1 were not absolutely linked to each other, nor were they linked to an HLA haplotype inherited from the mother. Upon stimulation by OKT3, lymphocyte preparations from OKT3-nonresponders failed to produce interleukin 2 (IL 2) and to display IL 2 receptors. OKT3 unresponsiveness was due to defective monocyte help: thus, responsiveness to OKT3 of T cells from an OKT3-nonresponder was restored by the addition of monocytes from an HLA-identical sister who had a normal response to OKT3. Inversely, T cells from the OKT3 responder had no reactivity to OKT3 when cultured in the presence of monocytes from an HLA-identical, OKT3-nonresponsive sister. Unresponsiveness to OKT3 could not be overcome by the addition of phorbol myristate acetate to the cultures. These data on a familial, non-HLA-linked deficiency of monocytes to exert their auxiliary function provide better insight into the mechanism of anti-T3-induced T cell activation.     

Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.     

Accumulation of eosinophils and monocytes in lymphoid organs of chick-embryos. II. Effect of mitogenic stimulation.

The mitogen effect on migration of eosinophils and monocytes was studied in embryonic chickens. On the 13th embryonic day, chickens were injected with mitogens, such as concanavalin A (Con A), phytohemagglutinin-P (PHA-P), and lipopolysaccharide (LPS), into the allantoic cavity, and the mitogenic effect was estimated from the relative frequencies of eosinophils and monocytes by enumerating the number of oxidase positive cells (OPC) in the spleen, thymus, and bursa of Fabricius. Splenic frequencies of OPC increased in the embryos treated with mitogens. Similar influences were also detected in the thymic OPC. Higher responses were seen on the 18th embryonic day in the number of splenic OPC when embryos were treated with Con A or PHA-P than with LPS. These findings suggest that Con A and PHA-P are preferential OPC accumulation promoters. However, bursal frequencies of OPC in the cortex were low after mitogenic stimulations when compared with controls, although appreciable responses were detected in the bursal medulla after LPS stimulation. These results suggest that the migration pattern in the population of eosinophils and monocytes is affected not only by T cell mitogens but is also derived from LPS stimulation.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Mitogenic Activity of a Water-Soluble Adjuvant (Bu-WSA) Obtained from Bacterionema matruchotii

Butanol-extracted water-soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was cultured with peripheral blood mononuclear cells (PBM) in the presence of sub- and/or supra-optimal mitogenic concentrations of concanavalin A (Con A). The addition of Bu-WSA resulted in increased tritiated thymidine incorporation above that produced by Con A alone. Bu-WSA by itself is not mitogenic for PBM and in fact produced a decrease in thymidine uptake compared to the control. We investigated the response of subpopulation(s) of PBM to Bu-WSA, Con A and a mixture of Bu-WSA and Con A. Separation of PBM into purified T cells, B cells and macrophages showed that cell-cell cooperation of T cells with B cells or macrophages is necessary for the observed synergistic effect of Bu-WSA with Con A. A marked increase in thymidine incorporation by the mixture of T and B cell populations occurred, while only a small amount of thymidine was incorporated when the B cell population was absent. Mitomycin treatment revealed that the response could be ascribed to the T-cell response with a B-cell helper effect. Moreover, Con A and Bu-WSA appeared to act on the same T cell population. This model may provide unique information about the activation of human peripheral blood T cells compared with the activation of these cells by other mitogens.     

Identification of human peripheral T cell antigens.

A new type of differentiation antigens on human T cells was demonstrated by using a heterologous anti-human T cell serum (ATS). This type of antigen, referred to as human peripheral T cell antigen (HPTA), was found on peripheral T cells and medullary thymocytes, but not on cortical thymocytes and B cells. The percentage of ATS-reactive lymphocytes in human peripheral lymphoid organs was correlated with that of cells rosetting with sheep erythrocytes, but contrasted with the number of B cells defined by the presence of a complement (C) receptor or by rabbit anti-human B cell serum (ABS). ATS also reacted with T cells purified by nylon fiber column filtration but ABS did not. Chronic lymphocytic leukemia cells rosetted with either sheep erythrocytes or erythrocyte-antibody-complement complexes were lysed by ATS and ABS, respectively. Mitogenic responses of blood lymphocytes to phytohemagglutinin (PHA) and concanavalin-A (Con A) were abrogated by treating them with ATS and C, whereas ABS suppressed only their response to Con A. Although numerous thymus cells rosetted with SRBC, only 14% were reactive with ATS. Quantitative absorption studies demonstrated that HPTA content of the thymus cells was much lower than that of lymph node cells. Anatomical localization of ATS-reactive lymphocytes in human lymphoid organs studied by immunofluorescence indicated that they were present in the thymus-dependent paracortical areas of lymph node and in the medullary region of thymus. ABS, on the other hand, did not stain thymocytes but reacted selectively with the cells located in the lymphoid follicles of lymph node. These data, together with that from cell suspension studies, confirmed that HPTA were shared between medullary thymocytes and peripheral T cells.     

Visualization of cholecystokinin receptors on a subset of human monocytes and in rat spleen

Direct radioreceptor binding experiments and Scatchard analysis reveal CCK receptors on elutriator purified human peripheral blood monocytes, but not on purified human T cells. The monocyte receptors have a single class of high (0.1 nM) affinity binding sites. A structure-function analysis of monocyte binding by different CCK analogs correlates well with previously demonstrated chemotactic responses in monocytes and receptors in brain tissue. Biochemical cross-linking indicates that the monocyte CCK recognition molecule is comparable in molecular size to that in brain membranes. Utilizing a novel fluoresceinated Texas Red-CCK conjugate we have visualized that up to 20% of human peripheral monocytes bear receptors for CCK. A discrete and anatomically significant distribution of CCK receptors in rat spleen is shown by film autoradiography of tissue sections. A more detailed microscopic analysis identifies a dendritic population of monocyte-derived cells within the periarteriolar lymphocyte sheath (PALS) of the white pulp as the CCK receptor-bearing cell in spleen. The anatomical localization of receptor-bearing cells within the PALS region suggests a role for CCK in the antigen processing and sensitization phases of the immune response via regulatory effects of this peptide on a specific, local macrophage-related cell population.     

Mitogen responsiveness and inhibitory activity of mesenteric lymph node cells

Summary Blood-, lymph node-, and tumour-infiltrating lymphocytes (PBL, LNC, and TIL, respectively) from patients with colonic neoplasms were tested for responsiveness to phytohaemagglutinin (PHA). All populations responded, with LNC and PBL showing comparable reactivities while TIL were less reactive as assessed by incorporation of ³H-thymidine. Increased mitogen responsiveness was observed for T cells enriched by SRBC rosette formation or passage through nylon columns. Mitomycin C-treated LNC and TIL inhibited PHA induced ³H-thymidine incorporation of admixed autologous PBL, suggesting the presence of suppressor cells. Suppressor activity resided primarily in the SRBC rosetting population and was dose-dependent, with increasing numbers of LNC giving greater diminution of PHA response. Suppression by LNC was apparent only when they were added to PBL responders within 6 h of the initiation of stimulation assays, in common with the effects of Concanavalin A (Con A)-induced suppressors on PBL phytomitogen responsiveness. Con A-induced and LNC-suppressor activity could be reversed by addition of lymphocyte-conditioned medium (CM) containing T cell growth factor (TCGF; interleukin IL-2). These data provide further evidence that the suppressor phenomena observed in this system are a function of activated T cells present both in drainage lymph nodes and at the tumour site.     

Cellular origin of histamine-releasing factor produced by peripheral blood mononuclear cells

Histamine releasing factors (HRF) are a group of cytokines that release histamine and other mediators from mast cells and basophils. It has been speculated that HRF might play a major role in the pathogenesis of allergic diseases. Most investigators have studied PBMC as a source of HRF. This study was undertaken to investigate the cellular origin of HRF. Peripheral blood was processed to isolate and purify monocytes, T cells, CD4- T cells, CD8- T cells and B cells by using plastic adherence, 2-aminoethylisothiomonium-treated SRBC rosetting and negative selection with the use of mAb OKM1, OKT11, OKT8, OKT4, and OKB7 plus C. Highly purified subpopulations of PBMC were cultured alone or in the presence of Con A for 24 h. Supernatants were harvested, dialyzed, and assayed for HRF activity in the basophil histamine release test. We found that all subpopulations of PBMC including T cells, CD4- T cells, CD8- T cells, B cells, and monocytes produce variable quantities of HRF. The spontaneous production is very high in B cells but only barely measurable in T cells and monocytes. The synthesis of HRF by B cells was confirmed by abolishing the release of the activity after treatment of B cells with OKB7 mAb and C. Stimulation of cell populations by Con A significantly enhances HRF production by PBMC and T cells but not by B cells and monocytes. In mixing experiments, unstimulated monocytes + B cells showed synergism, but other combinations demonstrated an additive effect. This is the first demonstration of HRF production by human peripheral blood B cells. The results of this study also suggest that histamine releasing cytokines are of multiple cellular origin. This perhaps contributes to their molecular heterogeneity.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

A distinct "slow" cellular pathway involving soluble mediators for the T cell-instructed induction of monocyte tissue factor activity in an allogeneic immune response

Allogeneic stimulation of human lymphoid cells initiates a collaborative cellular pathway that relatively rapidly induces in monocytes the synthesis and cell surface expression of tissue factor, the initiating cofactor of the extrinsic coagulation pathway. T cells are required for the monocyte procoagulant generation, because the addition of autologous or allogeneic T cells fully reconstitutes the activity in otherwise nonresponding highly purified monocytes. Despite a strict T cell requirement, only low T cell to monocyte ratios are necessary for maximal PCA response. Our results further demonstrate that the collaborative signal from allogeneically stimulated T cells to effector monocytes is transferred by a soluble mediator rather than direct cell to cell contact. Other aspects of the present study include the observation that among normal peripheral blood lymphoid cells, monocytes elicit the strongest allogeneic PCA response. This response is clearly exceeded by that induced upon stimulation with Daudi lymphoblastoid B cells. Our data demonstrate the existence of a second distinct cellular pathway that mediates the lymphoid procoagulant response. This pathway differs from the previously characterized response to bacterial LPS in respect to: a) kinetics of T cell triggering; b) mediation by a soluble product; c) lack of genetic restriction of T cell; monocyte collaboration; and d) deficient capacity for direct T cell induction of the monocyte PCA response.     

The role of nonactivated and interferon-gamma activated monocytes in regulating normal and SLE patient B cell responses to TNP-Brucella abortus

We have previously characterized the human B cell response to trinitrophenol (TNP)-Brucella abortus (Ba) response as being T cell independent. In this report we examine the role of monocytes in the TNP-Ba antibody response of human peripheral blood mononuclear cells (PBMC). Depletion of monocytes by sequential adherence to plastic and Sephadex G-10 passage did not result in decreased plaque-forming cell responses to TNP-Ba, suggesting that monocytes were not required. On the contrary monocytes were probably inhibitory because their removal resulted in enhanced responses. This was confirmed by showing that adding monocytes back reconstituted the inhibition. When interferon-gamma (IFN-gamma), a potent activator of monocytes, was added to TNP-Ba-driven PBMC cultures, marked inhibition (greater than 90%) of the responses ensued. This IFN-gamma-mediated suppression was monocyte dependent because it was completely abrogated by monocyte, but not T cell depletion. Previously, we described a concanavalin A (Con A), T cell inhibition pathway of the TNP-Ba response. Both the Con A and IFN-gamma pathways were tested for their ability to inhibit systemic lupus erythematosus (SLE) patient responses to TNP-Ba. The B cell response of SLE patients was inhibitable by both pathways. In all of the patients, the inhibition was complete (greater than 95%) when IFN-gamma was added to the cultures. In the presence of Con A, greater than 95% inhibition was observed in six of 10 patients, the remainder being inhibited to a lesser extent. Thus the hyperactive B cells from SLE patients can be down-regulated, particularly in the presence of IFN-gamma.     

Phenotypic markers for the feline monocyte: rosette formation with human erythrocytes and a monoclonal antibody which binds myeloid cells

A small population of cells with the ability to form rosettes with human erythrocytes was found in feline peripheral blood leukocytes (PBL) (10%) and bone marrow (9%), but not in purified granulocyte preparations, thymus, and lymph node tissues. The morphologic appearance and ability to phagocytize latex beads indicated these cells were monocytes. A monoclonal antibody, CM277, with a binding specificity for feline peripheral blood phagocytes was also characterized. Immunofluorescent microscopy revealed CM277 to bind specifically to monocytes and polymorphonuclear neutrophils. The binding of CM277 to monocytes was also shown by human erythrocyte-rosette formation wherein there was a high degree of correlation between these two phenotypic markers for cells ingesting latex beads. Monocytes, polymorphonuclear neutrophils, and T lymphocytes of the cat rosette with guinea pig erythrocytes (GPE) and using CM277 we were able to determine the contribution of the former two cell types to the GPE-rosetting population. Monocytes and polymorphonuclear neutrophils comprised the majority of the GPE-rosetting cells in fresh PBL (greater than 60%), but after culturing overnight, there was a substantial decrease in these cells (less than 35%). In contrast, GPE-rosetting T lymphocytes comprised approximately 10% of the cells in fresh PBL, and after in vitro culture for 1 day they constituted 35-45% of all cells. The removal of monocytes by human erythrocyte-rosetting did not affect the pokeweed mitogen-induced synthesis of Ig, but did lead to an increased production of interleukin 2. Removal of the GPE-rosetting population from PBL resulted in a marked decrease in interleukin 2 production, pointing to a positive contribution of GPE-rosetting T lymphocytes to the synthesis of this lymphokine.     

Microtubules and microtubule-associated proteins in resting and mitogenically activated normal human peripheral blood T cells.

The binding of an appropriate ligand to its specific receptor on the membrane of T cells triggers a cascade of events involved in T cell activation. An important yet unanswered question is how the mitogenic signals are transmitted through the cytoplasm and into the nucleus. The present study was carried out to determine changes in the microtubule (MT) system, following T cell activation. Fluorescence microscopy was employed to examine the organization of the microtubule network in human peripheral blood T cells in response to four different mitogens (phytohemagglutinin, concanavalin A, anti-CD3, and phorbol 12-myristate 13-acetate). The microtubules increase in length, number, and complexity of distribution 20 h after mitogenic stimulation. Using an in situ direct analysis protocol consisting of selective extraction of cells with detergent and Ca2+, 11 protein species, which fulfill the operational definition of microtubule-associated proteins (MAPs), were identified in resting human T cells. Alterations in the expression of these protein species were studied following mitogenic stimulation. These alterations in MAPs expression were also found in purified blast cell fractions indicating that they were specific changes occurring in activated T cell populations. These observations suggest a role for MT and MAPs in the cascade of human T cell activation.     

Enhancement of murine B cell responses to lipopolysaccharide by supernatants of irradiated peripheral blood leukocytes

Summary At low cell density, the proliferative response of B cells to lipopolysaccharide (LPS) is not detectable. We investigated under these experimental conditions the role of several cell populations on the LPS-induced B-cell proliferation. The addition to murine B cells of irradiated peripheral blood leukocytes (PBL) from the C3H/ HeJ mouse strain, or of culture supernatants of these cells, efficiently restored a response to LPS. Similar results were also obtained with irradiated PBL from other mouse strains and from rabbits. The activities of the culture supernatants were not significantly modified when the PBL were depleted of adherent cells or of Thy-1.2 positive cells, thus suggesting that the active factors were secreted neither by T cells, nor by monocytes.Abbreviations BSS balanced salt solution - ConA concanavalin A - EBMR enhancement of B-cell mitogenic response - J-B, J-T, J-Th, J-MØ, J-PBL, J-RBC splenic bone marrow-derived lymphocytes, splenic thymus-derived lymphocytes, thymocytes, splenic macrophages, peripheral blood leukocytes, red blood cells, obtained from the LPS-non-responding C3H/ HeJ-Pas mouse strain - R-PBL peripheral blood leukocytes obtained from the LPS-responding C3H/ He-Pas mouse strain - LPS lipopolysaccharide - MO macrophages - PBL peripheral blood leukocytes     

Antigen-specific and nonspecific mediators of T cell/B cell cooperation. III. Characterization of the nonspecific mediator(s) from different sources.

T cell-containing lymphoid populations produce a nonantigen-specific mediator(s) (NSM) which can replace T cell helper function in vitro in the response of B cells to sheep red blood cells (SRBC), but not to the hapten-protein conjugate, trinitrophenyl-keyhole limpet hemocyanin, (TNP-KLH). NSM produced under three conditions: 1) stimulation of KLH-primed cells with KLH; 2) allogeneic stimulation of normal spleen cells; and 3) stimulation of normal spleen cells with Con A (but not PHA) are indistinguishable on the basis of their biologic activity and m.w., estimated as 30 to 40,000 daltons by G-200 chromatography. Production of NSM is dependent on the presence of T cells. The action of NSM on B cells responding to SRBC in the presence of 2-mercaptoethanol is unaffected by severe macrophage depletion. Extensive absorption of NSM with SRBC failed to remove its activity, confirming its nonantigen-specific nature.     

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.     

Human peripheral blood monocytes release a 30,000 dalton factor (30 KD MF) that stimulates immunoglobulin production by activated B cells

Supernatants (SN) of well-washed adherent human monocytes, obtained from T cell-depleted peripheral blood mononuclear cells, contain a 30,000 dalton protein (30 KD MF) that increases immunoglobulin (Ig) synthesis by EBV-activated B cells two- to fourfold. This factor is released spontaneously during the first 20 hr after monocytes are placed in culture. SN containing 30 KD MF are inactive in the thymocyte co-stimulator assay, under conditions that will detect as little as 0.5 U of purified IL 1. The addition of autologous T cells to isolated adherent monocytes, previously depleted of T cells, suppresses the release or activity of this B cell stimulator in a dose-dependent manner. In addition, 30 KD MF stimulates a two- to fourfold increase in IgA production by cells of an EBV-transformed B cell line (JB/FF line) without increasing incorporation of [3H]thymidine. In contrast, stimulation of this B cell line with up to 10 U of purified IL 1 increases IgA synthesis by less than 50%, and addition of up to 100 U of recombinant IL 2 causes no change whatsoever in IgA production. However, co-stimulation with 30 KD MF and recombinant IL 2 or recombinant gamma-interferon induces more Ig production than is caused by the monocyte factor alone. These observations suggest that the monocyte, in addition to acting as an antigen-presenting cell and source of IL 1, facilitates B cell differentiation by producing a factor which acts both independently and in synergy with cytokines produced by T cells to stimulate Ig production by B lymphocytes.