Preliminary characterization of accessory cells in the nonadherent fraction of human blood mononuclear cells

We investigated why peripheral blood mononuclear cells rigorously depleted of adherent cells by sequential incubation on plastic and nylon wool remained fully responsive to both antigenic and mitogenic signals. Nylon wool nonadherent cells (NWNA) depleted of cells expressing HLA-DR by monoclonal antibody and complement lysis did not respond to tetanus toxoid (TT) or suboptimal concentrations of phytohemagglutinin (PHA). Addition of adherent accessory cells to these NWNA HLA-DR- cells reconstituted the response to stimuli. NWNA, fractionated by discontinuous density gradient centrifugation, contained high density cells which were unresponsive alone to optimal concentrations of both TT and PHA. All the lower density fractions contained cells which were accessory for higher density cell responses to stimuli. The lowest density fraction was approximately 30% monocytes (esterase and peroxidase positive) and less than or equal to 3% B lymphocytes (surface IgG bearing). The other low density fractions contained large granular lymphocytes but rarely monocytes and no B lymphocytes. Depletion of OKT3+, OKM1+, and Leu-11+ cells from lower density cells by monoclonal antibody and complement lysis did not abolish their accessory activity, but depletion of HLA-DR+ cells or gamma irradiation of these cells decreased their accessory activity for PHA and eradicated accessory activity for TT. Thus, the responsiveness of NWNA to soluble antigenic and mitogenic signals is due, in part, to the presence of low density cells which are radiosensitive and phenotypically HLA-DR+ OKT3-OKM1-Leu-11-. Accessory activity in NWNA seems to reside, therefore, in a cell which is not a typical monocyte, natural killer cell, nor B or T lymphocyte.     

Distinct role of neonatal and adult monocytes in the regulation of the in vitro antigen-induced plaque-forming cell response in man

Mononuclear cells from human cord blood (CBMC) are able to mount an antigen-specific IgM plaque-forming cell (PFC) response after primary in vitro stimulation with the T cell-dependent antigen ovalbumin (OA). The antigen dose-response relationship for the induction of PFC in cultures of CBMC is represented by a bell-shaped curve comparable to that found for mononuclear cells from adult peripheral blood (adult PBMC). The dose of OA optimal for the induction of a response in cultures of CBMC consistently, however, is 100-fold lower than the antigen dose optimal for adult PBMC (0.03 microgram OA/ml vs 3.0 micrograms OA/ml). Results obtained from co-culture experiments in which semiallogeneic combinations of parental/neonatal lymphocytes and monocytes were stimulated with a variable dose of OA indicate that the adherent cell (AC) plays a pivotal role in the establishment of the optimum antigen dose. From experiments using antigen-pulsed AC, it was concluded that neonatal and adult AC differ in their antigen handling capacity. In the presence of the prostaglandin synthetase inhibitor indomethacin the antigen dose-response relationship for the induction of PFC in cultures of CBMC shifts to an "adult type" of curve. From pulsing experiments it emerges that indomethacin affects the interaction between antigen and monocytes. Indomethacin causes an enhancement of the expression of HLA-DR at the surface of neonatal as well as adult AC; this can be down regulated by the addition of prostaglandin E2 (PGE2). The addition of PGE2 to cultures of adult PBMC leads to a shift of the optimal antigen dose for induction of PFC toward lower concentrations. Although higher levels of PGE2 were measured in the supernatant of cultured neonatal AC compared with adult AC, it seems unlikely that this observation can explain the distinct antigen dose-response relationship for the induction of a PFC response in cultures of CBMC.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

Delineation of suppressor and helper activity within the OKT4-defined T lymphocyte subset in human newborns

Lymphocytes taken from the cord blood of newborns have active suppressor activity. Using in vitro PWM-stimulated cocultures, unfractionated T cells from newborns potently suppressed the expected immunoglobulin G (IgG) synthesis of their mothers' peripheral blood lymphocytes (PBL). Using positive and negative selection techniques, we characterized the active suppressor cell as expressing the OKT4+T8- phenotype. This cord blood lymphocyte subset suppressed maternal IgG synthesis after depletion of maternal suppressor cells, implicating the ability of newborn T cells to suppress directly rather than by inducing adult suppressor activity. Sublethal amounts (1500 rad) of gamma-irradiation fully abrogated the suppressor activity of cord blood T lymphocytes. Radioresistant cord T cells provided T cell help. Irradiation of cord OKT4+ and OKT8+ populations and their subsequent culture with maternal B cells determined that helper activity was a radioresistant subpopulation of the OKT4+ subset. These results indicate significant differences in the functional properties of T cell subsets from adults and newborns. Population studies determined that cord blood lymphocytes had a greater proportion of OKT4+ cells and lower proportion of OKT8+ cells than PBL from unrelated adults. The mothers tested had similar proportions of OKT4+ cells as their babies, and these levels are significantly higher than those of unrelated adults.     

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.     

Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.     

Immature T lymphocytes in human cord blood identified by monoclonal antibodies: a model for the study of the differentiation pathway of T cells in humans

The reactivity of human cord blood lymphocytes was assessed against a panel of monoclonal antibodies (MoAb). The mean proportion of OKT3+ cells (pan-T) was significantly lower in cord blood (52 +/- 13.8%; mean +/- SD) compared with that of adult blood (75 +/- 8.9%) and paralleled well with the E-rosette-forming capacity (50 +/- 16.3%). Both the proportions of OKT4+ cells (helper/inducer phenotype) and of OKT8+ cells (suppressor/cytotoxic phenotype) were significantly reduced in cord blood (43 +/- 11.8% vs 50.3 +/- 7.4% and 20 +/- 10.3% vs 25.6 +/- 6.0%, respectively), while the overall OKT4/OKT8 ratio was increased compared with adult blood (2.87 +/- 1.83 vs 2.04 +/- 0.61). Unlike adult blood, in 30 of the 35 samples of cord blood an overlap was observed between the total proportion of OKT4+ and OKT8+ cells (65 +/- 15.2%) and that of OKT3+ cells (52 +/- 14.3%). Although small numbers of cells coexpressing both antigens were occasionally found, double-staining analysis showed that the overlap in cord blood was mostly due to an expanded proportion of OKT3 (Leu-4)-/OKT8 (Leu-2)+ cells. Relevant proportions of OKT6+ (common thymocyte antigen) and OKT10+ (thymocytes, activated T cells, precursor cells) cells were found in cord blood as opposed to adult blood (10.8 +/- 8.6% vs 0.6 +/- 0.6% and 67 +/- 18.0% vs 8 +/- 2.1%, respectively), while terminal deoxynucleotidyl transferase-positive cells were observed only in two samples of cord blood. A small proportion of T cells (E-rosette+) reacted with the MoAb OKIa1 (HLA-DR). Finally, the proportion of cord blood cells recognized by the MoAb Leu-7 (HNK-1 clone) was almost negligible compared with adult blood (2.8 +/- 2.4% vs 15 +/- 7.5%). These data confirm the immaturity and heterogeneity of cord blood lymphocytes and demonstrate the presence at birth of circulating lymphocytes which express a surface phenotype reminiscent of that found in the late stages of intrathymic differentiation and in some human T-cell leukemias. Human cord blood may thus represent a suitable model for the study of the differentiation pathway of normal and pathological T-cells in humans.     

Inability of newborns' or pregnant women's monocytes to suppress pokeweed mitogen-induced responses

Although an excess of human adult blood adherent cells inhibits the pokeweed mitogen- (PWM) induced normal adult lymphocyte proliferation and B cell maturation into immunoglobulin-containing cells (ICC), adherent cells collected from newborn infants or pregnant women at time of delivery were unable to exert a similar suppressor activity. After activation by Concanavalin A (Con A), newborns' and pregnant women's adherent cells acquired a suppressor activity comparable to that of control adult adherent cells. The adherent suppressor cell was shown to be radioresistant (3000 rad), indicating its probable monocytic origin. Both monocyte-suppressor activities (MSA) observed in adulthood (spontaneously) and in the neonatal period (after activation) were dependent on prostaglandin E2 (PGE2) secretion, because they were abolished by indomethacin or a specific anti-PGE2 antiserum. Expression of MSA appeared to be under a negative regulation exerted by naturally occurring T suppressor lymphocytes present in the blood of newborns or pregnant women, because incubation of adult monocytes or Con A-activated newborn monocytes with newborns' or pregnant women's T lymphocytes resulted in a dramatic decrease of their MSA. These results strongly suggest that the lack of MSA in the neonatal period and in late pregnancy is a consequence of activation of T suppressor lymphocytes.     

Lymphocyte subpopulations in the neonate: identification of an immature subset of OKT8-positive, OKT3-negative cells

T cell subpopulations of lymphocytes from cord blood (CBL) of 24 newborns and from peripheral blood (a-PBL) of 24 healthy adult volunteers were assessed in T cell-enriched, T cell depleted and unseparated lymphocyte fractions by using OKT3, 4, 6, and 8 monoclonal antibodies. The results show that T cell-enriched CBL include adult numbers of OKT3+, OKT4+, OKT6+ and OKT8+ lymphocytes whereas the T cell-depleted fraction consists of a high percentage of OKT8+, OKT3-, non-E rosette-forming cells bearing a PNA receptor. The presence of the PNA receptor and the lack of the OKT3+ antigen strongly support the hypothesis that the subset of OKT8+ cells in cord blood includes immature T lymphocytes that may represent an intermediate stage between thymocytes and mature peripheral T cells.     

Defective IFN-gamma production in the human neonate. I. Dysregulation rather than intrinsic abnormality

Cord blood leukocytes (CBL) stimulated with PHA, Con A, or with the monoclonal antibody OKT3 proliferate normally but produce very low titers of IFN-gamma. This defect was not observed with maternal leukocytes collected at the time of delivery, indicating that the defective production of IFN-gamma in CBL is not a mere consequence of a hormonal change associated with labor. CBL produced large amounts of IFN-gamma (comparable to those observed in adult control and in mothers) after stimulation with staphylococcal enterotoxin A (SEA). Furthermore, gamma-irradiation with as little as 500 or 1000 rad, or incubation at 37 degrees C for 24 hr, reversed the defect in PHA-induced IFN-gamma secretion. This finding indicates that the defective secretion of IFN-gamma of CBL is not intrinsic, but rather is the consequence of a subtle dysregulation. We could not find evidence for a defective accessory function with cord blood monocytes, because the addition of adherent cells from adult donors did not reverse the defect. In co-cultures of adult leukocytes and CBL, PHA-induced IFN-gamma secretion was comparable to that of adult cultures tested alone. Nonirradiated CBL were not able to suppress IFN-gamma secretion by irradiated autologous leukocytes. Together, our results suggest that the defective PHA-induced IFN-gamma secretion of CBL is the result of an original type of dysregulation and is associated with an excessive sensitivity to suppressive signals rather than excessive suppressor function.     

Accessory cell competence of ovine oligodendrocytes in mitogenic activation of human peripheral T cells

The multiple sclerosis (MS) plaque is characterized by mononuclear inflammatory cell infiltration, demyelination, loss of oligodendrocytes (OGC), and proliferation of astrocytes. Although antigen-specific, Ia-dependent cellular immune mechanisms have been sought in plaque pathogenesis, Ia-independent T cell activation has not been actively investigated. We examined a potential role of OGC in accessory cell-dependent T cell mitogenesis with the anti-T3 monoclonal antibody OKT3. OGC isolated from ovine white matter on sucrose density gradients were uniformly negative for esterase activity, unlike ovine monocytes. Purified human T cells did not exhibit significant proliferation in 3-day cultures with OKT3, autologous peripheral blood adherent cells (PBAC), or ovine OGC. When T cells were cultured with either PABC or OGC in the presence of OKT3, brisk mitogenesis was observed. Thus, OGC have the capacity to function as accessory cells in the mitogen-induced proliferation of T cells.     

Role of class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes

The capacity of peripheral blood monocytes and B lymphocytes to support staphylococcal protein A (SpA)-induced proliferation of autologous and allogeneic T cells, as well as the role of major histocompatibility complex (MHC) class I and II molecules in this activation process, were investigated. Highly purified peripheral T lymphocytes did not proliferate in response to SpA, but their response was reconstituted by both irradiated (or mitomycin C-treated) monocytes and B lymphocytes. The effect of B cells on the SpA-induced T-cell response could not be explained by a contamination of residual accessory cells because long-term continuous B-cell lines restored SpA-induced T-cell DNA synthesis as effectively as did monocytes. Support of SpA responsiveness by B cells could not be accounted for by polyclonal binding of SpA to cell surface immunoglobulins, since the ability of SpA-unreactive and SpA-reactive B cells was comparable. The cells from two human leukemic lines--K562 and Raji--showed the same ability in supporting the pokeweed mitogen-induced T-cell response, but the class II-positive Raji cells were much more effective than class II-negative K562 cells in restoring the T-cell responsiveness to SpA. Monoclonal antibodies specific for monomorphic determinants of MHC class II antigens, as well as their F(ab')2 fragments, consistently inhibited the SpA-induced proliferative response, whereas antibodies specific for MHC class I antigens were without effect. The antibodies specific for class II antigens appeared to act at the level of accessory cell, since pretreatment with these antibodies inhibited the ability of SpA-pulsed monocytes or Raji cells to present SpA to autologous or allogeneic T lymphocytes, respectively. These data indicate that either monocytes or normal and lymphoblastoid B cells can act as accessory cells for the proliferative response of human T cells to soluble SpA and that monomorphic determinants of MHC class II molecules play an important role in this activation process.     

OKT3 monoclonal antibody induces production of colony-stimulating factor(s) for granulocytes and macrophages in cultures of human T lymphocytes and adherent cells

OKT3 monoclonal antibody (mab) recognizes a membrane antigen associated with the T cell antigen recognition receptor, and is known to be mitogenic and to induce lymphokine production. Our studies demonstrate the ability of OKT3 mab to induce from cultures of human T lymphocytes supplemented with adherent cells the production of colony-stimulating factor(s) for granulocytes and macrophages (GM-CSF) and interferon-gamma (IFN-gamma), an inhibitor of clonal growth of hematopoietic progenitor cells. As has been shown for the mitogenic and IFN-gamma-inducing activity of OKT3 mab, the induction of GM-CSF release in cultures of T cells is strictly dependent on the presence of adherent cells. However, the concentrations of OKT3 mab required for optimal GM-CSF production (50 ng/ml) were found to be 80-fold higher than those sufficient for maximal IFN-gamma production, proliferation, and interleukin 2 production. IFN-gamma activity induced by OKT3 mab partially inhibited colony and cluster formation from progenitor cells of granulocytes and macrophages in vitro. Therefore, neutralization of the IFN-gamma by monoclonal anti-human-IFN-gamma antibody before assay of conditioned medium in bone marrow cultures significantly enhanced the detection of GM-CSF. Kinetic studies demonstrated maximal cumulative GM-CSF production in response to optimal OKT3 mab concentrations on days 4 through 6 in cultures of T cells supplemented with 15% adherent cells. Highly enriched OKT4+ and OKT8+ T cell subsets co-cultured with adherent cells in the presence of OKT3 mab both produced GM-CSF and IFN-gamma and showed similar dose-response curves to OKT3 mab. The requirement for the presence of adherent cells could not be overcome by the addition of purified interleukin 1 or macrophage supernatants. Studies using irreversible inhibitors of DNA (mitomycin C) or protein biosynthesis (emetine-HCl) revealed the necessity of intact DNA synthesis and translation in mononuclear cells to produce GM-CSF in response to OKT3 mab. Loss of GM-CSF production was observed when either adherent cells or T lymphocytes were treated with emetine before co-culture with untreated cells of the other population in the presence of OKT3 mab. In contrast, mitomycin C reduced GM-CSF production significantly when T cells, but not adherent cells, were pretreated. These results suggest that T lymphocytes and adherent cells closely cooperate in the production of GM-CSF induced by OKT3 mab.     

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.     

Ia- and IgG-Fc receptor-positive accessory cell sustains peripheral T lymphocyte but not thymocyte mitogenesis induced by OKT3 monoclonal antibody

The proliferative responses of human peripheral blood mononuclear cells (PBL) and thymocytes to OKT3 monoclonal antibody have been investigated. The PBL response to OKT3 was maximal after 72 hr while that of thymocytes was inappreciable at all times measured. Unlike phytohemagglutinin, OKT3 was unable to elicit the mitogenesis of adherent cell-depleted T cells in spite of the presence of exogenously added Interleukin 1 and/or Interleukin 2. The addition of autologous or heterologous adherent cells restored the OKT3 mitogenic response of peripheral purified T cells but not of thymocyte cultures. The adherent cell population that was able to sustain the OKT3-elicited T-cell mitogenesis was constituted by Ia-, Fc receptor-positive cells. These data suggest that the adherent cell-T cell interaction is mediated via the Fc portion of the OKT3 molecule. Furthermore, unlike peripheral T cells, T3-positive thymocytes, which represent the more mature. PHA-responsive subset within the thymus, are unable to cooperate with accessory cells when pulsed with OKT3 monoclonal antibody.     

Dextran-sulfate: a mitogen for human T lymphocytes

Dextran-sulfate (DxS) induced proliferation of human peripheral blood T lymphocytes but not of adult or neonatal B lymphocytes. The mitogenic activity on T cells by DxS required the presence of accessory cells because DxS was unable to trigger T cells to DNA synthesis in the absence of accessory cells. In addition, DxS stimulated OKT4+8- T cells to produce interleukin 2, a process that also occurred only in the presence of accessory cells. Cyclosporin-A strongly suppressed T cell proliferation induced by DxS by rendering T cells unresponsive to interleukin 2 and by inhibiting the synthesis of this T cell growth factor by OKT4+ T cells. These results indicate that DxS is a mitogen for human T lymphocytes but not for adult or neonatal B lymphocytes. The mechanism by which DxS triggers T cells is discussed.     

The regulation of gamma-interferon production by interleukins 1 and 2

Purified interleukins 1 and 2 (IL-1 and IL-2) were used to investigate their role in the production of gamma-interferon (gamma-IFN). Macrophage depletion from human peripheral blood mononuclear leukocytes (PBML) inhibited gamma-IFN production. Addition of purified IL-1 partially restored IFN production of macrophage-depleted PBML induced by three T cell mitogens (phytohemagglutinin, PHA; concanavalin A, con A; and staphylococcal enterotoxin A, SEA), but had no effect on induction of IFN production by undepleted PBML. Therefore endogenous IL-1 production by macrophages is probably one of the mechanisms by which they act as accessory cells for IFN production by lymphocytes. A monoclonal antibody 9.6 which binds to the sheep erythrocyte (E) receptor found on human T cells inhibited IFN production. Addition of IL-2, but not IL-1, was found to reverse this inhibition. Prostaglandin E2, a macrophage product, inhibited gamma-IFN production induced by PHA, Con A, and OKT3 but usually not SEA. This inhibitory effect was reversible by the addition of IL-2 but not IL-1. In the absence of mitogen IL-1 alone rarely induced any IFN production, although some IFN was produced by PBML from a small minority of donors. Without mitogen IL-2 induced IFN production only at very high concentrations and the added presence of IL-1 did not enhance this induction.     

Effector activity of OKT4+ and OKT8+ T-cell subsets in lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

The role of OKT4+ and OKT8+ T-cell subsets was studied in lectin-dependent cell-mediated cytotoxicity (LDCC) against adherent HEp-2 human epipharynx carcinoma target cells. LDCC was evaluated by detachment from the monolayer of [3H]thymidine prelabeled HEp-2 cells in a 24-hr assay with a concanavalin A (Con A) dose of 25 microgram/ml at effector:target cell ratios of 5:1, 25:1, and 50:1. Under these conditions but without Con A considerable natural cell-mediated cytotoxicity (NCMC) was not elicited; however, the cytotoxicity was significantly augmented in the presence of Con A (=LDCC) by sheep erythrocyte rosette-forming T lymphocytes and by both OKT4+ and OKT8+ T-cell fractions. LDCC activity by isolated OKT8+ T cells was superior to that by OKT4+ T cells and unfractionated T lymphocytes. By contrast, addition of either OKT4+ or OKT8+ T cells together with unfractionated T lymphocytes, or OKT4+ and OKT8+ T cells mixed at ratios of 1:1, 1:2, and 2:1, to target cells did not result in major differences in comparison of LDCC activities by these mixed effector cell populations with each other or with that by unfractionated T lymphocytes. Parallel studies were carried out to determine the effect of OKT4+ and OKT8+ T-cell subsets on the Con A-induced proliferation of peripheral blood mononuclear cells (PBMC). While OKT8+ T cells inhibited the mitogenic response to Con A, OKT4+ T lymphocytes had no major effect. A higher responsiveness of the OKT8+ to OKT4+ T-cell subset in LDCC to HEp-2 targets and in Con A-induced lymphocyte proliferation is suggested.     

Regulation of the synthesis of the third component of complement and factor B in cord blood monocytes by lipopolysaccharide

We compared the regulation of C3 and factor B synthesis in cord blood and adult monocytes by using techniques for identification and quantification of newly synthesized proteins, lipopolysaccharide (LPS) from several Gram-negative organisms, and precursors of LPS. Synthesis of C3 and factor B in cord blood monocytes was unaffected by lipid A (the active moiety of LPS extracted by the Westphal procedure). In contrast, adult monocytes increased C3 synthesis by 11.5-fold and factor B synthesis by 3.1-fold in response to LPS. This difference in cord blood monocyte response to LPS was specific in that other LPS-induced monocyte functions (superoxide production and phagocytosis) were stimulated comparably in both cord blood and adult monocytes by LPS. To characterize further this regulatory difference, the roles of LPS precursors, arachidonic acid metabolites, and of factor(s) released by adult monocytes were examined. Precursors of the lipid portion of LPS (lipid X and lipid Y), LPS isolated by trichloroacetic acid extraction, and endotoxin-associated protein (EAP) increased C3 and factor B synthesis in cord blood monocytes. Inhibitors of the lipoxygenase pathway (dexamethasone, ETYA) but not of the cyclooxygenase pathway (indomethacin) abrogated the response of adult monocytes to lipid A and EAP and of cord blood monocytes to EAP. Finally, co-incubation of adult monocytes and cord blood monocytes in LPS-containing medium resulted in enhancement of C3 and factor B synthesis in cord blood monocytes. These data suggest that the difference in LPS response between cord blood and adult monocytes may result from differences in lipid processing or protein recognition of LPS, differences in the production of lipoxygenase pathway products, and/or one or more regulatory factors. The availability of human mononuclear phagocytes which exhibit distinct differences in biosynthetic responsiveness to LPS should permit investigation of the molecular mechanism(s) by which LPS affects C3 and factor B gene expression.     

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.