Subsets of human large granular lymphocytes (LGL) exhibit accessory cell functions

The present study shows that human large granular lymphocytes (LGL) depleted of OKT3 (T lymphocytes) and Leu-M1-positive (monocytes) cells exhibit accessory cell function for the T lymphoproliferative responses to the soluble stimulants Staphylococcus protein A (SpA) or Streptolysin O (SLO), as well as to surface antigens in the autologous and allogeneic mixed leukocyte reaction (MLR). Fractionation of LGL into subsets according to their reactivity with alpha OKT11, alpha DR, and alpha OKM1 MoAb led to the identification of the subset(s) of LGL with OKT11+, DR+, OKM1+ phenotype as the antigen-presenting cell (APC), whereas the DR-, OKM1- subset(s) of LGL was completely ineffective. Furthermore, virtually all the natural killer (NK) activity of LGL was associated with OKT11+ and OKM1+, DR+ LGL that exerted the observed APC function, suggesting that NK-active cells may also act as effective APC for T lymphocyte activation. These results indicate that human LGL with NK activity may exert other noncytotoxic functions and may play a major role in immunoregulation.     

Agar human T cell colony growth promoted by a B + null cell-derived lymphokine distinct from IL 2

Human T cell agar colonies can be grown under PHA stimulation from either mature T cells or their E rosette-negative (E-), OKT3- peripheral blood and bone marrow precursors. Colonies comprise a majority of mature E+, OKT3+ cells and a minor (5 to 10%) population of immature E-, T3-, T8-, T4-, DR+, T10+, RFB1+ cells, which upon replating in subculture, can generate secondary colonies of OKT3+, E+, OKT4+, OKT8+ cells. Secondary colony formation can serve as a test for growth requirement of colony precursors, because it depends on the presence of both PHA and a colony-promoting activity (CPA) recovered in PHA-stimulated B + null or T + adherent cell supernatants. CPA production by B + null cells was not affected by their treatment with OKT3 or D66 (T11-like) monoclonal antibodies (MAB) + complement but was abolished by an anti-HLA-DR MAB + complement. However, B cells sorted by panning with the same anti-HLA-DR MAB did not release CPA, demonstrating the requirement of both B cells and null cells for CPA production. Neither IL 2 nor IL 1 could account for B + null cell-derived CPA.     

A prostaglandin-mediated suppressive activity of cord as compared to maternal or other adult adherent cells in OKT3 antibody-induced proliferation

In a previous study we reported that cord blood lymphocytes show lower OKT3 responses as compared to their mothers and to other, unrelated adults. In the study reported here, we investigated the interactions between lymphocytes and adherent accessory cells in OKT3-stimulated cultures of newborn (cord), maternal, and other adult peripheral blood mononuclear leukocytes (PBML) and determined the following. (1) Removal of adherent cells (AC), by two cycles of plastic adherence or by nylon wool columns, impaired the OKT3-induced proliferation of maternal/adult cells, but significantly enhanced the OKT3 responsiveness of cord cells. (2) Addition of indomethacin, and other prostaglandin (PG) synthesis inhibitors, caused a more than twofold augmentation of cord PBML OKT3 responses, but had only a small, if any, enhancing effect on maternal/adult PBML. Cord PBML cultures deprived of AC were no longer enhanced by indomethacin. (3) Exogenous PGE2 (1.4 X 10(-6) through 1.4 X 10(-9) M) strongly inhibited OKT3-induced proliferation of maternal, cord, and adult PBML, at a wide range of antibody concentrations (5-100 ng/ml). However, an obvious difference in the extent of PG-mediated inhibition was observed among these three populations, and the order of PG sensitivity, from most to least sensitive, was cord greater than maternal greater than adult. (4) Purified interleukin-1 (IL-1) could not replace the accessory function of AC in the OKT3-induced proliferation of maternal/adult lymphocytes. In contrast, IL-1 increased by greater than 50% the OKT3 responsiveness of cord PBML in the absence, but not in the presence, of cord monocytes. Our observations strongly argue for a distinct, predominantly suppressive function of cord monocytes as compared to maternal/adult monocytes in OKT3-induced mitogenesis, and indicate prostaglandins as major mediators of this suppression.     

Stimulation and inhibition of human T cell subsets by wheat germ agglutinin

Wheat germ agglutinin (WGA), a tetravalent lectin, has both stimulatory and inhibitory effects on human T lymphocytes. It has been suggested that these actions are related and that WGA selectively stimulates a suppressive subset of T cells. We studied the ability of WGA to stimulate and inhibit subpopulations of human peripheral blood mononuclear cells (PBMC) known to have helper or suppressor activity. Fresh human PBMC were depleted of either T4+ or T8+ cells by using antibody-mediated complement lysis. The resultant cell populations were stimulated with WGA, and the proliferative response was assessed by [3H]thymidine incorporation, IL 2 receptor expression, the ability to elaborate IL 2 in culture supernatants, and the susceptibility to inhibition by the monoclonal antibody anti-Tac. Similar experiments with cells from a WGA-responsive continuous T cell culture were also performed. WGA inhibited phytohemagglutinin (PHA)-induced proliferation of PBMC depleted of either T4+ or T8+ cells. WGA also inhibited PBMC that had been depleted of adherent cells and Ia+ cells and then induced to proliferate with a combination of TPA and PHA. Our findings indicate that WGA induces IL 2-dependent proliferation in a small proportion of both T4+ and T8+ lymphocytes. We also provide evidence that the inhibitory activity of WGA is not mediated by a T4+, T8+, or Ia+ cell, suggesting that WGA acts directly on the proliferating cell rather than selectively stimulating a suppressive subpopulation.     

Activation of human T lymphocytes by 12-O-tetradecanoylphorbol-13-acetate: role of accessory cells and interaction with lectins and allogeneic cells

Stimulatory effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on human T lymphocytes have been investigated. TPA was found to stimulate highly purified T cells (obtained by a three-step isolation procedure involving plastic adherence, nylon wool passage and Ig-anti-Ig column passage) in the absence of accessory cells (stimulation index of 5 to 10), whereas phytohemaglutinin (PHA) and concanavalin A (Con A) did not. This response was, however, increased by the addition of autologous adherent cells. Addition of TPA, but not adherent cells, induced T-cell proliferation in response to the nonmitogenic lectin, wheat germ agglutinin (WGA), while both adherent cells and TPA restored T-cell proliferation to mitogenic lectins such as PHA and Con A. Furthermore, TPA greatly increased the mixed-lymphocyte response of purified T cells to otherwise nonstimulating allogeneic cells such as T lymphocytes or tumor cells from some patients with chronic lymphocytic leukemia. These results suggest that TPA can directly act on human T cells to render them reactive to a variety of stimuli.     

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34

Human large granular lymphocytes (LGL), which are known to be responsible for natural killer (NK) cell activity, also produced a variety of lymphokines including interleukin 2 (IL 2), colony stimulating factor (CSF), and interferon (IFN) in response to phytohemagglutinin (PHA) or concanavalin A (Con A). Human peripheral blood LGL, which were purified by removal of monocytes adhering to plastic flasks and nylon columns, followed by separation on a discontinuous Percoll gradient, and additional treatment with anti-OKT3 and Leu-M1 plus complement, were more potent producers of these lymphokines than unseparated mononuclear cells (MNC), nylon column-eluted cells, or purified T lymphocytes. Moreover, IL 2 production by LGL could be further distinguished in that it was not enhanced by the addition of macrophages or macrophage-derived factor, i.e., IL 1, whereas addition of macrophages did potentiate IL 2 production by T lymphocytes. Further analysis of cells in the LGL population using various monoclonal antibodies revealed that removal of cells with OKT11 or AF-10, a monoclonal antibody against human HLA-DR antigen, decreased IL 2 production, whereas removal of OKT8+, OKM1+, Leu-M1+, or Leu-7+ cells led to enhanced IL 2 production. The LGL population is therefore heterogeneous and includes at least three functionally and phenotypically distinct subsets. An atypical T cell subset (OKT3-, Leu-1-, OKT11+) rather than the myeloid subset of LGL (Leu-M1+ or OKMI+) was the source of LGL-derived IL 2, whereas the latter subset and/or another subset of OKT8+ cells appear to regulate this IL 2 production. In addition to performing NK activity, LGL on a per cell basis seem to be more effective than T lymphocytes in producing lymphokines, namely, IL2, CSF, and IFN.     

rIL 2-induced proliferation of human circulating NK cells and T lymphocytes: synergistic effects of IL 1 and IL 2

Cells participating in the rIL 2-induced proliferation of resting PBMC were identified by using different methods of cell purification. NK cells recovered in the light density fraction of Percoll gradients responded, as already known, directly to rIL 2 by strong proliferation. In contrast, large T lymphocytes co-purifying with NK cells, and small T cells sedimenting in the high density area of the Percoll gradients, were virtually unresponsive when cultivated in the sole presence of rIL 2. However, the addition of either irradiated autologous monocytes or highly purified IL 1 allowed both kinds of T cells to undergo cell division. Stringent elimination of possibly contaminating NK cells (NKH-1+) and/or activated T cells (TNKTAR, Tac+, HLA-DR+) from the high density T cells by complement lysis did not impair rIL 2-induced cell proliferation, indicating entire responsiveness of these cells to the synergistic action of IL 1 plus IL 2. Both high density CD4+ and CD8+ participated in this phenomenon, with an apparent advantage for CD4+ cells. All Tac+ cells emerging in a 6-day culture of these cells expressed the WT31 antigen, which indicates that T cells involved in rIL 2-induced proliferation are conventional mature T cells. The relative precursor frequencies of NK cells, large T lymphocytes, and small T lymphocytes that proliferated in response to rIL 2 were analyzed by limiting dilution analysis. The frequencies of clonal growth of NK cells and low density T lymphocytes were approximately the same (1/103 vs 1/185), whereas that of high density T cells was four times lower (1/458). Thus, we clearly demonstrate that resting T cells, defined as such by morphological, density, and phenotypic criteria, are able to proliferate in response to IL 2 in the presence of IL 1 without antigenic or mitogenic triggering.     

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.     

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.     

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.     

Correlation of biophysical properties and cell surface antigenic profile of Percoll gradient-separated human natural killer cells

Human peripheral blood nylon wool nonadherent lymphocytes were fractionated according to cellular density on discontinuous Percoll gradients. The various fractions of cells obtained by this procedure were then analyzed to correlate morphology, cytotoxicity, light scatter properties and antigenic profile. The majority of natural killer (NK) cell activity was manifested by light density lymphocytes, which demonstrated relatively high light scatter characteristics, large granular lymphocyte morphology, and expressed the phenotypes: Leu 11+(B73.1+), Leu 7-; Leu 11+(B73.1+), Leu 7+, and Leu 11+(B73.1+), Leu 2a+ (low density), Leu 4-. The denser fractions of Percoll (fractions 3-4) tended to show lower NK activity, fewer large granular lymphocytes, lower light scatter profiles, and to selectively localize the subpopulations of lymphocytes with the phenotypes: Leu 11-, Leu 7+, Leu 4+, and Leu 11-, Leu 7+, Leu 2a+ (high density).     

Noncytotoxic functions of natural killer (NK) cells: large granular lymphocytes (LGL) produce a B cell growth factor (BCGF)

Highly purified human large granular (LGL), depleted of any detectable contaminant T and B cells or monocytes, were found to be potent producers in vitro of a soluble B cell growth factor (BCGF) able to sustain proliferation of B cells activated by anti-mu. Activation by lectins (phytohemagglutinin, PHA, concanavalin A, Con A; and pokeweed mitogen, PWM) was required to induce the production of high levels of this BCGF from cultured LGL. Production of BCGF was also detected after the binding of LGL with natural killer (NK)-sensitive (K562) but not with NK-resistant (RL male 1) target cells. In contrast to T cells, LGL did not need the additional presence of accessory cells to reach optimal production of BCGF by 72 hr of culture. The subpopulation of LGL responsible for the production of BCGF had phenotypic characteristics associated with NK cells (3G8+, HNK1+/OKT11+, DR-, OKT3-, Leu-M1-), and separated cells with these markers exerted high levels of NK activity. Selective production of BCGF also was obtained from cytotoxic clones derived from LGL. A partial characterization of the LGL-derived BCGF was performed by gel filtration. BCGF activity was detected in fractions with estimated m.w. of 20,000 and 45,000. The LGL-derived BCGF activity was resistant to reduction with 2-mercaptoethanol and was stable at -20 degrees C for months. Conversely, heating (56 degrees C for 1 hr) or digestion with trypsin greatly reduced the LGL-derived BCGF activity. These findings strongly suggest that LGL including those with NK activity can play an important positive role in the early events of the B cell-mediated immune response.     

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.     

Human T cell clones exerting multiple cytolytic activities show heterogeneity in susceptibility to inhibition by monoclonal antibodies

Human cytotoxic T cell clones (CTL) were obtained by limiting dilution after in vitro priming against an allogeneic Epstein Barr virus (EBV)-transformed B cell line (B-LCL) BSM. Three OKT3+, OKT8+ E rosette-forming (RFC) but EA gamma-RFC- clones with cytotoxic activity against the stimulator cell and one "non-cytolytic" clone were expanded for over 50 generations and further characterized. Clone G9 showed allospecific lysis of Cw3+ lymphocytes and B cell lines. Three cytolytic clones (G9, D11, and A3) showed cytotoxicity to the stimulator B-LCL, to the human plasma cell leukemia-derived line LICR-LON-HMY2 and to short-term cultured melanoma cells (O-mel). Four other EBV-transformed B-LCL unrelated to the stimulator B-LCL were not lysed. These clones also exerted cytotoxic activity against NK-sensitive target cells (TC), e.g., the erythroleukemia cell line K562. Other NK-sensitive TC, e.g., lymphoma-derived Daudi cells, were killed provided they were pretreated with phytohemagglutinin (PHA). Cytolytic activity against the B-LCL cell LICR-LON and O-mel, but not against K562 or PHA-treated target cells, was inhibited by monoclonal anti-HLA ABC antibodies (MCA). The cytolytic activities of OKT3+,8+ clones G9 and A3 but not that of OKT3+,8+ clone D11 were inhibited by OKT8. Another MCA, 13.3, directed against the murine glycoprotein T-200, inhibited the cytolytic activity of clone D11 against K562 but not against the stimulator cells. Clone G9 was not inhibited by MCA 13.3. The four clones, including the OKT4+ "non-cytotoxic" clone K12, exerted lytic activity against TC that are normally resistant to lysis provided these TC were pretreated with PHA. The TC specificity range of the clones was confirmed by cold target inhibition experiments. A correlation between blocking of lytic activity by cold TC and the percentage of conjugate formation with the particular cold TC was observed. Because these clones also show differential susceptibility to inhibition of lysis by various MCA, it is concluded that human cytotoxic T cell clones can exert multiple lytic activities, i.e., the operationally defined lytic mechanisms differ at least at certain stages of the lytic cycle.     

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.     

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.     

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.     

Interleukin-2-induced production of interferon-gamma by resting human T cells and large granular lymphocytes: requirement for accessory cell factors, including interleukin-1

Between 5 and 20% of normal human lymphocytes were found to synthesize interferon-gamma (IFN-gamma) in primary cultures with recombinant interleukin-2 (rIL-2). After 22 hr, IFN-gamma-producing cells included CD5+ T lymphocytes, CD16+ large granular lymphocytes (LGL), and a population of CD5-, CD16- blast cells. Only a small proportion (0-7%) of IFN-gamma-synthesizing cells expressed HLA-DR. The production of IFN-gamma by all rIL-2-responding lymphocyte subsets was shown to require the presence of DR+ accessory cells, probably including nonadherent, esterase-negative monocytes and/or dendritic cells. Accessory cell function in lymphocyte preparations depleted of DR+ cells, or in purified (greater than or equal to 95%) suspensions of LGL, was fully replaced either by addition of 2% autologous, adherent monocytes or by monocyte culture supernatant. The activity of monocyte supernatant was greatly reduced by treatment with antiserum specific for human interleukin-1 beta (IL-1 beta), although a combination of rIL-1 beta and rIL-2 failed to stimulate IFN-gamma production in DR- lymphocytes. These results indicate that rIL-2-induced IFN-gamma synthesis in both T cells and LGL requires the synergistic activity of IL-1, and possibly of one or more other monokines, as yet unidentified.     

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.     

Evidence for interactions between monocytes and natural killer cells in the regulation of in vitro hemopoiesis

The role of accessory cells in modulation of in vitro hemopoiesis was studied using C-mediated lysis with mAb of differing specificities. One such antibody, 25E11, which bound to all NK cells and a subset of monocytes, consistently led to enhancement of in vitro hemopoiesis when 25E11+ cells were depleted by complement or the FACS. In cultures maximally stimulated by erythropoietin and human placental conditioned medium, depletion of 25E11+ cells from beta-thalassemic PBMC resulted in up to a fourfold enhancement of multi-potential and pure erythroid colonies and a twofold enhancement of non-erythroid colonies when compared with C-only treated cells. This enhancement of in vitro hemopoiesis was also observed with cells from normal bone marrow, cord blood, and peripheral blood. The enhancement of in vitro hemopoiesis was abrogated by removal of either adherent cells or monocytes using Leu M3 antibody and C lysis. The data presented herein suggest that NK cells and a subset of monocytes may exert a negative regulatory control on both granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.