Monocyte-depleted Peripheral Blood Research Articles

Differential production of IL-10 by T cells and monocytes of HIV-infected individuals: association of IL-10 production with CD28-mediated immune responsiveness.

Immune unresponsiveness in HIV-1 infection can result from impaired signals delivered by the costimulatory CD28-B7 pathway and the altered production of immunoregulatory cytokines, in particular IL-10, whose production is altered in HIV-1 infection. In this study we investigate IL-10 regulation in T cells and monocytes from HIV+ individuals, and its association with CD28-mediated T cell proliferation. IL-10 production as analysed in T cell- and monocyte-depleted peripheral blood mononuclear cells (PBMC), and by intracellular staining at the single-cell level, reveals a defect in IL-10 production by CD4+ and CD8+ T cells, whereas monocytes constitute the major IL-10-producing cell type. To investigate the impact of IL-10 on immune responsiveness, CD28-mediated proliferative responses in HIV+ individuals were correlated with PHA-induced IL-10 production. CD4+ T cells expressed CD28, yet exhibited markedly reduced CD28-mediated cell proliferation. This CD28-mediated CD4+ T cell proliferation was found to be inversely associated with the levels of PHA-induced IL-10 production and could be restored, at least in part, by anti-IL-10 antibodies. These results suggest that IL-10 production is differentially regulated in T cells and monocytes of HIV+ individuals, and that IL-10 may have a role in inducing immune unresponsiveness by modulating the CD28-B7 pathway.

Comparative Analysis of Total and Integrated HIV-1 DNA in Peripheral CD4 Lymphocytes and Monocytes After Long Treatment with HAART

Objective: To determine the level of total and integrated HIV-1 DNA load in CD4 lymphocytes and monocytes of patients undergoing HAART treatment for at least 2 years.Methods: CD4 lymphocytes were isolated by subjecting monocyte-depleted blood samples to immune-purging carried out with M-450 Dynabeads. Monocytes were separated by blood through a combined procedure of cell adherence to dishes and complement induced immune lysis with anti-CD3 Mab. HIV DNA in CD4 lymphocytes and monocytes was quantified by polymerase chain reaction (PCR) based limit dilution assay with two PCR protocols, specific for total (LTR PCR) and integrated (Alu PCR) forms of HIV DNA. The replication competence of the provirus harboured in monocyte-depleted peripheral blood mononuclear cells (PBMC) and adherent monocytes was assayed by measuring HIV-1 p24 antigen produced by in-vitro cultures established with these cells.Results: The CD4 lymphocytes of all patients contained a consistent number of HIV DNA copies. Most patients were also positive for HIV DNA in monocytes. The Alu PCR analysis detected, integrated provirus in CD4 lymphocytes of 9 patients and in the monocytes of only three. Four patients had replication-competence virus in their PBL. The monocytes of all patients did not produce virus in vitro.Conclusion: The HIV infection of CD4 lymphocytes and monocytes is maintained even after HAART related, apparent, and durable suppression of viral replication. We suggest that the viral persistent infection of monocytes may play a role in maintaining the residual HIV activity found in patients undergoing HAART.

Immunoregulating properties of peptides related to tumor rejection antigens: effect on human monocytes and natural killer cells.

The authors analyzed the effect of several 15-amino acid peptides with sequences related to tumor-rejection antigens, tyrosinase, and the MAGE family on peripheral blood mononuclear cells from healthy donors cultured for periods of 1 to 7 days. Some of these peptides promoted stimulation of monocytes, manifested by phenotypic changes, release of interleukin (IL)-1a, IL-6, and tumor necrosis factor-alpha, and induction of nitric oxide synthase on differentiated CD14++/+ CD16+ DR++ monocytes. An increase in the percentage of cytotoxic monocytes (CD14+/- CD16+) containing granule-associated DNase activity was also observed. Active peptides induced the release of IL-2 and interferon-gamma. Nonspecific natural killer and lymphokine-activated killer cell-mediated cytotoxicity was also observed against classical target cell lines (K-562 and Daudi) and allogenic melanoma cell lines AC and BB, together with an increase in granule-associated DNase in the natural killer cell-enriched population. Monocytes were needed to enhance this innate response, because peptides failed to induce the release of IL-2 on monocyte-depleted peripheral blood mononuclear cells. Data show an enhancement of the rapid innate immune response by peptides related to tumor rejection antigens and suggest that they could also determine the nature of a slow and more definitive specific immune response against tumor cells.

Dendritic cells that process and present nominal antigens to naive T lymphocytes are derived from CD2+ precursors.

Dendritic cells (DC) are potent APC that, in mature form, can be distinguished from other mononuclear cells on the basis of their distinct morphology, absence of lineage markers, and dense expression of MHC and costimulatory molecules. While comparing different DC preparation methods, we observed that DC derived from cultured PBMC that had been depleted of CD2+ cells before culture were functionally distinct from DC derived from PBMC that had not been depleted of CD2+ cells. Thus, both types of DC stimulated allogeneic T cells to proliferate in the MLR, but only DC derived from CD2+ precursors could sensitize naive T cells to soluble Ags such as keyhole limpet hemocyanin and HIV gp160 glycoprotein. Subsequent studies confirmed the existence of CD2+ and CD2- DC precursor populations among HLA-DRbright, lineage-negative PBMC. Immediately after their isolation, these populations were morphologically similar to one another by light and electron microscopy, and neither had substantial Ag-presenting activity. After culture for 24 to 48 h with supernatant from PHA-activated PBMC, both populations developed dendrites, formed clusters with T cells, and stimulated allogeneic T cell responses in the MLR as well as autologous T cell responses to tetanus toxoid, a recall Ag. However, CD2+ DC precursors alone gave rise to APC that presented soluble Ags to naive CD4+ T cells, a property that could be inhibited by Abs to CD4, CD11a, and CD28 on T cells or CD86 on DC. The expression of CD54 and CD86 on CD2+ DC precursors was increased markedly after their culture and differentiation, while the expression of these molecules on CD2- DC precursors was not remarkably changed. These findings reveal the existence of two functionally distinct populations of DC, each derived from a phenotypically distinct precursor present in monocyte-depleted peripheral blood.

Interleukin-12 decreases human immunodeficiency virus type 1 replication in human macrophage cultures reconstituted with autologous peripheral blood mononuclear cells.

In vitro interactions between interleukin (IL)-12, interferon (IFN)-gamma, and human immunodeficiency virus (HIV) type 1 infection in human macrophages were examined. Macrophages were infected with HIV-1 and cocultured with autologous monocyte-depleted peripheral blood mononuclear cells (PBMC). The addition of autologous PBMC to HIV-1-infected macrophages resulted in an expansive increase in reverse transcriptase (RT) activity; however, when both autologous PBMC and IL-12 were added, RT activity decreased (75%-90%) and high levels of IFN-gamma (9-16 ng/mL) were detected. The addition of anti-IFN-gamma antibodies blocked the IL-12-induced decrease in RT activity. Surprisingly, exogenous IL-12 added to HIV-infected macrophage cultures without autologous lymphocytes resulted in a 50%-60% reduction in RT activity and no detectable increase in IFN-gamma. The addition of anti-IFN-gamma did not inhibit this IL-12-mediated effect. These results suggest that IL-12 is capable of indirectly down-regulating HIV proliferation in macrophage cultures reconstituted with autologous PBMC and of directly suppressing HIV replication in purified macrophage cultures.

Molecular analysis of highly enriched populations of T-cell-depleted monocytes

CD4+ T lymphocytes and monocytes/macrophages are important components of the immune system. Blood monocytes are usually targeted for studies of the human macrophage lineage cells because of their accessibility through blood sampling. Most separation techniques currently available to obtain human monocytes either require large volumes of blood or do not yield a monocyte fraction sufficiently depleted of other cell types. We have developed a simple strategy to isolate a highly enriched population of monocytes from small volumes (< 6 ml) of peripheral blood by using an anti-CD14 monoclonal antibody and magnetic microspheres. Yields of monocytes ranged from 75 to 80% of CD14+ cells in peripheral blood. CD4+ T cells were subsequently selected from the monocyte-depleted peripheral blood by using an anti-CD4 monoclonal antibody and immunomagnetic beads. The effectiveness of immunomagnetic selection to yield a monocyte population highly depleted of T cells was analyzed by using a sensitive molecular strategy based on PCR amplification and detection of T-cell receptor (TCR) gene rearrangements. The relative frequency of rearranged TCRs within the monocyte population was compared with the frequency of rearranged TCRs within the CD4+ T-cell fraction from the same individual. Molecular analysis indicated that a viable monocyte population which contains fewer than 2% residual T lymphocytes can be consistently selected from small aliquots of blood.

Diminished cell-cell binding by lymphocytes from healthy, elderly humans: evidence for altered activation of LFA-1 function with age.

We studied the kinetics of cell-cell adhesion by monocyte-depleted peripheral blood lymphocytes with a stirred-cuvette aggregometer using samples from healthy young (mean age = 25 years) and healthy elderly (mean age = 75 years) human donors. This was a very reproducible assay, as there was virtually no intraindividual variability and very little interindividual variability among donors of the same age group. In all cases, aggregation of cells began immediately upon addition of the protein kinase C activator phorbol myristate acetate (PMA) or the calcium ionophore ionomycin, and continued until reaching an asymptotic limit by 20 minutes or less. Unstimulated cells did not aggregate during this time. The rate of aggregate formation and total amount of aggregation (using young donors' cells) varied between PMA- and ionomycin-stimulated cells, suggesting different mechanisms for initiating cellular aggregation, or possibly, different peripheral blood lymphocyte subsets affected by these activating agents. However, for both stimuli, the rate of aggregation and the total amount of cellular aggregation were significantly lower with the elderly donors' cells. Further, aggregation was Ca2+/Mg2+ dependent, and the reaction required metabolically active cells, as the reaction was inhibited by the addition of sodium azide and 2-deoxy-D-glucose in both donor groups. Pretreating cells with the actin polymerization inhibitor cytochalasin B resulted in 35-40% inhibition of aggregation among young donors' cells, although, interestingly, there was no apparent effect upon the cells capable of forming aggregates in the old donor group. In both donor groups, aggregation by activated cells could be partially blocked by pretreating cells with monoclonal antibodies directed against the alpha and beta chains of the integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18). These results show that there is diminished cell-cell binding among lymphocytes from healthy, elderly humans, and this is partly due to altered activation of LFA-1 function with age.

Human, recombinant interleukin-2 induces in vitro histamine release in a dose-dependent manner.

We previously observed that human, recombinant interleukin-2 in a pharmacologic dose (200 u/ml) induced histamine release from monocyte-depleted peripheral blood mononuclear cells in vitro. Therefore, we studied the role of various pharmacologic doses of rIL-2 on in vitro histamine release. Peripheral blood mononuclear cells (5 x 10(6) cells/ml), which also contain basophils, from 13 patients scheduled for elective colorectal cancer surgery and 10 age and sex matched healthy volunteers were stimulated with rIL-2 in concentrations of 0, 50, 100, 200, 450, 900, 1,800 and 3,600 u/ml, respectively, for 1, 24 and 48 hours under standard conditions. Histamine was analysed in supernatants using the glass fiber method. Simultaneously, total cell-bound histamine was analysed in lysate from 5 x 10(6) mononuclear cells from all patients and volunteers, thus allowing determination of percent histamine release. Supernatant histamine concentration from unstimulated cells was 17.2 +/- 1.5 ng/ml in patients compared to 7.9 +/- 1.0 ng/ml in volunteers (#p < 0.05) after 1 hour stimulation, and no further increase was observed after 24 and 48 hours, respectively. Histamine concentration increased significantly in the supernatant from cells stimulated by rIL-2 in a dose-dependent manner both in patients and volunteers. Total cell-bound histamine was 49.3 +/- 4.1 ng/ml in patients compared to 78.5 +/- 7.7 ng/ml in volunteers (p < 0.05). Therefore, both spontaneous and rIL-2-induced histamine release was significantly enhanced in cancer patients compared to volunteers (*p < 0.05). These data suggest that rIL-2 in high pharmacologic doses stimulates in vitro histamine release in a dose-dependent manner in both cancer patients and volunteers. This may in part explain the severe toxicity observed during high-dose rIL-2 therapy.

Natural killer and lymphokine-activated killer activity in HLA-B8,DR3-Positive subjects

The haplotype HLA-B8,DR3 is over-represented in several autoimmune diseases, implying that genes predisposing people to these disorders are linked to this haplotype. In these diseases, various dysfunctions reflecting an impairment of the immune system have been found. Several reports indicate also that in HLA-B8,DR3-positive healthy subjects similar disorders may be demonstrated. In the present work, we have evaluated NK and LAK activity in these subjects. The study has been performed on monocyte-depleted peripheral blood MNCs by using the K-562 cell line as a target for NK activity and the HL-60 cell line for as a target LAK activity. LAK cells were obtained by incubating MNCs for 3 days with 100 U/ml of rIL-2. The results of our experiments demonstrate that NK cell activity is significantly decreased in healthy subjects bearing the HLA-B8,DR3 haplotype. Since the number of circulating CD16+ cells is not significantly different between HLA-B8,DR3-positive subjects and negative ones, it is unlikely that this defect is due to a decreased number of NK cells in effector cell preparations. The observation that the treatment with rIL-2 can restore the killer activity of MNCs from these subjects suggests instead that the reduced NK activity may be due at least in part to the imbalance of cytokine network that has been demonstrated in HLA-B8,DR3-positive subjects. Finally, since a decreased NK activity has been reported in the autoimmune diseases linked to this haplotype, our results support the suggestion that immunologic changes observed in autoimmune diseases reflect systemic regulatory disorders that have a genetic basis.

Secondary cytokine production by lymphoid cells used in cellular immunotherapy

Interleukin-2 (IL-2) has been used extensively in cellular immunotherapy trials as a systemic activator of the immune system as well as an ex vivo stimulant for lymphoid cell function. Despite the measurement of several in vitro and in vivo immunologic parameters related to cellular immunotherapy, determinants of successful cellular immunotherapy remain unknown. To further delineate the consequences of exposing peripheral blood lymphocytes to high concentrations of IL-2, we assessed the supernatants of IL-2-activated peripheral blood lymphocytes for production of tumour necrosis factor (TNF) and interferon-γ (IFN-γ). Exposure of normal monocyte-depleted peripheral blood mononuclear cells (PBMC) to IL-2 caused a dose-dependent increase in secretion of TNF and IFN-γ which increased linearly after 48 h in culture. Analysis of positively selected, highly purified PBMC subpopulations exposed to IL-2 revealed that TNF-α and TNF-β were produced by both CD3 + and CD16 + subpopulations but not by CD22 + cells. These studies were extended to supernatants obtained from PBMC cultures used in the adoptive cellular immunotherapy of patients with advanced cancer. Patients treated with lymphokine-activated killer (LAK) cell immunotherapy were classified as responders ( N = 14) or non-responders ( N= 17) to therapy. We found no significant difference in the production of TNF between responders and nonresponders (22 ± 9 U ml −1 vs. 20 ± 6 U ml −1), P > 0.05. However, LAK cell supernatants harvested from non-responders contained a significantly higher level of IFN-γ (232 ± 94 U ml −1) compared with responders (42 ± 14), P < 0.05. Furthermore, the linear association between IFN-γ and TNF-α production was different between these two response groups ( r s = −0.19 for non-responders and r s = 0.48 for responders). These results suggest that secondary cytokine production by adoptively transferred lymphocytesmay play an important role in the host response to cellular immunotherapy.

Adhesion characteristics of human interleukin 2-activated natural killer cells

Culture of human monocyte-depleted peripheral blood mononuclear cells with recombinant IL2 (rIL2) induced adherence to plastic by 24 hr and subsequent proliferation in a subpopulation of lymphocytes with phenotypic and functional characteristics of activated natural killer (NK) cells. Purified human NK cells activated in the presence of IL2 for 24 hr upregulated the expression of the CD11c (p150,95) and CD11a antigen but not other cellular adhesion molecules (CAM). After further incubation with IL2, NK cells displayed upregulation of all of the antigens in the CD11/CD18 family of CAM. The process of adhesion was strictly dependent on culture in the presence of IL2, divalent cations, and active cellular metabolism. Adhesion also was dependent on expression of CAM on the cell surface, since monoclonal antibodies to CAM inhibited adhesion of activated NK cells to varying degrees (from 50 to 80%). An antibody (LeuM5) to the CD11c antigen (p150,95) gave the highest level of inhibition, and anti-CD11a (LFA-1) also was inhibitory, while anti-CD56 (NKH1) or anti-CD11b did not interfere with adhesion to plastic. Anti-CD11c was also the most effective in initiating the detachment of adherent-phase NK cells. Antibodies to CD18 or CD2 antigen also inhibited binding of NK cells to plastic. The blocking effects of anti-CD2 and anti-CD11a were additive in this system. On the surface of plastic-adherent and motile NK cells, all CAM except the CD56 antigen had a polar or bipolar distribution, as determined by staining with anti-CAM antibodies. Surface antigens CD11b, CD11c, CD2, and CD18 on nonadherent NK cells were clustered at the cellular poles by both immunofluorescence and immunogold electron microscopy, whereas CD11a (LFA-1) and CD56 antigens were distributed diffusely. CAM, especially CD11c, were also detected in cytoplasmic granules by immunostaining in IL2-activated NK cells. Thus, CAM may be stored in granules, allowing for their rapid transfer to the cell membrane in response to activation. Our results indicate that CAM are upregulated in IL2-activated NK cells and that some of these molecules (e.g., CD11c) play an important role in the development of plastic adherence by a subpopulation of these cells.

Humoral modulation of lymphokine‐activated killer (LAK)‐cell induction in humans: IgG‐related and non‐IgG inhibitors in sera from cancer patients

Soluble inhibitors of lymphokine-activated killer (LAK) cell induction were characterized and purified from serous fluids from healthy donors or from patients with advanced cancer. Inhibitory activity in sera from cancer patients was partially absorbed with protein A agarose or anti-human IgG agarose. Following absorption, residual inhibition varied with individual sera, suggesting the presence of IgG-related and non-IgG inhibitory factors, and the proportions varied in the patient population. IgG, purified by affinity chromatography from ascitic fluids, from plasma of cancer patients, and from plasma of healthy donors, significantly inhibited IL-2 induction of LAK cells in a dose-dependent manner. Irrespective of the sources, inhibitory activity resided in the high-molecular-weight IgG fraction composed of IgG aggregates and immune complexes, but not monomeric IgG. Commercially prepared human IgG in aggregated form, but not in monomeric form, inhibited LAK cell induction in a dose-dependent manner. In contrast, neither bovine IgG nor human albumin affected LAK cell induction, even at higher concentrations. Aggregated human IgG inhibited LAK cell induction in unfractionated peripheral blood mononuclear cells (PBMC) but not in monocyte-depleted peripheral blood lymphocytes (PBL). Despite extensive (greater than 99%) depletion of IgG by protein-G affinity chromatography, the serous fluid from cancer patients displayed significant inhibitory activity. Fractionation of the IgG-depleted inhibitory materials by Sephacryl S-300, high-pressure ion-exchange column (HPIEC) or gel-permeation chromatography (HPGPC) demonstrated a 65-kDa inhibitor, distinct from IgG. Affigel-blue affinity chromatography of the 65-kDa fraction depleted albumin but did not remove the inhibitory activity, suggesting that the 65-kDa inhibitor is not serum albumin nor an albumin-bound component. These results suggest that serous fluids from patients with advanced-stage cancer contain 2 distinct regulators for LAK cell induction: (I) aggregated IgG and (2) a 65-kDa inhibitor, distinct from albumin.

Endogenous interleukin 6 plays an obligatory role in interleukin 4-dependent human IgE synthesis.

The lymphokine interleukin (IL) 4 plays a crucial role in the regulation of IgE synthesis. In the present study, the cellular and cytokine requirements for the IL4-dependent induction of IgE synthesis in humans were analyzed. Recombinant IL4 could induce IgE synthesis by peripheral blood mononuclear cells and autologous T/B cell mixtures, but not by highly purified B cells. IgE induction by IL4 was strongly decreased in monocyte-depleted peripheral blood mononuclear cells. These results show that the induction of IgE synthesis by recombinant IL4 is T cell dependent and optimal in the presence of monocytes. IL5 and IL6, but not IL2, IL1 and tumor necrosis factor-alpha, strongly up-regulated the IL4-dependent synthesis of IgE, with modest effects on cell proliferation. An anti-IL6 polyclonal antibody strongly inhibited IL4-driven IgE production. Endogenous IL6 plays, therefore, an obligatory role in the IL4-dependent induction of IgE. However, a combination of IL4, IL5 and IL6 (with or without IL1) at optimal concentrations could not induce IgE synthesis by purified normal B cells, indicating that cytokine-mediated signals, although essential, are not sufficient for the IL4-dependent induction of IgE synthesis.

Selective proliferation of natural killer cells among monocyte-depleted peripheral blood mononuclear cells as a result of stimulation with staphylococcal enterotoxin B.

In vitro stimulation of monocyte-depleted peripheral blood mononuclear cells with staphylococcal enterotoxin B (SEB) resulted in selective proliferation of cells which express the phenotypic and functional characteristics of natural killer (NK) cells. This culture system provides an easy method for obtaining highly purified NK cells, by sequential incubation of monocyte-depleted cells with SEB and then with interleukin-2 (IL-2). After culture for 4 to 5 days in the presence of SEB, 98 to 100% of the cells expressed the CD16 (Leu11) and HNK-1 (Leu19) antigens. This purification occurred through the death of lymphocytes lacking NK cell markers and marked proliferation of NK cells themselves, which leads to an enrichment of the NK cell population. Activation of NK cells was detected by the appearance of the gamma interferon receptor and IL-2 receptor antigens. This homogeneous population showed the morphology of large granular lymphocytes, were potent effectors of cell-mediated cytotoxicity against K562 and Daudi tumor cell lines, and were able to kill gram-positive and gram-negative bacteria. IL-2 was necessary to maintain the activation and proliferation after SEB stimulation for 4 days. Moreover, the maximum frequency of binding to K562 cells (60.6%) was similar to that recently found (58 +/- 3%) (P. Garcia-Peñarrubia, F. T. Koster, and A. D. Bankhurst, J. Immunol. Methods 118:199-208, 1989) with fresh and highly purified NK cells. This method can be used as a source of highly purified NK cells to study their functional properties and applications to the treatment of cancer.

Monocyte-dependent, serum-borne suppressor of induction of lymphokine-activated killer cells in lymphocytes from melanoma patients.

Both phytohemagglutinin-induced cytotoxicity and recombinant-interleukin-2 (rIL-2)-induced lymphokine-activated killer (LAK) activity against noncultured melanoma cells were significantly reduced when peripheral blood mononuclear cells (PBMC) from patients with metastatic melanoma were incubated in RPMI medium 1640 and 10% autologous human serum instead of 10% fetal calf serum, while serum from either healthy donors or patients with primary melanoma did not affect the level of cytotoxicity. The serum-mediated suppression was not restricted by major histocompatibility complex and was time-dependent. Addition of 10% human serum from the patients with metastatic melanoma [HS-Pt(m)] to the culture of PBMC with rIL-2 at the same time or 1 day after incubation significantly inhibited LAK activity. However, addition of 10% HS-Pt(m) 2 or 3 days after incubation did not inhibit LAK activity. Incubation of PBMC for 2 h with a high dose (10(4) U/ml) of rIL-2 in the presence of 10% HS-Pt(m), followed by incubation in the absence of either rIL-2 or HS-Pt(m), did not affect LAK cell activity. These results suggest that HS-Pt(m) inhibits the early stage of LAK cell differentiation, rather than the binding of rIL-2 to PBMC or a later stage in the differentiation. In contrast to PBMC, monocyte-depleted peripheral blood lymphocytes exhibited comparable levels of LAK activity when cultured with rIL-2 either in 10% fetal calf serum, 10% human serum from healthy donors or 10% HS-Pt(m). Addition of purified autologous monocytes to the culture of monocyte-depleted peripheral blood lymphocytes with rIL-2 suppressed LAK cell induction when 10% HS-Pt(m) was present. Thus serum-mediated suppression of LAK cell induction is largely dependent on the presence of monocytes, which may produce a secondary inhibitor that acts on lymphocytes. Addition of indomethacin to the culture did not reverse this monocyte-dependent serum-mediated suppression in a majority of cases, suggesting that prostaglandin E2 does not have a major role in the suppression.

Production of tumor necrosis factor/cachectin by human T cell lines and peripheral blood T lymphocytes stimulated by phorbol myristate acetate and anti-CD3 antibody.

The induction of mRNA synthesis and accumulation of TNF/cachectin and lymphotoxin (LT) mRNAs in T leukemic cell lines and freshly isolated T cells were studied by Northern blot analyses. Without stimulation, TNF mRNA was barely detected in four T cell lines (CEM, KE4, MT-1, and SKW-3) and not detectable in Molt-4 and Jurkat cells, while a considerable amount of TNF mRNA was observed in HSB-2 cells. When stimulated by PMA, these T cell lines accumulated varying levels of TNF mRNA. All seven T cell lines expressed LT mRNA when unstimulated and responded well to PMA by increased accumulation of LT mRNA. The calcium ionophore A23187 by itself had no effect on TNF and LT mRNA accumulations in these cell lines. The CD3+ T cell lines did not respond to anti-CD3 mAb T3-II alone. However, A23187 and mAb T3-II further elevated TNF and LT mRNA accumulations in PMA-treated T cell lines. Synergism between PMA and mAb T3-II was modest in the CD3+ cell lines. A slight difference in kinetics of TNF and LT mRNA accumulations was noted. In addition, heterogeneities in TNF and LT expressions by these cell lines in responses to PMA and other stimuli were observed. In monocyte-depleted peripheral blood T cell populations. PMA was able to induce both TNF and LT mRNA syntheses. This effect was potentiated markedly by the addition of anti-CD3 mAb T3-II. This synergistic response to anti-CD3 mAb and PMA provided further evidence that T cells were the source of TNF synthesis in these cultures. There was a difference in the kinetics of TNF mRNA accumulation and that of LT mRNA. Maximal accumulation of TNF mRNA occurred at 4 h while 8-18 h was required for maximal LT mRNA accumulation. IL-2 mRNA accumulated at an intermediate peak time of 4-8 h. Western blot analyses and cytotoxicity assays with L cells as targets indicated that these T cell lines and peripheral blood T cells secreted TNF. These results provide further evidence that human T cells are capable of making TNF as well as LT under appropriate stimulations. Their productions are an integral part of T cell response to activation signals. In addition, it appears that the production of these two closely related molecules is independently regulated.

The bovine autologous mixed leukocyte reaction: A proliferative response of non-T cells under the control of monocytes

The culture of bovine monocyte-depleted peripheral blood mononuclear cells (PBM) (responder) with irradiated autologous undepleted PBM (stimulator) resulted in a strong proliferative response, which was called the bovine autologous mixed leukocyte reaction (MLR).A comparative study was made between this autologous MLR and the bovine allogenic MLR, with respect to the inductive requirements and the nature of responding and stimulator cells.In both the autologous and allogenic MLR, the monocyte was responsible for inducing proliferation: low concentrations of purified monocytes (> 95 % pure) induced strong proliferative responses, while monocyte-depleted PBM (< 0.2 % monocytes) were unable to induce proliferation.Both MLRs were blocked by monoclonal antibodies specific for class II antigens of the major histocompatibility complex.The magnitude of the autologous MLR was regulated by monocytes: unirradiated monocytes induced or suppressed autologous proliferative responses depending on their concentration in the culture. Proliferating cells, identified by quantifying DNA with chromomycin A3, were all within the population of large cells.In the allogenic MLR, 50 % of these large cells were T cells, as defined by monoclonal antibodies.In contrast, all of the proliferating cells in the autologous MLR were non-T cells.

HLA-linked nonresponsiveness to Cryptomeria japonica pollen antigen. I. Nonresponsiveness is mediated by antigen-specific suppressor T cell.

The objective of this study was to elucidate the cellular mechanism of IgE nonresponse to the Cryptomeria japonica (Japanese cedar) pollen antigen (CPAg), which was shown in our previous study to be HLA-linked (1). We established an assay system for the measurement of small amounts of anti-CPAg IgE antibody, both in an antigen-specific and isotype-specific manner, and a culture system to induce antigen-driven IgE antibody synthesis in vitro. By using these methods, we clarified that the function of the HLA-DR molecule in the CPAg-driven IgE response is similar to that of I-A or I-E molecule in mice, namely the product of immune response genes (Ir-genes), because anti-HLA-DR monoclonal antibody blocked the response, and the interaction between monocyte and monocyte-depleted peripheral blood lymphocytes (PBL) to respond to CPAg was restricted by HLA-DR. Furthermore, PBL from nonresponders revealed a specific IgE response to CPAg when the Leu-2+3- T cell fraction was depleted, thereby suggesting that even nonresponders have Leu-2-3+ T cell and B cell clones specific for CPAg, and they apparently show no response due to the presence of CPAg-specific Leu-2+3- suppressor T cells. This suppressor T cell fraction abolished the IgE response of the autologous B + monocyte + Leu-2-3+ T cell in a CPAg-specific manner. The current cellular analysis together with our previous genetic analysis strongly suggest that the HLA-linked IgE nonresponse to CPAg is mediated by CPAg-specific suppressor T cells. The HLA-linked gene controlling the nonresponsiveness to CPAg is thus designated as the immune suppression gene for CPAg (Is-CPAg). Mapping of Is-CPAg within HLA-DQ subregion is discussed.

Modulation of natural killer cell function by human alveolar macrophages

Lymphocyte natural killer (NK) activity appears to be important in resistance against viral infection and malignancy. Since pulmonary alveolar macrophages (PAM) derived from nonsmokers suppress NK activity in vitro, we asked if PAM derived from smokers modulated NK activity in a similar fashion. Bronchoalveolar lavage cells (BLC) were obtained from healthy nonsmokers and healthy smokers by bronchoalveolar lavage. Increasing numbers of BLC were cocultured with a fixed ratio of monocyte-depleted peripheral blood lymphocytes (PBL) and Cr 51-labeled K562 tumor target cells (PBL:K562, ratio 25:1; BLC:PBL ratios ranging from 0.02:1 to 1:1). Increasing numbers of BLC progressively suppressed the ability of either autologous or homologous PBL to lyse tumor targets. Smoker and nonsmoker BLC inhibited NK activity of homologous lymphocytes with similar potency. Suppression of NK activity by BLC was not reversed by the addition of indomethacin or catalase to cell cultures. Furthermore, suppression could not be reproduced by performing cytotoxicity assays in the presence of BLC culture supernatants. Lymphocytes boosted to high levels of NK activity by preincubation with α-interferon or interleukin-2 were still susceptible to suppression by BLC. Thus, PAM derived from smokers efficiently suppress lymphocyte NK activity, and the degree of suppression increases as the ratio of PAM to lymphocytes increases. Since smoking causes a bronchoalveolar accumulation of PAM, these inhibitory cells may result in profound local suppression of NK activity in smoker's lungs.

Concanavalin A induction of suppressor activity in the T-helper subset defined by monoclonal antibodies.

Human monocyte-depleted peripheral blood mononuclear cells were separated in T4+ and T8+ populations by a panning technique. Petri dishes coated with goat anti-mouse antibodies were plated by peripheral blood mononuclear cells coated by monoclonal antibodies, either T4 or T8. The cell populations were separated into adherent and non-adherent populations based on binding to the goat anti-mouse-coated plastic dishes. The purity of the adherent populations was 96%. T4+ and T8+ populations were used as effector cells in the concanavalin A-induced suppressor test. The T4+ population revealed a pronounced suppressor activity similar to that exhibited by the T8+ population. This finding was independent of two different sources of monoclonal antibodies, T4/T8 and OKT4/OKT8. The registered suppressor activity in the monoclonal antibody-defined helper population could not be explained either by a switch of the membrane phenotype from T4+ to T8+ cells or by an increased interleukin 2 consumption of the concanavalin A-treated cells.