SYNERGY BETWEEN SUBPOPULATIONS OF MOUSE SPLEEN CELLS IN THE IN VITRO GENERATION OF CELL-MEDIATED CYTOTOXICITY : Evidence for the Involvement of a Non-T Cell

Two subpopulations separated from normal spleen have been shown to synergize as responding cells in the in vitro induction of specific cell-mediated cytotoxicity during the mixed lymphocyte culture (MLC). The synergizing populations are a nylon wool column-adherent and a nylon wool column-nonadherent fraction, enriched for B lymphocytes and T lymphocytes, respectively. When a mixture of these fractions is used as the responding cell population in MLC, greater cytotoxicity is generated than would be expected from the sum of activities generated in the two subpopulations sensitized separately. The synergy appears to occur at the sensitization rather than the effector phase. The synergizing cell which is contained in the nylon-adherent subpopulation is distinct from the cytotoxic effector T lymphocyte, is resistant to lysis by rabbit antimouse brain serum, and is unresponsive to phytohemagglutinin; its synergizing function could not be replaced by either plastic-adherent spleen cells or peritoneal exudate cells. These results suggest a role of a non-T-cell nonmacrophage population in the generation of cytotoxic activity.     

Two-stage separation of immature and mature T lymphocytes from myeloerythroid clonogenic bone marrow cells by apheresis and elutriation centrifugation

Because graft-versus-host disease remains a major complication in allogeneic bone marrow transplantation, a number of techniques capable of removing mature T lymphocytes from bone marrow cells have been attempted. The authors describe a simple two-step procedure using counterflow centrifugation elutriation (CCE) that eliminated 95 to 98 percent of the mature T lymphocytes and greater than 97 percent of the T lymphocyte colony-forming units (CFU-T) while concentrating the bone marrow myeloid colony-forming cells. Viability was greater than 98 percent, and 72 to 98 percent of the total cells separated were recovered. Lymphocyte depletion was substantiated by both morphologic and phenotypic criteria using monoclonal antibodies to T lymphocytes, as well as by responsiveness in mixed-lymphocyte cultures and to mitogens. In addition, this technique separated the lymphoid colony-forming cells from the larger myeloid colony-forming cells. It was concluded that this simple two-step CCE procedure can be used to separate T lymphocytes and CFU-T from myeloid colony-forming cells and offers a means of purging T lymphocytes from large numbers of marrow cells that may be required for human allogeneic bone marrow transplantation.     

Factors affecting the autologous mixed lymphocyte reaction in kidney transplantation.

In long-term well adapted kidney transplant recipients we have found a close correlation between the T helper (TH):T suppressor/cytotoxic (TS/C) subset ratios and the presence of T cells that respond in the autologous mixed lymphocyte reaction (AMLR). In 21 recipients with T cell E rosette levels ranging between 53 and 86% and TH:TS/C ratios between 0.15 to 2.10, ratios of greater than 0.8 correlated with AMLR responses (13/13), and ratios of less than 0.8 with AMLR nonreactivity (7/7). By contrast, the allogeneic MLR showed no apparent correlation with the TH:TS/C ratios or with the AMLR pre- or postoperatively. It was found that the AMLR in 22 of 23 normal individuals was markedly inhibited by autologous T cells obtained from peripheral blood lymphocytes, exposed to 3,000 rad (Tx) and added as a third component to the cultures. In contrast, 13 of 13 kidney transplant recipients failed to exhibit this Tx AMLR inhibitory cell population. The "naturally occurring" T inhibitory cells, fractionated by an affinity column chromatography procedure into x-irradiated TH and TS/C subsets, inhibited the AMLR to the same extent as unseparated Tx cells. In cell interchange studies performed in four of five HLA identical donor-recipient pairs the Tx cells of the (normal) donor inhibited the recipient AMLR (immunosuppressed), but recipient Tx cells failed to inhibit the donor AMLR. Finally T cells, primed in AMLR and allogeneic MLR for 10 d were tested for AMLR or allogeneic MLR inhibitory activity. Allogeneic MLR primed x-irradiated cells, inhibited both the AMLR and allogeneic MLR while AMLR x-irradiated primed cells inhibited neither reaction. The Tx AMLR inhibitor found in normal peripheral blood, appears to be a cell that is highly sensitive to the effects of biologic or pharmacologic immunosuppressive agents.     

Antigen-specific immunosuppression in visceral leishmaniasis is cell mediated.

Visceral leishmaniasis is associated with an antigen-specific immunosuppression during the acute disease. Patients become responsive to Leishmania antigen in both in vivo and in vitro assays after successful antimony therapy. The cell type involved in the suppression of lymphocyte reactivity to Leishmania antigen was studied by selective depletion of mononuclear cell (MNC) populations and in co-cultivation experiments. Adherent cells were depleted on plastic and by passage on nylon wool columns. High-avidity Fc+ cells were depleted by adherence to BSA-anti-BSA complexes and OKT4+ and OKT8+ cells were depleted by treatment with monoclonal antibody (anti-OKT4+ and OKT8+) and complement. Depletion of MNC preparations of adherent cells, high-avidity Fc+ cells, OKT4+ cells and OKT8+ cells failed to restore the lymphocyte reactivity to Leishmania antigen. Antimony therapy was associated with restoration of the proliferative responses of unseparated MNC (before treatment 460 +/- 76 cpm and after treatment 4,293 +/- 1,442 cpm). Co-culture of frozen cells obtained before chemotherapy with autologous MNC obtained after treatment reduced the response of posttreatment cells to Leishmania antigen by 80%. We conclude that the antigenic specific suppression of lymphocyte proliferation in visceral leishmaniasis is cell mediated.     

Human antibody-dependent cellular cytotoxicity. Isolation and identification of a subpopulation of peripheral blood lymphocytes which kill antibody-coated autologous target cells.

Antibody-dependent cellular cytotoxicity (ADCC), has been shown to be independent in vitro of thymus-derived lymphocytes, but the precise nature of the effector lymphocyte has not been fully clarified. To further study the identity of the ADCC effector cell type(s), peripheral blood leukocytes were purified by Ficoll-Hypaque density centrifugation and fractionated into surface immunoglobulin-positive [Ig(+)] and surface immunoglobulin-negative [Ig(-)] populations by chromatographic separation on Sephadex G-200 anti-human immunoglobulin columns. After column fractionations, the ADCC effector activity against antibody-coated autologous lymphocytes was predominantly and consistently found in the Ig(-) fraction. This latter population was then further fractionated, by rosetting techniques, into two subpopulations, The first was depleted by lymphocytes with surface receptors for sheep red blood cells [E(+)]and the second was depleted of lymphocytes with receptors for sheep red blood cell-antibody-complement [EAC-(+)]. Analysis of these populations showed that ADCC effector activity was predominantly a property of the Ig(-) lmyphocytes which are E(-) but EAC(+). These lymphocytes have been referred to as "null lymphocytes" and probably represent a subset of bone marrow-derived (B) cells. In addition, variable and low levels of ADCC activity were observed in some Ig(+) populations (B cells). Further purification of the null cell population by filtration over nylon wool columns to reduce the number of contaminating latex ingesting monocytes did not reduce ADCC effector activity. Isolated null cell ADCC effector activity was inhibited by either rabbit anti-human F(ab)2 or normal pooled rabbit gamma globulin, but not by rabbit F(ab)2 anti-human F)ab)2 or media. This supports the contention previously suggested in studies using unfractionated lymphocyte populations that the ADCC effector cell recognizes the Fc portion of the antibody molecule. The variable and low level of activity noted in the Ig(+) populations is unexplained but possibly due to a variable population of null cell-derived Ig(+) lymphocytes within the whole Ig(+) population. In conclusion, these experiments demonstrate that, in vitro, the major ADCC effector activity of circulating human peripheral blood lymphocytes resides in the Ig(-), E(-), EAC-(+) subpopulation termed "null cells." Since it has been noted that in certain disease states, such as immunodeficiency syndromes, autoimmune disorders, and neoplasms, the percentage of this population of lymphocytes in the peripheral blood is elevated, it is speculated that these cells, perhaps through their ADCC function, may play an important pathophysiologic role in these diseases.     

Functional heterogeneity of human monocytes--with a special reference to flow cytometric assays

A large number of human mononuclear cells were simultaneously separated into fractions enriched in B cells, T cells, large granular lymphocytes (LGL) and monocytes by centrifugal elutriation. Lymphocyte populations were analyzed using monoclonal antibodies. In particular, highly enriched natural killer cells, Leu7+ cells, were collected in the intermediate fractions. Monocytes, which were identified as esterase positive cells, and Leu M3 cells were collected at higher counterflow rates and in the final fraction. The purity of monocytes in the final fraction was 81%. The oxidative metabolic activity (H2O2 production) and non-specific esterase activity of individual monocytes was estimated in the analysis of functional heterogeneity of monocytes using flow cytometry. 2',7'-dichlorofluorescein diacetate (DCFH-DA) and fluorescein diacetate (FDA) were used as indicators in the measurement of H2O2 generation and esterase activity. Intracellular generation of a fluorescence product (H2O2 Production; average percentage of fluorescence positive cells) of monocytes in the stimulation of phorbol myristate acetate (PMA, 100 ng/ml) was greater in larger than smaller cells. H2O2 production gradually increased from 6% and 25-38% and 60% in the intermediate and final fractions respectively. Furthermore, the average fluorescence intensity of the large monocyte population in the final fraction was 1.13-1.31 fold more active than that of the smaller cells. Thus, the functional heterogeneity of human monocytes was further confirmed in the assays of H2O2 production exposed to PMA and FDA hydrolysis using flow cytometry. Furthermore, the CCE system can isolate lymphocyte subsets and LGL.     

Emergence of insulin receptors upon alloimmune T cells in the rat.

Insulin, as well as other ligands which increase intracellular guanosine 3',5'-cyclic monophosphate (cGMP), augments thymic-derived (T)- lymphocyte effector activity as revealed by alloimmune lymphocyte-mediated cytotoxicity. The observation that insulin binds only to monocytes among circulating nonimmune human mononuclear cells fosterd reexamination of the mechanism by which insulin augments T-lymphocyte function. This report concerns a test of the hypothesis that the T cell is directly affected by insulin and that an insulin receptor emerges upon T lymphocytes consequent to immune activation. Spleens were removed from rats skin grafted across a major histocompatibility barrier. Lymphocytes were harvested from Ficoll-Hypaque density gradients and subsequently enriched for T cells by passage over one or two nylon wool columns. This population was composed of more than 98% T cells as assessed by surface marker techniques (Ig staining, erythrocyte antibody, and erythrocyte antibody complement rosetting, anti-T staining). There was no loss of augmentation of lymphocyte-mediated cytotoxicity induced by insulin, carbamycholine, and 8-bromo-cGMP in the purified cells when compared to unfractionated cells 7 days after transplantation. 125I-insulin bound saturably to the allostimulated T-enriched lymphocytes with maximum binding at 12.8 +/- 0.2 pg and a dissociation constant at equilibrium of 1.3 nM. In contrast, insulin receptors were not present on nonimmune T-enriched cells or on T cells from animals that received syngeneic grafts. The affinity of the lymphocyte insulin receptor was similar to that of more conventional insulin-sensitive tissues e.g., liver, adipocyte. After 89% of T cells from spleens on day 7 were lysed with anti-thy 1.1 antibody and complement, the ability to measure specific insulin binding was lost. These data confirm a physiologic role for insulin in T-lymphocyte effector function and describe the emergence of insulin receptors concomitant with cell sensitivity to ligand. Such receptors may play a role in hormonal modulation of the immune response.     

Human bone marrow lymphocytes. Cytotoxic effector cells in the bone marrow of normal individuals.

This study was undertaken to determine the capability of lymphocytes in the bone marrow of normal individuals to mediate nonspecific killer cell functions in assays of phytohemagglutinin (PHA)-induced cellular cytotoxicity, and antibody-dependent cellular cytotoxicity (ADCC) against 51Cr-labeled chicken erythrocyte target cells. Relatively pure mononuclear cell suspensions were obtained from bone marrow aspirates in 30 normal volunteers by sucrose gradient centrifugations and from the peripheral blood of the same individuals by Hypaque-Ficoll density centrifugations. At an effector: target ratio of 10:1, the PHA-induced cellular cytotoxicity of peripheral blood was 78.8 +/- 1.3%, while that of bone marrow was not significantly less at 66 +/- 9% (P greater than 0.1). At low effector:target ratios, the ADCC of bone marrow was negligible, while at higher effector:target ratios (20:1) bone marrow ADCC was 69 +/- 3.7%, which was comparable to that of peripheral blood. The lymphocytes themselves in the mononuclear cell suspensions of both peripheral blood and bone marrow were capable of cytotoxicity activity since depletion of monocytes from the suspensions by adherence to rayon wool and G-10 Sephadex columns did not remove the cytotoxic activity. Blocking of the Fc receptor on the effector cells by the addition of aggregated gamma globulin to the cultures suppressed the ADCC but not the PHA-induced cellular cytotoxicity of both peripheral blood and bone marrow, indicating that ADCC is dependent on an Fc receptor on the effector cell in both compartments. These studies demonstrate that the bone marrow of normal humans contains populations of lymphoid cells which have highly efficient killer cell capacities. It is uncertain what portion of these cells arise in the bone marrow and what portion enter the bone marrow parenchyma as part of the recirculating lymphocyte pool. These findings have relevance in the clearer understanding of the killer cell potential of grafted human marrow, as well as the bone marrow sequestration of functionally capable lymphocyte subpopulations in disease states and during chemotherapy.     

Impairment of the autologous mixed lymphocyte reaction in atopic dermatitis.

The T cell proliferative response to autologous non-T cells is termed the autologous mixed lymphocyte reaction (AMLR). Recent studies have suggested that the AMLR represents an inducer circuit for the activation of T8+ suppressor/cytotoxic effector cells. Since atopic dermatitis (AD) patients are deficient in T8+ cytolytic T cell function, we investigated the AMLR in AD. When sheep erythrocytes were used to separate T cells from non-T cells, the AMLR was found to be significantly decreased (P less than 0.001) in AD patients (n = 11; delta cpm = 1,550 +/- 393) when compared with normal control subjects (n = 13; delta cpm = 25,819 +/- 4,609). To exclude the possibility that these results were an artifact of the sheep erythrocyte separation, T cells were also separated on a fluorescence-activated cell sorter after treatment of peripheral blood lymphocytes with the OKT3 monoclonal antibody. AD T cells separated by the latter method were also found to have a significantly reduced AMLR response when compared with similarly treated normal T cells. Co-culture studies using cells from AD patients and their HLA identical siblings indicated that the defect resided at the responder T cell level rather than at the stimulator non-T cell level. Co-culture studies revealed no evidence for excessive suppressor cell activity resulting in the decreased AMLR. However, enumeration of T cells reactive with the monoclonal antibody T29, which recognizes a subset of T cells proliferating in the AMLR, demonstrated that AD patients (n = 8; % T29 = 2.5 +/- 0.7) had a significantly decreased (P less than 0.001) number of circulating T29+ T cells when compared with normal controls (n = 8; % T29 = 10.4 +/- 0.8). These studies suggest that a deficiency of T4+ T29+ cells contributes to the deficient AMLR in AD and possibly underlies the abnormalities of T8+ effector cells present in this disease.     

Idiotype-like determinants on human T lymphocytes alloactivated in mixed lymphocyte culture

T cells alloactivated in 5-d MLC with an HLA-DR-different stimulator acquire the capacity of stimulating the autologous mixed lymphocyte response (AMLR). We have demonstrated that activation of AMLR by allosensitized T cells is determined by the expression of the idiotype receptor for the stimulating HLA-DR alloantigen. This has been shown in experiments in which purified, OKT-3-positive T cell suspensions were first primed for 9 d with AMLR-activated T lymphoblasts, then tested in secondary AMLR with autologous lymphoblasts sensitized to various HLA- DR alloantigens. Accelerated memory responses were induced only by autologous lymphoblasts that had been sensitized against the same HLA- DR specificity as the primary AMLR stimulators. This response was not inhibited by a mouse monoclonal antibody recognizing Ia-like determinants, and was not triggered by human allogeneic resting peripheral blood lymphocytes. Thus, recognition of alloactivated T lymphoblasts in secondary AMLR seems to be specific for the idiotype- like determinants expressed by the autologous stimulators.     

The nylon wool adherence of rat mononuclear cells: physical, chemical and biological influences

The application of mononuclear cell populations to a nylon wool (NW) column is a common early procedure in the selection of T lymphocytes for further study. The technique as presently employed in various laboratories does not appear to be standardized, and surprisingly little is known about the effects of physicochemical alterations and biologic mediators on the interaction of lymphocytes with the NW substrate. In this study the factors controlling the adherence of rat splenocytes to NW were examined. NW adherence was shown to be independent of loaded cell concentration, column packing or pH, but was very dependent on NW column size, wash volume, incubation time and ambient temperature. The addition of protein to media did not alter lymphocyte NW adherence, and the interaction did not appear to depend on intact cellular glycolysis or protein synthesis or on microtubular or microfilament function. Because of the theoretical importance of surface adherence to cell motility, the effects of various agents that alter lymphocyte migration were tested in the lymphocyte NW assay. Of the positive chemokinetic factors tested, only casein altered (decreased) NW adherence. Of the negative chemokinetic principles tested only the human lymphokine LyMIF altered (increased) NW adherence. The studies show that the NW-nonadherent cell pool may be a heterogeneous population depending on the physical conditions of the assay, and the NW adherence of rat splenocytes is not an all-or-none phenomenon but can be altered by physical and biological factors. This makes the standardization of the assay of critical importance, particularly if one wishes to compare results of subsequent experiments between laboratories.     

Comparative functional analysis of lymphocytes and monocytes from plateletapheresis

Large numbers (2.9 +/- 1.2 X 10(9)) of mononuclear cells can be obtained from incidental samples collected during routine plateletapheresis. We conducted studies comparing characteristics and functions of mononuclear cells derived from venous blood samples and from routine plateletapheresis in the same normal donors. Cell viability was similar in both samples (96 +/- 1% plateletapheresis vs 97 +/- 2% venous blood). Higher concentration of monocytes were observed in the plateletapheresis samples (32.3 +/- 6%) than in the venous blood (14.3 +/- 4%). The procedure of plateletapheresis does not seem to alter lymphocyte or monocyte function. Thus, the functional integrity of these cell populations was demonstrated in terms of natural killer cell activity, blastogenic response to mitogens, local graft-versus-host reactions, monocyte-mediated antibody-dependent cellular cytotoxicity against human red cells, monocyte-mediated tumor cell cytotoxicity, latex phagocytosis, and monocyte-dependent lymphocyte blastogenesis. We conclude that monocytes and lymphocytes obtained during routine plateletapheresis are functionally intact.     

Human endothelial cell-lymphocyte interaction. Endothelial cells function as accessory cells necessary for mitogen-induced human T lymphocyte activation in vitro.

Mitogen-stimulated human T cell activation is absolutely dependent on the participation of a nonresponding accessory cell. In populations of human peripheral blood mononuclear cells, monocytes function as the requisite accessory cells. The possibility that cultured endothelial cells (EC) might also function as accessory cells was studied by examining the potential of endothelial cells to restore mitogen responsiveness to monocyte-depleted human T cells. Highly purified T cells were prepared by isolating cells rosetting with sheep erythrocytes and removing monocyte contamination by glass adherence and nylon wool column passage. When cultured at low cell density, T cells failed to respond to stimulation with various mitogenic lectins, whereas co-culture with monocytes restored responsiveness. Similarly, EC obtained from umbilical vein, pulmonary artery, and ovarian vein restored the capacity of T cells to respond to mitogens. Mitogen-stimulated T cell activation required viable endothelial cells. Moreover, effective endothelial T cell cooperation appeared to involve the establishment of cell-to-cell contact between EC and responding T cells. Accessory cell function was not a nonspecific property of all tissue culture cells as evidenced by the finding that human foreskin fibroblasts, lung fibroblasts, and HeLa cells were unable to restore responsiveness to monocyte-depleted T cells. These observations indicate that endothelial cells can support the induction of mitogen-induced T cell activation and suggest that cells lining blood vessels may play an active role in the initiation of immune responses in vivo.     

Enrichment of dog leukocyte subpopulations using density gradients

Density gradient methods have been used for the enrichment of leukocyte subpopulations from human blood. We have adapted a three step method utilizing centrifugation on Hypaque-Ficoll (HF), double density HF (ddHF) and Percoll density gradients to separate lymphocytes, monocytes and basophils from dog blood. After HF separation, both Basenji-Greyhound (BG) and mongrel dogs had similar percentages of lymphocytes and monocytes, but BG dogs had significantly greater (p greater than 0.025) numbers of granulocytes. When cells from HF gradients were spun directly on Percoll, the presence of granulocytes decreased the purification of leukocyte subpopulations. An intervening separation on a ddHF gradient removed granulocytes without a selective loss of lymphocytes or monocytes. When cells from ddHF gradients were further separated on Percoll, 2 distinct cell layers resulted. Layer 1 was monocyte rich with 72.4 +/- 6.5% monocytes, 26.6 +/- 6.2% lymphocytes, and 0.8 +/- 1.8% granulocytes. Layer 2 was lymphocyte rich with 74.3 +/- 11.1% lymphocytes, 21.4 +/- 7.8% monocytes and 3.9 +/- 4.2% granulocytes. Viability as determined by Trypan blue exclusion was above 90%. Using cAMP-Phosphodiesterase (PDE) as a marker, higher PDE activity was present in the monocyte rich layer. This was similar to that reported in humans. Basophil numbers were enhanced by the use of a 1-step separation on a ddHF gradient. The percentage of basophils was increased 10-fold over baseline levels, with a viability of 90%. These techniques can be used to purify various canine leukocyte subpopulations and provide cells for biochemical analysis.     

Cultured human monocytes synthesize and secrete alpha2-macroglobulin

alpha2-Macroglobulin levels in the supernates of cultures of different subpopulations of human peripheral blood mononuclear leukocytes were assayed by a radioimmunoassay. Unfractionated mononuclear leukocytes produced greater amounts of the macroglobulin (4.0 vs. 0.8 ng/10(6) cells) than did subpopulations enriched in T or B+T lymphocytes, by passage through nylon wool or cotton wool columns, respectively. Still higher concentrations of alpha2-macroglobulin (40 ng/10(6) cells) were measured in the supernates of glass-adherent mononuclear leukocyte cultures. These results suggest that cells of monocyte-macrophage lineage are mainly, if not exclusively, responsible for the appearance of alpha2- macroglobulin in the supernate of human peripheral blood leukocyte cultures. The de novo synthesis and release of alpha2-macroglobulin by cultured monocytes was demonstrated by immunoprecipitation of radioactivity from supernates of 32S-methionine-labeled glass-adherent cells. Antiserum against purified alpha2-macroglobulin was used in both Ouchterlony double diffusion and double antibody precipitation tests. SDS-polyacrylamide gel electrophoresis of immunoprecipitates showed that most of the radioactivity comigrated with authentic alpha2-macroglobulin subunit at about 160,000 daltons.     

Isolation of monocyte-depleted and monocyte-rich fractions from human mononuclear cells

Utilizing a recently reported characteristic of monocytes to aggregate in cold, a new procedure for obtaining monocyte-rich and monocyte-depleted mononuclear fractions from human blood is described. After aggregation in cold and adherence to plastic, a fraction containing 88% monocyte (LeuM3 CD14+) is obtained. After treating the supernatant, which separates from the aggregates, with a monocyte lysosomotropic agent (L-Leucine Methyl Ester), a fraction containing 0% monocyte (LeuM3 CD14+) and 88% lymphocytes (Leu4 CD3+ and Leu 12 CD19+) is obtained. In round-bottom culture wells, this monocyte-depleted fraction produced immunoglobulins in response to pokeweed mitogen (5,826 +/- 2,356 IgM micrograms/ml), to a degree not significantly different that that produced in the presence of monocytes (7,426 +/- 3,347 IgM micrograms/ml). This suggests the presence of cells in the lymphocyte fraction, that are not monocytes but are capable of antigen presenting.     

Autologous mixed lymphocyte reaction in man. X. T-T autologous mixed lymphocyte reaction in young and aging humans

T cells upon activation with mitogens or autologous non T cells express surface HLA-DR antigens and are capable of stimulating autologous T cells in the autologous mixed lymphocyte reaction (T-T AMLR). We have examined T-TA AMLR, using T-non T AMLR activated-(TA) T cells as stimulators in young (21-32 yr) and aging humans (62-84 yr). In aging subjects a significantly (p less than 0 . 01) higher proliferative response was observed in T-TA AMLR as compared to simultaneously studied young subjects. In allogeneic MLR, no significant difference was observed between young and aging subjects. The increased T-TA AMLR could be a mechanism responsible for deficient T-non T AMLR reported in aging humans.     

Human monocyte-derived soluble product(s) has an accessory function in the generation of histamine- and concanavalin A-induced suppressor T cells.

We have analyzed the cellular interactions required for the generation of histamine- and concanavalin A (Con A)-induced suppressor T cells by employing a co-culture assay and techniques for fractionation of human blood mononuclear cells (PBMC). PBMC cultured in the presence of histamine (0.1 mM-1 mM) or Con A (20 micrograms/ml) for 24 h, mitomycin treated and subsequently combined with autologous mitogen-stimulated mononuclear cells, significantly suppressed a subsequent blastogenic response. PBMC fractionated over nylon wool columns and depleted of adherent cells and enriched for T cells (NWNA-T) were unable to generate suppressor activity. However, suppressor cell function by NWNA-T cells was reconstituted by the addition of autologous monocytes. In both the histamine and ConA suppressor systems, the requirement for monocytes in the activation process was enhanced by suspending the NWNA-T population in supernatants derived from allogeneic monocytes stimulated with heat-killed Staphylococcus albus. These crude supernatants contained leukocytic pyrogen (LP) and lymphocyte activating factor (LAF). Sequential purification and separation of the crude supernatants using gel-filtration, immunoadsorption, and isoelectric focusing demonstrated that only those fractions containing LP and LAF were capable to reconstituting NWNA-T cell histamine and Con A-induced suppressor activity. Thus, these studies suggest that the accessory role of supernatants derived from activated monocytes in the generation of suppressor cells may be mediated by LP/LAF. Further studies are in progress to explore the mechanism by which soluble factors stimulate suppressor T cells.     

HLA-DR human histocompatibility leukocyte antigens-restricted lymphocyte-monocyte interactions in the release from monocytes of acidic isoferritins that suppress hematopoietic progenitor cells

Acidic isoferritins, which under normal conditions are released from monocytes and macrophages, have a suppressive effect in vitro on granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cells. Cell interactions modulating the release of acidic isoferritin-inhibitory activity (AIFIA) from human monocytes were investigated using the bone marrow granulocyte-macrophage progenitor cells as a target cell assay for assessing AIFIA. Monocytes, in the absence of T lymphocytes, released AIFIA when allowed to condition culture medium at 10(4) or higher concentrations of monocytes/ml. However, subpopulations of T lymphocytes modulated the release of AIFIA from monocytes. OKT8+- and OKT4+-T lymphocytes were obtained from E-rosette-positive lymphocytes by using T lymphocyte subset-specific monoclonal antibodies in either a complement-dependent cytotoxicity test to select negatively for the cells or by selection using a "panning" procedure. OKT8+-T lymphocytes suppressed completely and OKT4+-T lymphocytes enhanced the constitutive release of AIFIA from monocytes. OKT4+ lymphocytes also induced the release of AIFIA from concentrations of 10(3) monocytes/ml which did not release measurable amounts of AIFIA by themselves. The release of AIFIA from monocytes involved HLA-DR+-monocytes and -T lymphocytes. Pulsing monocytes with monoclonal antibodies to framework determinants on HLA-DR molecules, in the absence of complement, did not influence the constitutive release of AIFIA. Pulsing monocytes or T lymphocyte subpopulations with such antibodies, in the absence of complement, blocked the suppressing and inducing activities of the appropriate subpopulations of T lymphocytes. Monoclonal antibodies to common determinants shared by HLA-A, B, and C molecules did not block these cellular interactions. Treating monocytes and T lymphocytes in a complement-dependent cytotoxicity test with dilutions of the anti-HLA-DR antibodies that did not block the cellular interactions removed the populations of monocytes constitutively releasing AIFIA and the T lymphocyte subsets modulating this release. Modulation of the release of AIFIA from monocytes by T lymphocyte subpopulations required the use of autologous cells, cells from HLA-identical siblings, or unrelated donors matched for HLA-DR. Matching for only one HLA haplotype gave partial responses and this was seen in testing cells from related individuals as well as among unrelated test combinations. These cellular interactions were not detected with HLA-DR-incompatible cells differing for two HLA-DR antigens. Admixture of such HLA-DR- incompatible allogeneic cells did not interfere with the regulation of AIFIA release in the autologous cell interactions. Thus, release of AIFIA from monocytes is restricted genetically by HLA-DR at the level of T lymphocyte-monocyte interactions. The genetic determinants on the HLA-class II molecules that induce stimulation in vitro in mixed lymphocyte culture (i.e., HLA-D), however, were not involved in this effort.     

Interaction of lactoferrin, monocytes, and T lymphocyte subsets in the regulation of steady-state granulopoiesis in vitro.

Colony-stimulating activities (CSA) are potent granulopoietic stimulators in vitro. Using clonogenic assay techniques, we analyzed the degree to which mononuclear phagocytes and T lymphocytes cooperate in the positive (production/release of CSA) and feedback (inhibition of CSA production/release) regulation of granulopoiesis. We measured the effect of lactoferrin (a putative feedback regulator of CSA production) on CSA provision in three separate assay systems wherein granulocyte colony growth of marrow cells from 22 normal volunteers was stimulated by (a) endogenous CSA-producing cells in the marrow cells suspension, (b) autologous peripheral blood leukocytes in feeder layers, and (c) medium conditioned by peripheral blood leukocytes. The CSA-producing cell populations in each assay were varied by using cell separation techniques and exposure of isolated T lymphocytes to methylprednisolone or to monoclonal antibodies to surface antigens and complement. We noted that net CSA production increased more than twofold when a small number of unstimulated T lymphocytes were added to monocyte cultures. Lactoferrin's inhibitory effect was also T lymphocyte dependent. The T lymphocytes that interact with monocytes and lactoferrin to inhibit CSA production are similar to those that augment CSA production because their activities are neither genetically restricted not glucocorticoid sensitive, and both populations express HLA-DR (Ia-like) and T3 antigens but not T4 or T8 antigens. These findings are consistent with results of our studies on the mechanism of lactoferrin's inhibitory effect with indicate that mononuclear phagocytes produce both CSA and soluble factors that stimulate T lymphocytes to produce CSA, and that lactoferrin does not suppress monocyte CSA production, but does completely suppress production or release by monocytes of those factors that stimulate T lymphocytes to produce CSA. We conclude that mononuclear phagocytes and a subset of T lymphocytes exhibit important complex interactions in the regulation of granulopoiesis.