Nonadherent Population Research Articles

Peripheral blood mononuclear cell-mediated cytolytic activity during cytomegalovirus (CMV) infection of guinea pigs.

Inbred Strain-2 guinea pigs exhibited endogenous peripheral blood mononuclear cell (PBMC)-mediated cytolytic activity against xenogeneic MA104 targets and guinea pig cytomegalovirus (gpCMV)-infected syngeneic and allogeneic targets. This endogenous cytolysis was unaffected by monoclonal T-cell antibody depletion but was diminished by removal of plastic adherent cells. In nonadherent effector populations, cytolysis was mediated predominately by large granular lymphocytes (LGL). During gpCMV infection, cytolysis of both target types was augmented (MA 104 for 3 weeks and gpCMV targets for 10-14 weeks). Augmented cytolysis of gpCMV targets was MHC-unrestricted and was diminished by removal of plastic adherent cells or monoclonal antibody depletion of T-cells, being found largely in LGL enriched populations. A role for this augmented activity in limiting gpCMV infection in inbred guinea pigs is suggested by the temporal association of augmented cytolysis of gpCMV targets with the cessation of viremia and clinical recovery.

The effect of age of cattle on the in vitro production on interferon by peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from normal cattle of different ages and from specific pathogen-free (SPF) calves, 2 to 4 weeks old, were cultured with bovine herpes virus type 1 (BHV1), parainfluenza-3 virus (PI3) and phytohaemagglutinin (PHA). The interferon (IFN) produced was characterized by acid stability and neutralizing antisera to recombinant bovine interferons. The virus preparations were presented either live or inactivated and as cell-bound virus or free virions. PBMC from cattle of all ages produced IFN-α when stimulated with live BHV1 and PI3 viruses. IFN-α was also produced with inactivated BHV1, even in cell cultures from SPF calves. However, inactivated PI3 virus failed to induce IFN in PBMC cultures from normal cattle, but approximately half of the animals, mostly calves, produced IFN-γ spontaneously in 48 h cultures in the absence of added antigen. PHA induced IFN-γ at an optimal concentration of 20 μg per ml after 3 days in culture. An age-related maturation of the IFN response was observed as PBMC from calves less than 2 weeks old produced little or no IFN when induced with either PHA or inactivated BHV1, although some IFN-α was produced in cultures containing live virus. Both adherent and non-adherent cells from adults and calves over 2 weeks old produced IFN on induction with inactivated BHV1 but only the non-adherent cell population produced IFN spontaneously or in response to inactivated PI3.

Effects of transforming growth factor-beta on human lymphokine-activated killer cell precursors. Autocrine inhibition of cellular proliferation and differentiation to immune killer cells.

With subpopulations of human lymphoid cells that were enriched for lymphokine-activated killer (LAK) cell precursors, studies were performed to examine the effects of transforming growth factor-beta (TGF-beta) on their IL-2-dependent growth and differentiation to killer cells. The majority of the LAK precursor cells appeared to reside in nonadherent, non-T, and non-B lymphocyte populations that expressed CD11 and CD16 Ag. These cells were induced to proliferate and become LAK cells by high concentrations of rIL-2 alone in the apparent absence of any prior activation with mitogen or Ag. The partially purified lymphocyte subpopulations generated varying but several-fold greater levels of LAK killing on a per cell basis than did unfractionated lymphocytes. The exogenous addition of TGF-beta to the LAK precursor cultures, markedly inhibited IL-2-stimulated growth as well as the development of LAK activity in a dose-dependent manner. The antimitotic effect of TGF-beta was reversible; inhibition of proliferation could be largely restored by increasing the concentration of IL-2 in culture. In contrast, TGF-beta inhibition of cytotoxicity was relatively independent of the concentration of IL-2. Further, LAK precursors constitutively expressed TGF-beta mRNA and high affinity receptor for TGF-beta. Activation of LAK precursors with IL-2 alone, resulted in a three- to fivefold up-regulation of intracellular TGF-beta mRNA and TGF-beta biologic activity secreted in the culture media. Furthermore, Northern blotting revealed that the resting LAK precursors did not express the Tac-mRNA. Receptor binding studies with 125I-IL-2 suggested the presence of a single class of IL-2R with an apparent Kd of intermediate range (beta-chain of IL-2R) on the unstimulated cells. Stimulation with high concentrations of Il-2 induced Tac-mRNA (both the 3.5- and 1.5-kb transcripts) and resulted in the expression of high affinity IL-2R (Kd approximately 10(-11) M) on these cells. Suppression of IL-2-dependent responses by TGF-beta was accompanied by a selective down-regulation of the 1.5-kb Tac-mRNA as well as by reduction in high affinity IL-2R. The results suggest a negative autocrine control of TGF-beta on IL-2-dependent growth and differentiation of human LAK cells, possibly related to regulate the killer activation function.

Emergence of a B lymphocyte population with ADCC effector function in mammary tumor bearing mice.

Differential expression of antibody dependent cellular cytotoxicity (ADCC) effectors was studied in normal Balb/cCrgI mice and those bearing a chemically induced 7, 12 dimethylbenzanthracene mammary adenocarcinoma. Depletion of macrophages from normal mouse splenocytes by Sephadex G-10 columns resulted in elimination of ADCC. Further separation of the normal G-10 nonadherent splenocytes on nylon wool columns did not result in any population with significant cytotoxicity. However, Balb/c mice bearing mammary tumors showed enhanced levels of ADCC which were not eliminated by macrophage removal. Lymphocytes from tumor bearers further separated on nylon wool yielded nonadherent and adherent populations both capable of effecting significant ADCC. Treatment of the nylon nonadherent cells of both normal and tumor bearing mice with anti-asialo GM1 (AGM1) and complement decreased the ADCC responses. The same treatment only marginally affected cytotoxic levels of nylon adherent cells from tumor bearers, indicating that these effectors are primarily of non-NK lineage. In addition, G-10 nonadherent, nylon adherent cells from tumor bearers separated on a fluorescence activated cell sorter based on the presence of surface immunoglobulins (slg) revealed that both the slg- and slg+ (98% pure) sorted cells were capable of functioning in ADCC. To determine whether in the tumor mice the 2% of slg- cells present in the slg+ sorted population were the ADCC effectors, mixing experiments were done in which up to 10% of slg- cells from tumor bearers were added to nylon adherent cells from normal mice. No significant increases in ADCC levels were found over that of normal mice. These experiments indicate that the 2% slg- cells were not the ADCC effectors nor were they inducing normal B cells to exert this type of cytotoxic reaction in vitro. To further substantiate the B cell lineage of the slg+ ADCC effectors, surface immunoglobulins were removed with protease treatment. After a 36 hr incubation, 92% of the cells had regenerated their slg. The results presented in this paper demonstrate that various splenic lymphoreticular populations from tumor bearers possess an enhanced cytolytic activity against antibody coated target cells. Among these is a unique nylon adherent slg+ cell that is capable of functioning as an ADCC effector.

Modulation of immunocompetent cell populations by benzo[ a]pyrene

These studies examined the cellular targets of benzo[ a]pyrene (BaP)-induced immunomodulation. The immunocompetence of murine mononuclear leukocytes was evaluated following in vivo exposure to BaP. In vitro cell separation and reconstitution techniques were used to evaluate T-dependent antibody (TDAb) production and delineate the immunomodulatory events. Reconstitution of cultures using combinations of adherent and nonadherent cell populations from BaP- and vehicle-treated mice demonstrated that both of these populations were sensitive to the immunomodulatory effects of BaP. Examination of the adherent cell population demonstrated that low numbers of BaP-exposed accessory cells enhanced in vitro TDAb responses. These responses were greater than those observed for analogous cultures containing vehicle-exposed accessory cells. Recombination of separated B cells and T cells from vehicle- or BaP-treated mice in cultures containing normal adherent cells demonstrated that both B cells and T cells were sensitive to the effects of BaP. Cultures containing BaP-exposed cell populations were incapable of supporting control level antibody responses. The results indicate that in vivo BaP exposure induces immunomodulation through effects on macrophages, B cells, and T cells.

Endotoxin-induced interferon-gamma production in culture cells derived from BCG-infected C3H/HeJ mice.

The cultured cells prepared from the spleens and peritoneal exudate cells of the C3H/HeJ strain of mice produce very little or no interferon (IFN) by stimulation of bacterial lipopolysaccharide (LPS). However, the cells taken from LPS-non-responder C3H/HeJ mice which had been infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) prior to the experiment were capable of producing IFN in culture in the presence of LPS. The peritoneal exudate cells of BCG-primed C3H/HeJ mice were separated into adherent cell and nonadherent cell populations by their adhesiveness to plastic culture dishes. IFN production required the presence of both these cell populations in the same culture, and the IFN activities produced were mainly IFN-gamma. The cultures with nonadherent cells and fixed adherent cells still produced IFN, but the cell cultures reconstituted with the BCG-primed cell population and unprimed cell population produce little if any IFN-gamma. Moreover, when both of the populations were cultured in Marbrook culture vessels separated by a membrane filter, the cultures produced very little or no IFN-gamma. These results indicate that there is a mechanism of IFN-gamma induction by LPS which requires the direct contact between adherent cells and nonadherent cells without the participation of any soluble factor(s) from the adherent cells. The producer cells were mainly in the nonadherent cell population. Previous treatment of nonadherent cells with anti-Thy-1.2 antibody, anti-Lyt-1.1 antibody, anti-L3T4 antibody, or anti-asialo-GM1 antibody and complement diminished the ability of the cells for LPS-induced IFN production with the help of adherent cells. Therefore, it is concluded that both T cells (presumably L3T4+T cells) and asialo-GM1+ natural killer cells in the BCG-primed C3H/HeJ cell cultures produced IFN-gamma in the presence of LPS, and the production was supported by the function of macrophages.

An investigation of the immune suppression induced by some saikosaponin derivatives

The relationship between lymphocyte activation by purified plant glycoside and the suppression of antibodies against sheep erythrocytes was investigated by an adoptive cell transfer system. Glycosides are one of the main components of Shôsaikotô — famous hematopoietic remedies of oriental medicine. In earlier reports, BALB/c mice treated with these drugs, suppressed the plaque-forming cell (PFC) response to T-dependent antigens. In contrast, the PFC response was enhanced for T-independent antigens. In this communication, an experiment was designed to determine the nature of the suppressive factor(s) produced in treated mice. Mice were orally or intraperitoneally administered 1 mg/kg of five types of purified SS derivatives every other day on five consecutive occasions. Spleen cells and serum were prepared from SS-treated mice and were then passively transferred to recipient mice. On the spleen cells (and not any of the sera) from drug-treated hosts effectively suppressed the PFC in recipients. The spleen cells of treated mice were further divided into adherent and non-adherent cells; each cell type was transferred into recipient animals. The observed suppressive was clearly detected in the non-adherent cell population. A subsequent analysis of the lymphocyte subpopulation revealed that Lyt.1.1 negative and Lyt.2.2 positive cells were responsible for this suppression. It was concluded that some SS derivatives elevate the activities of the major immunocyte population, with suppressor cell activity predominating.

Chemotactic effect of human recombinant interleukin 2 on mouse activated large granular lymphocytes.

Human recombinant interleukin 2 (hrIL-2) was demonstrated in vitro to be chemotactic for mouse large granular lymphocytes (LGL) activated in vivo by virus infection. Peritoneal exudate cells harvested from virus-infected mice were used as a source of LGL. LGL collected from mouse hepatitis virus-infected mice at 3 days postinfection were a source for NK 1.1 positive natural killer (NK)/LGL. LGL collected from mice treated with antiserum to gangliotetraosylceramide and infected with lymphocytic choriomeningitis virus for 7 days were used as a source for Lyt-2 positive cytotoxic T lymphocytes (CTL)/LGL. Both NK/LGL and CTL/LGL responded chemotactically to hrIL-2, purified IFN-beta, and to crude cell-free washout fluids collected from the peritoneal cavity of virus-infected mice. hrIL-2 had chemotactic activity for virus-elicited granular and agranular lymphocytes but did not attract the contaminating macrophages, in contrast to IFN-beta, which displayed chemotactic activity for virus-elicited granular and agranular lymphocytes as well as macrophages. The migration to hrIL-2 was inhibited by a monoclonal antibody (7D4) to the IL-2 receptor, but treatment with 7D4 did not affect migration in response to IFN-beta. Microscopic examination of Wright's-Giemsa-stained migrated NK/LGL and CTL/LGL revealed that the majority of migrated LGL in either LGL population had a blast cell morphology (enlarged cells with rich basophilic cytoplasm). The frequency of cells bearing the LGL morphology within the virus-elicited nonadherent peritoneal exudate cell population was on incubation in vitro, stabilized by either hrIL-2 or IFN-beta. These data suggest that another important immunomodulating function of IL-2 may be to attract activated NK/LGL and CTL/LGL to sites of inflammation.

Inhibition of macrophage accessory cell function in casein-treated B6C3F1 mice.

Humoral immunity of casein-treated B6C3F1 mice was evaluated. Splenocytes from casein-treated mice sensitized in vitro exhibited a marked suppression in their antibody response to the T-dependent antigen, SRBC (82%) and T-independent antigen, DNP-Ficoll (80%). In contrast, a control response to the polyclonal antigen, lipopolysaccharide (LPS), was observed. Cell fractionation and crossover reconstitution assays of adherent (ADH) and nonadherent (NAD) splenocyte populations from vehicle and casein-treated mice indicated that: 1) ADH splenocytes from casein-treated mice were responsible for suppression of humoral responses, 2) NAD splenocytes from casein-treated mice reconstituted with vehicle or naive ADH cells abrogated the immunosuppression, and 3) suppression of humoral responses in cultures containing casein ADH splenocytes was due to this cell populations inability to function as accessory cells in humoral responses rather than induction of suppressor macrophages. Results from in vivo studies with casein-treated mice sensitized with sheep red blood cells or dinitrophenyl-Ficoll paralleled the in vitro results.

Interleukin 3-dependent mouse mast cells express the cholera toxin-binding acidic glycosphingolipid, ganglioside GM1, and increase their histamine content in response to toxin.

The acidic glycosphingolipid, ganglioside GM1, which is the binding site for cholera toxin on many cell types, was identified by chemical and by flow cytometric analyses of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC). Ganglioside GM1 and other acidic glycosphingolipids were isolated from BMMC by chloroform/methanol extraction and chromatography on DEAE-Sephadex and were analyzed by thin layer chromatography. The presence of ganglioside GM1 in the BMMC extract was demonstrated by its co-migration with ganglioside GM1 standard in thin layer chromatography and by the binding of peroxidase-labeled cholera toxin B subunit to both molecules. As assessed by fluorescence flow cytometric analysis of the binding of fluorescein-conjugated cholera toxin B subunit, the majority of BMMC expressed ganglioside GM1 on their surface, and the total presentation per cell increased as cells progressed from the G1 to S to G2 + M phases of the cell cycle. The addition of increasing amounts of cholera toxin starting with 0.08 microgram/ml to BMMC cultured in 50% WEHI 3-conditioned medium containing IL 3 for 48 hr caused the adhesion of BMMC to the tissue culture flasks to increase in a dose-related manner, from less than 1% adherent cells in cultures without toxin to a plateau value of approximately 17% adherent in the presence of 1.25 micrograms/ml of toxin. The histamine content of BMMC increased from 26.7 +/- 3.59 ng/10(6) cells (mean +/- SD, n = 4) for control cultures to 201 +/- 17.4 ng/10(6) cells (mean +/- SD, n = 4) for nonadherent cells and to 588 +/- 89.4 ng/10(6) cells (mean +/- SD, n = 4) for adherent cells after 48 hr of culture in 0.31 microgram/ml cholera toxin, which was the optimal dose for nonadherent and adherent populations. The content of another preformed intragranular mediator, beta-hexosaminidase, did not increase appreciably in the presence of cholera toxin (n = 3). The increase in the histamine content of BMMC after the addition of 0.31 microgram/ml cholera toxin was detectable at 4 hr, plateaued by 24 to 48 hr, and gradually declined over the next 6 days. Cholera toxin also augmented the histamine content of BMMC in the presence of purified synthetic IL 3. Preincubation of whole cholera toxin with purified ganglioside GM1 inhibited the histamine-augmenting effects of cholera toxin on BMMC, indicating that the effect was not due to a contaminant, and neither the A nor B subunit of cholera toxin alone increased the histamine content of BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.

Dendritic cells in human peripheral blood: effective enrichment from the nonadherent cells

Dendritic cells (DC) from human peripheral blood were enriched using a method including adherence on plastic, depletion of phagocytic cells, flotation on density gradient column and panning on antibody-coated surfaces. The course of cell separation was evaluated by characterizing the morphological, antigenic and functional features of the different cell fractions. Using the population depleted of strongly adherent cells as the source, we were able to achieve a cell fraction containing 40–65% of DC (the main contaminants being monocytes and natural killer cells). Functionally these cells were highly stimulatory in autologous mixed leukocyte reaction. On the other hand, cells which primarily adhered well and were detached after overnight culture contained less than 5% of DC (the main contaminants being monocytes) after the same purification protocol. The calculated yields of DC were 1–2 × 10 6 and less than 0.5 × 10 6 from the nonadherent and adherent populations, respectively. Thus we concluded that the adhesiveness of DC from human blood is so weak that they can more efficiently and in a more reproducible way be enriched from the primarily nonadherent cell fraction.

T-cell surface molecules involved in the induction and expression of lymphokine-activated killing of autologous and allogeneic tumor targets.

The cellular interactions and the surface molecules involved in the generation and the expression of lymphokine-activated killer-cell (LAK) activities in vitro in blood mononuclear cells (BMN) from cancer patients and healthy individuals against autologous and allogeneic tumors were studied. The depletion of a plastic-adherent population(s) from BMN at the initiation of in vitro cultures in recombinant interleukin-2 (rIL-2) markedly interfered with the generation of LAK activities. Readdition of the same number of irradiated autologous plastic-adherent cells to the nonadherent population restored the generation of LAK. The requirement of the plastic-adherent population(s) in in vitro induction of LAK activities was observed only in autologous situations. Furthermore, selective modulations of CD3 and CD2 receptors on BMN with the appropriate monoclonal antibodies (MAb) during the induction phases of LAK responses profoundly inhibited the generation of LAK. Thus, unhindered expression of CD2 molecules and CD3 molecules were necessary for the maximum cytotoxic activation of non-antigen-driven effector cells in short-term cultures in rIL-2.

Investigations into the mechanism of immunosuppression caused by acute treatment with O,O,S-trimethyl phosphorothioate: Generation of suppressive macrophages from treated animals

At concentrations normally found in the spleen, macrophages from animals treated with O,O,S-trimethyl phosphorothioate (OOS-TMP) for 24 hr were previously shown to be immunosuppressive (Rodgers et al., 1985b). In addition, it was shown that macrophages from OOS-TMP-treated animals had a diminished capacity to present antigen (Rodgers et al., 1985c). In this report, it was shown that lowering the number of splenic adherent cells (95% macrophages by morphology) utilized in cell-mixing experiments to reconstitute the nonadherent splenic populations returned the humoral immune response to control levels. One day following acute administration of OOS-TMP, resident peritoneal cells were able to suppress the proliferation of P815 tumor cells. In addition, proliferative responses to concanavalin A and lipopolysaccharide were decreased at suboptimal concentrations of mitogen. Fresh supernatants from splenocytes cultured for 24 hr from OOS-TMP-treated animals blocked the generation of a humoral immune response. However, supernatants from splenocytes of control animals generated in the same manner did not block the generation of a humoral immune response. These data suggest that OOS-TMP induced the generation of suppressive macrophages which may potentially act through the release of labile factors which block proliferation or antigen- or mitogen-induced lymphocyte stimulation.

Glucocorticoid receptors in subpopulations of human lymphocytes defined by monoclonal antibodies

Glucocorticoid receptors (GR) were investigated in subpopulations of lymphocytes identified by monoclonal antibodies. Purified T (OKT 3+) and non-T lymphocyte subpopulations were isolated from human peripheral blood using Degalan bead columns coated with rabbit antihuman IgG. Purified subpopulations of OKT 4+ and OKT 8+ lymphocytes were obtained by coating the nonadherent population (T cells) from the first column with OKT 4+ or OKT 8+ and pouring it into a second Degalan column, coated with goat anti-mouse IgG. GR content and affinity were analyzed by a whole cell assay with [ 3H]dexamethasone as tracer. The numbers of GR in lymphocyte subpopulations (OKT 3+ cells, non-T cells, OKT 4+, and OKT 8+ cells) were nearly equal. It is concluded that the differential effects of glucocorticoids on the circulatory kinetics of OKT 4+ and OKT 8+ cells probably are not related to differences in glucocorticoid receptors of these T-cell subpopulations.

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis.

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.

Primary cellular target responsible for dimethylnitrosamine-induced immunosuppression in the mouse

The present studies were undertaken to identify the cellular targets responsible for immunosuppression by dimethylnitrosamine (DMN). The in vitro antibody responses of splenocytes from B6C3F1 mice exposed to 6 mg/kg DMN for 7 days to the T cell-independent antigen dinitrophenyl-Ficoll and the T cell-dependent antigen sheep erythrocytes were used for separation and reconstitution studies. The antibody-forming cell response of spleen cells from DMN-treated mice to the T-independent and T-dependent antigens was suppressed by 72% and 61%, respectively, when compared to vehicle controls. Whole spleen suspensions were fractionated into nonadherent populations by plastic adherence and Sephadex G-10 depletion of macrophages. Adherent antigen-presenting cells were obtained by incubating spleen suspensions in culture wells and removing nonadherent cells after 3 h. By combining vehicle and DMN nonadherent and adherent populations it was demonstrated that the population most affected by DMN exposure in both the sheep erythrocyte and dinitrophenyl-Ficoll responses was the nonadherent population. The lack of T cell dependence of the dinitrophenyl-Ficoll response was verified by elimination of T cells from whole spleen suspensions by monoclonal anti-thy 1.2 antibody plus complement treatment. These results indicated the B cell from DMN-treated mice as the splenic cell type responsible for suppressed antibody-forming cell responses to dinitrophenyl-Ficoll. B cells, T cells (prepared by cytotoxic elimination of B cells using anti-immunoglobulins) and adherent antigen-presenting cells (macrophages) from vehicle- and DMN-treated mice were fractionated and recombined and immunized with sheep erythrocytes (requires B cell, T cell, and macrophage cooperation). While splenic macrophages from DMN-treated mice supported control responses, T-helper activity was slightly reduced and B cell function was especially impaired. The conclusion that T cell function was less suppressed was supported by control responses of DMN-treated mice to concanavalin A but reduced responsiveness to lipopolysaccharide under conditions of limiting cell densities. These studies indicate that the primary cellular target of DMN exposure resulting in suppressed antibody responses is the B lymphocyte.

Cell-populations involved in the humoral leukocyte adherence inhibition reaction. The T8-suppressor/cytotoxic cells are both effectors and responders

In an attempt to identify the cell subpopulation involved in the humoral leukocyte adherence inhibition (H-LAI) reaction, mononuclear leukocytes were fractionated and the response with the different cell populations was measured in the H-LAI assay using sera from lung cancer patients and a lung cancer associated antigen. The monocytes were found to exhibit a suppressive effect on the H-LAI response. The B-lymphocytes seemed not to take an active part in the reaction. Depletion of the T-lymphocytes abolished the reaction. Subsequent studies on the T-lymphocyte subpopulations revealed that removal of the T 4-cells had little effect while removal of the T 8-cells resulted in complete abrogation of the H-LAI response. Direct blocking by monoclonal antibodies against the T 4 and T 8 surface determinants gave additional support for the T 8-cell dependency of the H-LAI assay. The cells that specifically were induced to loose adherence in the H-LAI assay upon incubation with serum from a lung cancer patient and lung cancer associated antigen were also analyzed. Compared to the total mononuclear leukocyte population, the relative content of monocytes and B-cells were lower in the non-adherent population. An increase was found in the relative number of T-cells. While the relative number of T 4-cells decreased, the content of T 8-cells was significantly increased. Furthermore, a direct correlation was found between the loss of adherence of the T 8-subpopulation and the index of the H-LAI response. The present study indicates the T 8-subpopulation of the T-lymphocytes to be involved both in the effector and responder side of the H-LAI reaction and gives implications for an immunological reaction behind the response of the H-LAI assay.

In Vitro Induction and Characterization of Mast Cells from Murine Peyer's Patches

The present study was carried out in vitro to determine whether murine Peyer's patches (PP) can be a source of mucosal mast cells, using a well-defined mast cell growth factor, interleukin 3 (IL-3). The non-T, non-B, non-adherent (null) cell population of PP contained the precursor cells for mast cells. The mast cells induced possessed toluidine blue and alcian blue (pH 3)-positive granules, which were heterogeneous and contained histamine. The IL-3-induced mast cells were IL-3-dependent, did not express Ia, T-cell, B-cell or macrophage markers, and released histamine in response to Ca ionophore, but not in response to compound 48/80. These cells bore IgE-specific surface receptors and intracellular 20 alpha hydroxysteroid dehydrogenase. The latter enzyme activity was induced by IL-3 in null cells. All these features of the mast cells support the view that these cells belong to a lineage of mucosal mast cells (MMC) and are no different from those of other lymphoid tissues, such as the bone marrow and spleen. In the analysis of MMC precursor frequency, PP had a similar frequency to that of other gut-associated lymphoid tissues (mesenteric lymph nodes and lamina propria), but was much lower when compared to those of the bone marrow and spleen. Furthermore, like IL-2, PP are capable of producing IL-3 in vitro by activating their helper marker-bearing T cells. Thus, under certain conditions, such as the initial participation by PP in MMC-associated intestinal immune responses, PP are likely to provide MMC in their microenvironment, like mucosal T and B cells.

Immunosuppression in experimental cryptococcosis in rats. Induction of afferent T suppressor cells to a non-related antigen.

To demonstrate the nature of the suppressor cells elicited in rats infected with Cryptococcus neoformans and immunized with human serum albumin (HSA), spleen mononuclear (SpM) cells were fractionated through a nylon wool column. The adherent and non-adherent populations were collected and transferred to syngeneic rats. In all cases, the non-adherent or T-enriched cells adoptively transferred suppression to HSA, however, the suppressive effects of the non-adherent cells were never as great as those of the unpassed population of SpM cells. The fractions adherent to nylon wool also diminished the delayed-type hypersensitivity response to HSA although this was not significant, but glass-adherent cells did exhibit significant suppressor activity. Immunized, non-infected rats were used as donor controls. Furthermore, we showed that the T-enriched-cells are sensitive to treatment with low doses of cyclophosphamide and that they bind HSA. These data indicate that immune suppression of the induction of the delayed-type hypersensitivity response to HSA in cryptococcosis can occur as a result of infection with C. neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells. Characterization of the non-adherent cells indicates that they are Ts1 cells.