Suppression of immune response by lung cells in experimental tuberculosis

In vitro proliferative response of lung cells from mice infected with Mycobacterium tuberculosis H37Rv against PPD and Con A was studied. It was shown that the infected lung contained immune T cells, but their response in vitro was totally inhibited by plastic and nylon wool adherent suppressor cells. The whole population of lung cells from infected, but not intact mice, efficiently suppressed the proliferative response of immune lymph node cells against various antigens (non-specific suppression). The inhibition of response again depended on the presence of plastic adherent lung cells. Our data suggest that at least two suppressor pathways are induced in the course of tuberculosis infection: one being specific for mycobacterial antigens and other non-specific. Both types of suppressor pathways depend on the plastic adherent lung cells from tuberculosis lesion.     

Leishmania mexicana and Leishmania tropica major: Adoptive transfer of immunity in mice

The transfer of lymphocytes from NIH mice infected with Leishmania tropica major to nonimmune syngeneic mice, which had received 600 rad irradiation, markedly altered the course of subsequent Leishmania mexicana and L.t. major infections in these animals, when compared with infections in irradiated mice reconstituted with lymphocytes from uninfected animals. Some resistance to L. mexicana could be transferred with unfractionated lymphocytes, mixed nylon wool adherent, and nonadherent lymphocytes and to a lesser extent by nylon wool nonadherent cells alone but not by the adherent population. When the recipients of “immune” lymphocytes were infected with L.t. major the resulting lesions were either small and did not ulcerate, or grew rapidly, ulcerated, and healed. The course of L.t. major in the recipients depended on whether the donor of “immune” lymphocytes had a healed or a healing lesion. Immunity to L.t. major was largely associated with the nylon wool nonadherent population.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Suppression of responses to cryptococcal antigen in murine cryptococcosis

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.     

Cell types required for H-2-restricted cytotoxic responses generated by trinitrobenzene sulfonate-modified syngeneic cells or trinitrophenyl-conjugated proteins.

Murine spleen cells were fractionated over nylon wool or Sephadex G-10 columns, and the cell types involved in the generation of trinitrophenyl (TNP)-specific, H-2 restricted (TNP-self) cytotoxic effector cells were studied from cultures stimulated with trinitrobenzene sulfonate (TNBS)-modified syngeneic cells, TNP-conjugated soluble proteins such as bovine gamma-globulin (TNP-BGG), or bovine serum albumin (TNP-BSA). Unfractionated or nylon nonadherent responding cells generated such effectors, irrespective of whether the cultures were stimulated with TNBS-modified cells or TNP-conjugated proteins. TNP-modified T lymphocytes, B lymphocytes, and phagocyte-enriched spleen cells were all capable of stimulating TNP-self effectors. TNP-self effectors. TNP-self as well as allogeneic cytotoxic responses were dependent on the presence of a radioresistant non-T cell that was removed by Sephadex G-10 fractionation and was replaced by irradiated, Thy 1.2-negative, glass adherent spleen cells, enriched in phagocytic cells. Results obtained by using glass adherent cells that were allogeneic or semi-syngeneic to the responding cells indicated that H-2 homology was not required for efficient glass adherent cell function, and that the H-2 restriction of TNP-self effectors is not determined by these glass adherent cells.     

Immunocompetent cells in the starfish Asterias rubens. An ultrastructural study

Cells from the axial organ of the starfish Asterias rubens were fractionated into two populations, adherent and non-adherent to nylon wool. In both populations the ultrastructural study revealed the presence of cells resembling the lymphocytes of the vertebrates, as well as phagocytic, peroxidase positive cells. The lymphocyte-like cells in the non-adherent population (average diameter 4 mu) have a high nucleo-cytoplasmatic ratio and are morphologically similar to Th lymphocytes while the adherent cells (average diameter 5.5 mu) are more similar to Bm lymphocytes. These observations are in line with the hypothesis that there exist, in the starfish, a primitive immune system with characteristics resembling those of the immune system of vertebrates.     

Homologous antigen for T cell receptor in axial organ cells from the asterid Asterias rubens.

The axial organ (A.O. cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated into two populations: adherent (B-like) and non-adherent (T-like) to nylon wool. The adherent cells resemble mammalian B lymphocytes and bear homologous human T cell receptor (beta chain) to a higher degree than T-like cells which resemble T lymphocytes.     

The role of adherent cells in the immunosuppressed state of mouse progeny transplacentally exposed to benzo(α)pyrene

In recent studies, we showed that murine fetal liver cells from progeny exposed to benzo(α)pyrene in utero by intraperitoneal injection of the dam at midpregnancy (12 d) suppressed cell proliferation in an allogeneic mixed lymphocyte response. On the other hand, fetal liver cells from the corn oil (vehicle for the carcinogen)-exposed progeny (control) appeared to enhance proliferation. Suppression or enhancement appeared to be mediated by fetal liver bearing CD8⁺ and Lyt 1⁺ (CD5⁺) cells. Despite these manifestations, the role of third-party cells needs to be considered. As a first premise, adherent cells were targeted as possible third-party cells. To test the role of the adherent cells, liver cells from benzo(α)pyrene-exposed fetuses were treated with ficoll-hypaque, and the interface cells were fractionated through glass wool or nylon wool. It is known that adhering cells, macrophages and B cells, readily attach to glass or nylon wool. The effluent cells and the adherent cells were cultured with syngeneic responder cells and allogeneic stimulator cells in a mixed lymphocyte response. The results showed that benzo(α)pyrene-effluent cells led to the enhancement of proliferation in the mixed lymphocyte response, while benzo(α)pyrene-adherent cells led to suppression. The effluent and adherent cells of corn oil controls did not modify cell proliferation in the mixed lymphocyte response. These data suggest that a third-party cell, reasonably the macrophage or possibly B cells or both, since these are adherent cells, play a decided role in mediating suppression in the benzo(α)pyrene-exposed progeny.     

Adherent cells (macrophages?) in tumor-bearing mice suppress MLC responses

The spleens of mice bearing transplanted methylcholanthrene (MCA)-induced fibrosarcomas (MCA-1425 and MCA-1460) were shown to contain cells capable of suppressing the generation of cytolytic T lymphocytes (CTL) in mixed leukocyte cultures (MLC). The suppressive activity was first detected 21 days after tumor transplantation. No suppression was seen with lymph node cells taken at the same time as the spleen cells. The cells responsible for the suppressive activity were adherent to nylon wool and plastic dishes and they were not lysed by anti-T-cell serum plus complement. The suppressor cells were phagocytic and were resistant to irradiation (3000 rads) in vitro. Spleen cells from tumor-bearing nude mice were as suppressive as were spleen cells from tumor-bearing conventional mice. We conclude from these findings that T cells were not involved either as inducers or as effectors of the suppression observed, although the responsible adherent cells may have exerted their effect by interacting with a T-suppressor cell population in the MLC mixtures. While spleen cells of tumor-bearing mice were suppressive when added at any time during the first 4 days of a 5-day MLC, they showed no effect on the cytotoxicity of fully differentiated CTL. Indomethacin reversed suppression, suggesting that prostaglandins may have been involved.     

Immunoregulation in disseminated histoplasmosis: Characterization of splenic suppressor cell populations

Potent immunosuppressor cell activity was induced during the course of disseminated histoplasmosis in C3H/Anf mice. Spleen cells from infected mice severely suppressed the primary antibody response in vitro of normal syngeneic spleen cells to both a T-dependent antigen (sheep red blood cells) and a T-independent antigen (trinitrophenyl-lipopolysaccharide) at Weeks 1 and 3 of infection, respectively. Likewise, marked suppressor cell activity was present within lymph nodes. In a kinetic study, suppressor activity was detected first on Day 2 and increased to the maximum level on Day 4 after inoculation of Histoplasma capsulatum. Two populations of spleen cells express suppressor function in this model. One population, identified as T cells, was nonadherent to nylon wool columns; its suppressor capacity was abolished by anti-Thy 1 and reduced greatly by low-dosage X-irradiation (500 R). Cells of the second suppressor population had macrophage-like properties; although poorly adherent to plastic surfaces, they adhered to nylon wool columns and could be removed from spleen cell suspensions by carbonyl iron treatment; high-dosage X-irradiation (3000 R) and mitomycin C treatment failed to abrogate suppression by these cells.     

Stromal cells derived from spleen or bone marrow support the proliferation of rat natural killer cells in long-term culture.

Rat nylon wool nonadherent bone marrow cells were propagated for up to 75 days in co-culture with stromal cells derived from either spleen or bone marrow. Interleukin (IL) 1 enhanced the ability of spleen stroma to support the long-term culture of natural killer (NK) cells, ostensibly by inducing these support cells to synthesize other cytokines. Flow cytometry studies indicated that the nylon wool separation procedure enriched the concentrations of mature NK cells from 7.9% to 38.1% for splenocytes and from 3.8% to 19.5% for bone marrow cells. Analyses of the adherent zones of suspended nylon screen NK cell cultures revealed substantial numbers of large granular lymphocytes that expressed NK 323+/MOM/3F12/F2- phenotypes. The presence of both mature and immature cells of the NK lineage in this matrix was inferred by the presence of both IL-2 receptor (IL-2R) positive and IL-2R negative, and OX-8+ and OX-8- NK 323+ cells over the greater than 4-month experimental period. Suspended nylon screen cultures displayed a greater potential for producing cytolytic cells than either co-cultures of bone marrow nonadherent cells on stroma monolayers or suspension cultures. The large granular lymphocytes produced in suspended nylon screen cultures could be transformed into active killers of YAC-1 targets by IL-2. In contrast to bone marrow nonadherent cells, more splenic nylon-wool-passed cells displayed a mature NK phenotype, but their proliferative potential and ability to be transformed into cytolytic cells by IL-2 decreased rapidly in culture. In the suspended nylon screen culture system, NK cells migrate from the underlying stroma in stages as they mature, retain their cytolytic potential, and manifest a capacity for self-renewal. Cultured cells were routinely dissociated into single cell suspensions via enzyme treatment and were reinoculated onto "fresh" nylon screen/stromal cell templates after passage through nylon wool columns. These co-cultures continued to generate cytolytic cells in numbers greater than those of the initial inoculum.     

Surface morphology of immunocompetent cells isolated from spleen of Bufo himalayanus (Günther)

Immunocompetent cells were isolated from spleen of B. himalayanus and studied surface morphology of the three different cell types--(i) plastic adherent; (ii) nylon wool adherent; and (iii) nylon wool non-adherent cells. As revealed by scanning electron microscopy, they resembled the macrophages, B and T cells, respectively. Presence of such cell types indicated that Bufo himalayanus possessed a well-organized immune system. Further work is needed to characterize the functional efficacy of these immunocompetent cells found in B. himalayanus.     

CD4+ suppressor cells differentially affect the production of IFN-gamma by effector cells of experimental autoimmune encephalomyelitis

Spleen cells from rats that have recovered from experimental autoimmune encephalomyelitis (EAE) suppress the production of IFN-gamma by effector T cells of EAE in an Ag-specific manner. These postrecovery suppressor cells also inhibit EAE in vivo. Fractionation of the postrecovery suppressor spleen cells on nylon wool and OX-8 coated plates yields a nylon wool-adherent CD4+ suppressor cell population that, when cocultured with effector T cells, suppresses IFN-gamma production by these effector cells. In contrast, the nylon wool-adherent, CD4+ postrecovery suppressor cell population fails to inhibit the production of IL-2 by the effector T cells. In further experiments, the effector T cell population was depleted of CD8+ cells and cocultured with the nylon wool-adherent, CD4+ postrecovery suppressor cells, and the supernatants were assayed for IFN-gamma and IL-2. IFN-gamma production was inhibited in these cultures but IL-2 production was not inhibited. Irradiated effector T cells were cocultured with CD4+ postrecovery suppressor cells, without myelin basic protein, in an effort to determine whether the mechanism of differential lymphokine suppression involved an anti-idiotypic response against effector T cells. No IL-2 was produced, indicating that there was no CD4+ suppressor cell mediated anti-idiotypic response against effector T cells. These studies suggest that the suppressor cell is a nylon wool adherent, CD4+ T cell that functions to down-regulate EAE effector T cells by differential inhibition of lymphokine production.     

BCG-induced suppressor cells. I. Demonstration of a macrophage-like suppressor cell that inhibits cytotoxic T cell generation in vitro.

Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.     

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.     

Suppressive effects of monocytic cells and transforming growth factor-beta on natural killer cell differentiation in autoimmune viable motheaten mutant mice.

Viable motheaten (mev) mice are homozygous for a recessive single gene mutation at chromosome 6. These mice develop numerous inflammatory and arthritic syndromes and exhibit abnormal B cell functions as well as lower T and NK cell activity. In this study, the differentiation of NK cells in mev mice was examined to elucidate the underlying basis for decreased NK activity. Although NK cells appear to be present in mev mice, their activity was demonstrable only when the spleen cells were enriched by nylon wool passage. Similarly bone marrow cells from these mice could be shown to contain precursors of NK cells when they were passed over nylon wool and transplanted into irradiated recipients. The adherent cells from both the spleen and bone marrow of mev mice suppressed the differentiation of NK cells from normal splenic populations. These suppressive adherent cells were F4/80(+), AsGm-1(+), Qa-5(+), and NK-1.1(+). They were not cytolytic when cultured in IL-2. Antibodies to a number of cytokines, such as IFN-alpha, -beta, and gamma, or TNF-alpha, could not reverse the suppressive effect of the adherent cells. Addition of anti-TGF-beta antibody could, however, overcome the suppression, suggesting that TGF-beta was partly responsible for the defective NK differentiation in the mev mice.     

Suppressor cell activity in tumor-bearing mice. I. Dualistic inhibition by suppressor T lymphocytes and macrophages.

Spleen cells from mice bearing methylcholanthrene-induced fibrosarcomas impaired mitogen responses of normal syngeneic lymphocytes. Nylon wool column and other depletion techniques were utilized to characterize the cellular source of suppressive activity in tumor-bearing host (TBH) spleens. Evidence is presented for two distinct suppressor cell systems operating in the spleens, but not lymph nodes, of BALB/c mice bearing transplanted tumors. Spleens from TBH were shown to have greatly increased numbers of macrophages over their normal counterparts. TBH macrophages were observed to have suppressive activity at low in vitro concentrations. Anti-Thy 1 serum treatment of TBH macrophages abrogated low dose inhibition but not suppression due to high numbers of macrophages. No functional difference was detected between anti-Thy 1 serum-treated TBH and normal splenic macrophages. In a macrophage-depleted culture system, mildly nylon wool adherent, anti-Thy 1 serum, and hydrocortisone succinate-sensitive suppressor cells could be detected. Soluble supernatant products of TBH spleen and thymus cells were also found to inhibit in vitro mitogen responses, whereas TBH macrophages and lymph node cells demonstrated no soluble suppressive activity. The major source of soluble inhibitor of DNA synthesis (IDS) seems to be an anti-Thy 1 serum, hydrocortisone-sensitive population.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

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

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

In vitro effect of rabbit anti sea star lymphocyte serum on axial organ cells

The axial organ (AO-cells) of the sea star Asterias rubens is a primitive immune organ. The total population was fractionated or not into two populations: adherent (B-like) and non adherent (T-like) to nylon wool. Rabbit anti sea star lymphocyte serum induces the proliferation of axial organ cells. The T-like antiserum stimulates the T-like cells exclusively; the whole axial organ cell antiserum only stimulates the whole axial organ cell population.