Class II HLA-DR antigens on non-autoimmune human thyroid cells stimulate autologous T cells with high suppressor activity

Human autoimmune thyroid cells "spontaneously" express MHC-class II antigens. These antigens have been assumed to trigger thyroid-specific helper T cell clones, leading in turn to the expansion of thyroid autoantibody-secreting B cells. Thyroid cells derived from non-autoimmune subjects do not express MHC-class antigens, but these can be readily induced with gamma-interferon. We have addressed the issue of whether it is sufficient for normal thyroid cells to bear class II antigens in order to trigger autologous T cells. We found that non-autoimmune thyrocytes expressing DR antigens fail to stimulate autologous resting T cells. However, proliferative activity and interleukin-2 secretion were observed when fresh T cells were first triggered by autologous non-T cells and then incubated with thyrocytes. More CD8 than CD4 cells proliferated in the T:thyrocyte cultures, but CD4 cells were necessary for the proliferation and interleukin-2 secretion. Addition of antibodies to thyroglobulin or to DR antigens inhibited T cell proliferation and interleukin-2 secretion, thus pointing to T cell recognition of both thyroid-specific and DR antigens. Evaluation of the function of the thyroid stimulated T cells revealed very potent suppressor but negligible helper and cytotoxic activities. It would seem, therefore, that DR-restricted T cell activation by autologous antigen on non-autoimmune thyroid cells does occur, but since it results in enhanced suppression, its nature seems protective, thus leading to maintenance of immunological self-tolerance.     

Mouse thymic virus-mediated immunosuppression: association with decreased helper T cells and increased suppressor T cells

Mouse thymic virus (MTV) is a herpesvirus which, when administered to newborn mice, induces an extensive but temporary thymic necrosis associated with immunosuppression. In the present study, the T cell subsets in the thymus of MTV infected newborn C57Bl/6 mice were evaluated at 4, 7, 14, 28, 56, and 84 days after infection, using labeled monoclonal anti-CD4 and anti-CD8 antibodies with two-color flow cytometry. At 7 and 14 days, the percentages of CD4+8- and CD4+8+ cells were significantly decreased whereas the percentage of CD4-8+ cell was increased. At days 28 and 56 percentages had returned to normal. These results indicate that the virus has an affinity for CD4+ T cells (helper cells and their precursors). Increased percentage of CD4-8+ T cells (suppressor cells) is also associated with depressed immune functions in MTV infected newborn mice.     

Antigen-binding cells in human fetal liver.

Antigen-binding cells (ABC) could be detected regularly by autoradiography among haemic cells in the liver of human fetuses ranging in age from 8 to 24 weeks. For radioiodine-labeled thyroglobulin, the antigen mainly used in these studies, counts of ABC ranged from 5.0 to 24.3 per 1,000 cells scanned. There was a trend for counts of ABC in liver to be highest at 10-12 weeks of fetal life. Binding of labeled thyroglobulin was inhibited by excess unlabeled thyroglobulin, but not by other protein antigens. Artifacts due to binding of antigen to normoblasts, which comprised 90% of the haemic cells in fetal lines, and to cells with 'sticky' surfaces were excluded as far as possible. There was no response by fetal liver cells to phytohaemagglutinin. Although there was only minimal inhibition of binding by anti-immunoglobulin sera of known potency, the ABC in human fetal liver were assumed to correspond to immunoglobulin-bearing precursors of B cells described by others in the liver of the fetal mouse.     

Characterization of human helper and suppressor factors with special reference to HLA-DR determinants

Human helper and suppressor cells can be induced entirely in vitro, by culturing lymphocytes with a small or large dose of antigen for 4 days in a Marbrook—Diener system. The corresponding helper or suppressor factor (vitro) can then be released by a second pulse of antigen for 1 day. However, if sensitization has taken place in vivo by a naturally encountered antigen, then helper or suppressor factor (vivo/vitro) can be released by a single pulse of antigen for 1 day from putative in vivo-induced helper or suppressor cells. Helper factor_vitro was compared with helper factor_vivo/vitro, stimulated by a streptococcal protein antigen derived from Streptococcus mutans. The results of affinity chromatography suggest that both helper factors have an antigen-specific binding site and bind to monoclonal antibodies directed against HLA-DR, MT, β₂M and μ chain and a `constant' part on helper factor (HF) determinants. The HF<sub>vivo/vitro</sub> unlike HF<sub>vitro</sub> may reflect in vivo-sensitization and thus assay putative in vivo-sensitized antigen specific helper cells. Similarly, suppressor factor <sub>vivo/vitro</sub> might be a measure of in vivo-induced suppressor cells. Like HF, suppressor factor (SF) has an antigen-specific binding site, HLA-DR, MT and β<sub>2</sub>M determinants and a `constant' portion of SF. The DR determinants were further characterized by immunoadsorption, using monoclonal antibodies to the α- and β-chain of DR antigen. The results suggest that helper and suppressor factor_vivo/vitro express similar Ia determinants. Both expressed an α-chain determinant, recognized by one of the two anti-α-chain antibodies (TAL/1B5), and a β-chain non-polymorphic determinant recognized by one of the four anti-β chain antibodies used (DA6.231). However, both HF and SF derived from DRw6- but not from DR4-typed lymphocytes expressed MT1 (DRw6,1,2) or MT2 (DRw6,3,5,8) determinants. As DR and MT determinants can be expressed on the same molecule, it is possible that both are involved in helper and suppressor activities.     

HLA-DR locus and maternal-foetal relation

Antigen HLA-A, B sharing couples have been previously observed in abnormal pregnancies of unknown etiology. We have determined the HLA-A, B, C and DR antigens of 20 couples with recurrent spontaneous abortions (RSAs), 32 control couples and 100 normal controls. The results showed that in couples with more than two idiopathic repeated abortions, there is no significant increase in the HLA antigen sharing couples. But we were able to observe, in the affected couples, a significant increase in HLA-DR antigen sharing as regards the control couples. We also find a significant increase in the DR5 antigen, in both wives and husbands, in couples with repeated abortions of unknown etiology as previously described for the Dw5 antigen.     

Idiotype-induced T cell stimulation requires antigen presentation in association with HLA-DR molecules

An important and yet unresolved question concerns the mode of T cell recognition of idiotypic epitopes on immunoglobulin molecules in humans. Results from murine and human studies show that some idiotype-specific T cells recognize conformational epitopes on immunoglobulin, and such T cells are not MHC-restricted. In the present study T cell stimulation induced by idiotypic determinants on the autologous monoclonal IgG (M-components) from patients with monoclonal gammopathies was studied. In parallel, T cell stimulation in response to a conventional antigen, purified protein derivative, was also examined. It is shown that, as with conventional antigen, idiotype-induced T cell stimulation requires the presence of antigen-presenting cells (APC; monocytes and/or B cells), and is MHC class II (DR)-restricted. B cells, but not monocytes, can present idiotypic determinants to T cells at very low antigen concentrations, while monocytes do so only when antigen is present at high concentrations. Antigen processing and presentation is abrogated by treatment of APC with chloroquine. In conclusion, our study demonstrates that human idiotype-specific T cells recognize processed idiotypic determinants presented by MHC class II (HLA-DR) molecules on APC, and that B cells require about 1000-fold less antigen than monocytes.     

Aberrations of suppressor T cells in human graft-versus-host disease.

To determine whether imbalances in immunoregulatory T-cell subsets exist in patients with graft-versus-host disease, we analyzed T cells in three patients with acute and in six patients with chronic graft-versus-host disease after bone-marrow transplantation. The normal human peripheral-blood T-cell compartment is composed of 80 per cent TH2-and 20 per cent TH2+ T cells, and defined by reactivity with subset-specific heteroantiserums. Human suppressor cells are TH2+, whereas helper cells are TH2-. Patients with acute and chronic graft-versus-host disease had abnormalities in these populations, and their T cells frequently bore la-like antigens. Patients with acute disease lacked TH2+ cells, and the reappearance of this subset preceded the cessation of disease activity. Chronic disease, in contrast, was more heterogeneous. Suppressor cells were lacking in two patients but increased in the other four. Two of these four patients had TH2+, la+ T cells, suggesting in vivo activation of suppressor cells. Studies showing that these TH2+, la+ cells actively suppressed the in vitro immune response support this hypothesis and suggest that the immunoregulatory cells may profoundly affect the overall immune response.     

Antigen-binding B cells and polyreactive antibodies

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigenbinding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and β-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%–57%) by both homogeneous and heterogeneous antigens. Both CD5⁺ and CD5^? antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5⁺ antigen-binding (85%) as compared to the CD5^? antigen-binding (50%) population. Comparison of CD5⁺ B cells that bound antigens with CD5⁺ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.     

Thymoma and hypogammaglobulinaemia with and without T suppressor cells.

Patients with acquired hypogammaglobulinaemia usually have near normal numbers of B cells and normal T cell function. When hypogammaglobulinaemia occurs in association with thymoma, then B cell numbers have been reported as low, and distinctive T cells are present which inhibit immunoglobulin production by normal cells. It has been suggested that these T cells are responsible for the observed hypogammaglobulinaemia. We report a patient with thymoma and hypogammaglobulinaemia who lacks these distinctive suppressor cells and has normal B cell numbers. It is therefore incorrect to propose a single pathogenic mechanism for hypogammaglobulinaemia in association with thymoma.     

Linkage and association analyses of the osteoprotegerin gene locus with human osteoporosis

 Osteoprotegerin (OPG), a secreted glycoprotein and a member of the tumor necrosis factor receptor superfamily, is considered to play an important role in the regulation of bone resorption by modifying osteoclast differentiation. Overexpression of OPG in mice has been reported to result in osteopetrosis, whereas targeted disruption of OPG in mice has been associated with osteoporosis. Accordingly, OPG could be a strong candidate gene for susceptibility to human osteoporosis. Here, we analyzed whether OPG is involved in the etiology of osteoporosis using both linkage and association analyses. We recruited 164 sib pairs in Gunma prefecture, which is located in the central part of Honshu (mainland Japan), for a linkage study, and 394 postmenopausal women in Akita prefecture, which is in the northern part of Honshu, for an association study. We identified two microsatellite polymorphisms in the linkage study, and six single-nucleotide polymorphisms (SNPs) in the OPG region for the association study. Although, no evidence of significant linkage between OPG and osteoporosis was found, a possible association of one SNP, located in the promoter region of the gene, was identified. A haplotype analysis with the six SNPs revealed that four major haplotypes account for 71% of the alleles in the Japanese population. Received: March 22, 2002 / Accepted: April 23, 2002     

Low T lymphocyte responsiveness to Mycobacterium leprae antigens in association with HLA-DR3

The type of leprosy which develops after infection with Mycobacterium leprae is influenced by the presence or absence of HLA-DR3, as has been demonstrated in an ethnic group originating from Surinam. In the present study we investigated in this same ethnic group the role of HLA-DR, and of HLA-DR3 in particular, in monocyte-T cell interactions during leprosy specific proliferative responses in vitro. HLA-DR3 heterozygous T cells from tuberculoid leprosy patients were cultured with antigen and either HLA-DR3 positive or HLA-DR3 negative homozygous HLA-DR compatible allogeneic monocytes as antigen presenting cells (APCs). T cell proliferative responses with DR3 homozygous monocytes as APCs, were observed to be decreased as compared to T cell proliferative responses with DR3 negative homozygous monocytes as APCs. Furthermore, although the leprosy specific monocyte-T cell interactions were shown to be restricted for HLA-DR, in the anti-MLW-1 response HLA-DR3 appeared to function as a restricting element poorly or not at all. These observations may imply, that an in vitro correlate has been found for an (HLA associated) genetic control of leprosy in vivo.     

Antigen-specific suppressor cells and suppressor factors in human filariasis with Brugia malayi

We investigated the mechanisms of specific immune unresponsiveness to microfilarial antigens. The blood of patients with obvious Brugia malayi infections contains an adherent cell type that specifically suppresses reactions to microfilarial antigens but not to other antigens. In the absence of continued stimulation by parasite antigens, this suppressor cell loses its functional activity after overnight culture in vitro. Furthermore, serums from patients with and without microfilaremia contain factors that also suppress reactions to filarial antigens in vitro. These results suggest that immune unresponsiveness in human beings with patent filarial infections is due to active suppression of immune responses directed against the parasite and not to an intrinsic inability of infected patients to react to parasite antigens.     

Characterization of efferent T suppressor cells induced by Paracoccidioides brasiliensis-specific afferent T suppressor cells.

Previously, we reported that Paracoccidioides brasiliensis culture filtrate antigen (Pb.Ag) when injected i.v. into mice induces antigen-specific suppressor cells which down-regulate the anti-P. brasiliensis delayed-type hypersensitivity (DTH) response. The suppressor cells are present in both spleens and lymph nodes of Pb.Ag-treated animals and suppress the afferent limb but not the efferent limb of the DTH response to P. brasiliensis. The suppressor cells induced by Pb.Ag are L3T4+ Lyt-1+2- I-J+ T cells and are considered to be equivalent to the Ts1 cells described for other antigen-specific suppressor cell pathways. This report provides data which show that Ts1 cells induced by Pb.Ag or a soluble factor derived from Ts1 cells (TsF1) stimulates the production of second-order or efferent suppressor cells. The second-order suppressor cells are detectable in spleens and lymph nodes of mice 7 days after injection of Ts1 cells or TsF1 and are specific in suppressing the paracoccidioidal DTH response. In addition, the second-order suppressor cells are T cells with an L3T4- Lyt-2+ I-J+ phenotype and are effective in suppressing only the efferent limb of the P. brasiliensis DTH response. On the basis of the characteristics defined in this study, the paracoccidioidal second-order suppressor cells are equivalent to the Ts2 cells described for other antigen-specific suppressor-cell pathways. Thus, the suppressive circuit induced by Pb.Ag is similar to the suppressor-cell pathways that regulate the DTH responses to azobenzenearsonate, 4-hydroxy-3-nitrophenyl acetyl, lysozyme, and Cryptococcus neoformans antigen. We propose that such a suppressor-cell circuit as defined here with the murine model could be responsible for the depressed cell-mediated immune responses observed in paracoccidioidomycosis patients who have antigen circulating in their sera.     

Revisiting and revising suppressor T cells.

A great deal of experimental evidence supports the phenomenon of immunological suppression. The molecular mechanisms to explain the phenomenology have, however, remained controversial. In this review, the data are reinterpreted in light of the recent advances in the understanding of T-cell subsets, the cross-regulatory properties of lymphokines and the differential presentation capacities of different antigen-presenting cell types.     

HLA-DR positive cells in the human placenta.

A population of heterogeneous HLA-DR positive cells has been identified in the human placenta and decidua using immunochemical and histochemical methods. These cells are found in three areas: the subepithelial layer of the amnion, the decidua, and more sparsely within the chorionic villous stroma. In addition to HLA-A, -B and -C antigens, they also express the leucocyte-common antigen, indicating their origin from bone marrow precursors. The majority have a characteristic stellate shape with many cytoplasmic processes. In the villous stroma these stellate cells can be distinguished from the Hofbauer cells (placental macrophages) by their morphology, stronger expression of HLA-DR and lack of lysosomal enzyme activity. In the amnion and decidua they cannot be clearly distinguished from tissue macrophages. By using monoclonal antibodies specific for foetal or maternal HLA-A or -B allotypes, the HLA-DR positive cells in the chorionic villi and the amnion have been shown to be foetal in origin. In contrast, most of the HLA-DR positive cells in the decidua are maternal; a few adjacent to the basal plate are foetal. The preponderance of these cells in those areas of the placenta where foetal and maternal tissues are in close proximity is striking. The possibility that some of the cells are equivalent to the dendritic cells that have been described in other tissues is discussed.     

Separation of human suppressor and helper T cells by concanavalin A-coated sheep erythrocytes

Normal human peripheral blood mononuclear leukocytes (PBML) were activated by concanavalin A (Con A). Con A-activated and non-activated T cells were separated by E (AET) rosettes (2-aminoethylisothiouronium hydrobromide treated sheep erythrocyte rosettes). Purified T cells were rosetted with Con A-coated sheep red blood cells (Con A-SRBC) at 37 degrees C resulting in Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted T lymphocytes in the T lymphocytes from Con A-activated and non-activated PBML were 44.4 +/- 5.4 percent and 16.0 +/- 7.5 percent (Mean +/- S.D.) while the Con A-SRBC non-rosetted T lymphocytes were 55.6 +/- 5.4 percent and 84.0 +/- 7.5 percent respectively. The Con A-SRBC rosetted and non-rosetted T cells were separated by Ficoll-Hypaque gradient centrifugation. Functional studies of Con A-SRBC rosetted and non-rosetted T cells were performed by in vitro tests using pre-amplified reverse hemolytic plaque assay for measuring numbers of immunoglobulin G (IgG) secreting cells and ELISA quantitation of IgG concentration. Both techniques were used to assess the suppressor and helper functions of the Con A-SRBC rosetted and non-rosetted T cells. The Con A-SRBC rosetted cells obtained from T cells of Con A-activated PBML showed strong suppressor activities to normal PBML in both pre-amplified reverse hemolytic plaque assay and sandwidh ELISA of IgG concentration, while the Con A-SRBC non-rosetted T cells demonstrated strong helper activities to normal PBML in both assay systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ identification of T lymphocyte subsets and HLA-DR expressing cells in the human skin tuberculin reaction

T lymphocyte subsets and HLA-DR expressing cells were studied with an immunohistochemical double staining technique in frozen sections of human skin 6, 48 and 96 hr after intradermal PPD injections. The number of lymphocytes reacting with monoclonal Leu 1 antibodies (all mature peripheral T cells) increased with time. The majority of the T lymphocytes at 48 and 96 hr reacted with Leu 3a ('helper/inducer' phenotype) antibodies and a few with Leu 2a ('suppressor/cytotoxic' phenotype) antibodies. Apposition of T lymphocytes of both subsets to HLA-DR expressing cells occurred in the dermis as well as in the epidermis. The study gives a morphological picture of the cell-mediated immune reactions in delayed type of hypersensitivity consistent with in vitro experiments on proliferative responses to soluble antigens.