T cells specific for lipid antigens

Lipid-specific T cells are important participants in human immune responses. Recognition of lipid antigens contributes to host defense against pathogens that can cause debilitating diseases, including mycobacterial, viral, and parasitic infections. Lipid-specific T cells also play important roles in various autoimmune diseases, atherosclerosis, and in tumor surveillance. A better understanding of the mechanisms that regulate lipid-reactive T-cell functions will enable the development of novel therapies across a wide range of diseases. In recent years, our laboratory has investigated lipid antigen specificities, mechanisms of lipid antigen presentation, molecular interaction of lipid antigens with CD1 antigen-presenting molecules, and the pathogenic and regulatory functions of lipid-specific T cells in a variety of disease settings. In this review, we present recent data that illustrate the critical role played by lipid-specific immune responses in host protection, with a particular focus on human studies.     

B-cell antigens within normal and activated human T cells

In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed ('occult') CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules.     

Distribution of class IIDQ antigens on normal and leukemic lymphoid cells

The distribution of DQ as well as DR antigens was examined on lymphoid and monocytic cells at different stages of differentiation. In the B cell series, both immature and differentiating (under Staphylococcus aureus or pokeweed mitogen stimulation) B cells often lacked DQ molecules; among surface IgM+ and IgD+ cells, both DQ and DR molecules were detected except on cells from 30% of the lymphoid malignancies studied. T cells expressed DQ molecules only after stimulation; a DQ-DR+ phenotype was observed in a large number of cells after allogeneic stimulation, in certain antigen-specific T cell lines as well as in T cell lymphomas, suggesting that class II antigens had a distinct pattern of regulation. In the monocytic lineage, DQ molecules were expressed by most lymph node monocytes and only a low percentage (20%) of circulating monocytes.     

Identification and partial characterization of Trypanosoma cruzi antigens recognized by T cells and immune sera from patients with Chagas'' disease.

Trypanosoma cruzi antigenic specificities involved in human T-cell and antibody responses were compared in chronic chagasic patients affected with cardiomyopathy (C) or with the indeterminate form (I), the asymptomatic form of chronic Chagas' disease. T-cell Western blotting (immunoblotting) was performed to identify the most active antigens in epimastigote extracts (EPI-Ag). The patterns of peripheral blood mononuclear cell (PBMC) and T-cell proliferative responses induced by fractions blotted to nitrocellulose were heterogeneous, but the computation of their frequency distribution disclosed some important antigen specificities. Molecules ranging from 100 to 150 kilodaltons (kDa) were frequently stimulatory to PBMC from I patients (5 of 8 cases) and were less so when confronted with C patient (1 of 7 cases) lymphocytes. In contrast, both groups of patients actively responded to fractions ranging from 48 to 57 and 28 to 32 kilodaltons (kDa). The Western immunoblotting patterns of antibody reactivity displayed by 17 C and 15 I patients were also similar, yielding outstanding staining in the molecular mass ranges of 70 to 80 and 43 to 57 kDa. The latter antigen complex was recognized by 100% of the 32 chronic Chagas' disease serum specimens tested and closely corresponded to the migratory position recognized by T cells of most patients tested. The identification of the active molecules contained in the 43- to 57-kDa region was sought, with a focus on GP57/51, an antigen with well-established serodiagnostic properties. Immunoblotting analysis of EPI-Ag with a monoclonal antibody to GP57/51 confirmed its presence within the predicted molecular weight region. Highly purified GP51 was then used to demonstrate directly its capacity to promote specific PBMC proliferative responses in vitro. Data computed from a survey with 12 patients have shown a linear correlation (r = 0.93) between PBMC responses to EPI-Ag and to purified GP51, suggesting that the immune response to this particular glycoprotein may be an important component of human immune responses against T. cruzi.     

Identification and partial characterization of diagnostically relevant antigens of Aspergillus fumigatus

Metabolic antigens of Aspergillus fumigatus, soil strain 2605 and sputum isolate, were evaluated for their diagnostic applicability using hyperimmune sera and sera of adults and pediatric patients of allergic bronchopulmonary aspergillosis. An indirect ELISA was standardised by using 2-10 micrograms/ml of coating antigen for detection of specific IgG and IgE antibodies in the sera of patients. The ratios of absorbance for specific IgE and IgG antibodies by ELISA (normal to patients) were observed to be in the range of 1:2 to 1:3 to 1:8 respectively. These antigenic preparations were further analyzed to identify and characterize the individual components by immunoblotting. This analysis indicated the presence of allergenic and antigenic determinants in the antigens of molecular weights 70, 34, and 28 Kd. The utility of the antigens of soil strain for diagnostic purpose is suggested.     

HLA Class I antigens on normal and leukemic cells (quantitative analysis)

A monomorphic anti-Class I monoclonal antibody, ST01, found in our laboratory, was used to quantify Class I antigens on normal and leukemic cells, using a "CELISA" technique. Saturation graphs were used to compare the quantity of Class I antigens on normal PBL (25 cases) with that on the following types of leukemic cells: a) common acute lymphoblastic leukemias (cALL) (11 cases), b) mature B lymphocytic proliferations (16 cases), c) T hemopathies (9 cases), d) non-lymphoid leukemias (9 cases). In most cases the quantity of HLA Class I antigens was greatly reduced. No correlation was found between the quantitative expression of Class I antigens and the stage of maturation in each cell type, nor was any correlation found between the quantitative expression of Class I antigens on the leukemic cells and the proliferation of leukemic cells in the peripheral blood.     

T cells specific for different antigens express different HLA-D region products

T cell lines and clones were analyzed for surface expression of Ia antigens using monoclonal antibodies (mAb) that detect monomorphic and polymorphic epitopes on Ia molecules encoded by the HLA-DR and HLA-DQ gene clusters. All mAb bound to B lymphocytes or lymphoblastoid cell lines of the same individuals from whom the T cells were derived. Three mAb detecting monomorphic epitopes, primarily associated with HLA-DR, bound to all T cells showing that each clone or line expressed some type of Ia. Three other mAb defining polymorphic epitopes associated with HLA-DR products showed differential binding patterns. Two reagents, R3 and E15/4 recognizing the supertypic specificity DRw52 (formerly MT2), bound to every alloreactive clone, whereas the 16.23 mAb, detecting a private DR3-associated epitope, failed to bind to any clone. In contrast, the 16.23 epitope was detected on high percentages of T cells specific for purified protein derivative of tuberculin (PPD) or tetanus toxoid (TT). Biochemical studies showed that the 16.23 and DRw52-like epitopes can be present on distinct DR molecules on B cell lines and this may also be the case for T cells. Three other mAb, detecting epitopes associated with HLA-DQ, also revealed differential binding patterns when tested on various T cells. Two failed to bind to any alloreactive clone and to only low numbers of PPD- or TT-specific T cell lines, whereas the third bound distinctly to a CD4+/CD8+ alloreactive clone. Biochemical analyses have shown that these DQ epitopes can be present on different molecules. Combined, these observations indicate that differential expression of Ia molecules encoded by both HLA-DR and DQ occurs between B and activated T cells as well as among T cell populations of the same individual. Whether these differences reflect quantitative variations in expression of given DR or DQ molecules or, alternatively, are due to differential class II gene expression in activated T cells remains to be determined.     

Identification, isolation and partial characterization of Mycobacterium tuberculosis glycoprotein antigens.

In Mycobacterium tuberculosis culture filtrates, three concanavalin A (ConA)-binding bands of 55, 50 and 38 kilodaltons (kD) were identified by labelling blotted proteins with a ConA-peroxidase conjugate. Binding was inhibited by the competitor sugar alpha-methyl mannoside and by reduction with sodium m-periodate. Bands of 55, 50 and 38 kD stained with Coomasie blue were sensitive to digestion with proteases, thus indicating that they are proteins. Glycoproteins were isolated by lectin affinity chromatography or by elution from nitrocellulose membranes. On the isolated form, the 55-50 kD doublet glycoprotein was 65.4% protein and 34.6% sugar. The purified 38 kD molecule was 74.3% protein and 25.7% carbohydrate. By immunoblot, antibodies against mycobacterial glycoproteins were demonstrated in immunized rabbits and in patients with pulmonary tuberculosis, but not in healthy individuals. Treatment with sodium m-periodate abolished binding of rabbit antibodies to the 38 kD glycoprotein. Reactivity of the 55-50 kD doublet glycoprotein was not altered by reduction. By immunoblot with monoclonal antibodies TB71 and TB72, a carbohydrate-dependent and a carbohydrate-independent epitope could be identified on the 38 kD glycoprotein.     

Abnormal expression pattern of Tac and T9 antigens onin vitro activation of leukemic T cells

Mitogen-induced T-lymphocyte proliferation is dependent on the presence of both interleukin 2 (IL-2) and transferrin, even though resting lymphocytes do not have receptors for either. Recently, it has been reported that IL-2 stimulates T-lymphocyte proliferation via IL-2 receptor by induction of transferrin receptor on these cells. Using leukemic T cells as a model of the monoclonal mature T cell, we examined those sequential steps of T-cell activation. Our studies revealed that (i) transferrin receptors do not always appear after IL-2 receptor expression, (ii) IL-2 up-regulates the expression of IL-2 receptor but not of transferrin receptor, and (iii) IL-2 can initiate DNA synthesis without altering transferrin receptor expression. Thus, the sequential activation steps reported for normal T cells were not observed in the present study on leukemic T cells. These data suggest that there are other pathways to DNA synthesis in leukemic T cells and even in normal T cells.     

Interactions of T lymphocytes with human vascular endothelial cells: role of endothelial cells surface antigens

We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.     

Circulatory antigens of Heymann nephritis. I. Identification and partial characterization.

Previously, we have isolated and characterized a complex glycoprotein antigen (gp600) from the rat kidney that can induce Heymann nephritis (HN) in the rat. A monospecific antibody against the gp600 was used as a probe to document the existence of cross-reactive antigens in normal rat serum. A competitive radioimmunoassay measured the concentration in normal rat serum as being 45.5 +/- 10.2 micrograms/ml (n = 17). Molecular exclusion gel chromatography of normal rat serum identified gp600 activity in three distinct peaks corresponding to the molecular weights of 150,000, 110,000 and 70,000, respectively. Soluble immune complexes of mean molecular weight 1.1 X 10(6) were formed when normal rat serum was reacted with affinity-purified 125I anti-gp600. Normal rat serum, electrophoresed in 8% SDS-PAGE gels, transblotted to nitrocellulose membrane and reacted with anti-gp600 by indirect immunoperoxidase technique, identified three to four bands in the molecular weight region of 66,000-80,000. Isoelectric focusing revealed these antigens to be anionic (pI of 4.5-5.5) in nature. We conclude that normal rat serum contains antigens that cross-react with gp600. Further, these antigens are anionic in nature and form soluble immune complexes with anti-gp600 in vitro. The relevance of these findings to the pathogenesis of HN is discussed.     

Stimulation of humoral immunity to surface antigens of leukemic cells by interferon-containing serum

C57BL/6 mice were immunized by a single injection of L-1210 leukemia cells and (CBA×C57BL/6)F₁ mice were immunized by a single injection of leukemia L-1210 and P-388 leukemia cells. For 8 days (including the day of immunization) the animals received daily intraperitoneal injections of 0.4 ml of allogeneic or syngeneic interferon-containing serum, whereas control animals received the same dose of normal serum from intact mice (the interferon-containing serum was obtained from (CBA×C57BL/6)F₁ mice 24 h after intraperitoneal injection of 2 mg tilorone hydorchloride). Some samples of interferon-containing serum were dialyzed for 48 h against physiological saline. The serum interferon titer was 512–1500 units/ml. On the 9th–10th day after immunization the mouse sera were put through the microcytotoxic test against leukemia cells. Definite stimulation of the cytotoxic activity of sera of mice receiving the interferoncontaining serum was discovered. The syngeneic interferon-containing serum produced a stronger immunostimulant effect than the allogeneic serum.Laboratory of Immunology of Leukemia, Central Institute of Hematology and Blood Transfusion, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 3, pp. 358–360, March, 1976.     

Preparation, derivation and partial characterization of organ-specific antigens from human brain

Fractional ethanol precipitation was used to prepare a concentrate of brain-specific components of thermostable (BE) antigens derived from heated extract of human brain. Chromatography on DEAE-cellulose yielded a well-defined acidic protein peak containing brain-specific material corresponding to a minimum of two brain-specific components.

Preparation of a protein fraction (RSP) from unheated human brain ribonucleoprotein is described. Chromatography of RSP on DEAE-cellulose permitted the isolation of an acid protein fraction (AP) containing brain-specific antigens identical with those from heated brain extracts. AP contained a minimum of three distinct antigenic components which emerged as a single peak on rechromatography, and were incompletely separable by immunoelectrophoresis or analytical ultracentrifugation.

Pronase digestion liberated material with the general properties of a glycolipid; digestion products lost ability to precipitate with antiserum to the intact material.

Neuraminidase treatment resulted in the appearance of at least one previously masked determinant, presumably related to oligosaccharide residues since the new determinant was destroyed by periodate treatment.

Periodate did not affect ability of AP to form immunoprecipitin arcs unless AP was previously desialized. A chloroform-soluble fraction liberated during acid hydrolysis of AP has not been identified.

The possible chemical nature and intracellular association of AP are discussed.

     

Comparison of multiple assays for detecting human antibodies directed against surface antigens on normal and malignant human tissue culture cells.

Four separate assays of human antibody reactivity to four separate normal and malignant human tissue culture cell lines from two patients have been evaluated using a single highly-reactive allogeneic serum. The visual and end-point cytolysis assay and the 51Chromium release assay were equally sensitive in measuring complement mediated antibody cytoxicity and both were far more sensitive than a trypan blue dye exclusion assay. The assay of antibody reactivity by hemadsorption technique was about 10 times more sensitive than any of the cytotoxicity assays. This latter assay measures only IgG antibody however. These assays showed that cell lines from different patients may differ greatly in 'reactivity' to an allogeneic serum and emphasized the importance of utilizing tumor and normal cells from the same patient when using tissue culture cells to search for tumor specific reactivity. These observations emphasize the importance of utilizing multiple assays against paired normal and malignant cells from the same patient to be certain of the specificity and magnitude of the measured antibody.     

The demonstration of cell surface antigens on T cells, B cells and accessory cells in paraffin-embedded human tissues

This paper describes a method for processing fresh tissue that allows immunohistological analysis on paraffin sections. The method is based on the use of periodate-lysine-paraformaldehyde fixation. The effects of variation in fixation time, concentration of paraformaldehyde, dehydration, clearing, wax embedding and enzyme treatment of cut sections were examined. An optimal processing procedure was established that retains good tissue morphology and allowed 21 out of 27 monoclonal antibodies tested to be used successfully on paraffin sections to identify all major cell subpopulations by their membrane antigenic characteristics. The value of this approach in studying the immunopathology of potentially dangerous infectious diseases and in leukaemia/lymphoma diagnosis is discussed.     

Detection, separation and characterization of organ specific antigens of human thrombocytes

By means of rabbit anti-human thrombocyte antibodies, two organ specific antigens of human thrombocytes were detected. Both antigens were found to be specific for human thrombocytes and did not cross-react with thrombocyte antigens of other species. Separation and purification of the antigens were achieved by ion exchange chromatography and polyacrylamide gel electrophoresis. Production of monospecific antisera against each antigen separately was obtained by using purified antigen preparations for immunization. Physicochemical studies showed that both antigens are thrombin resistant, trypsin sensitive, and denatured at temperatures of 100° but not at 56°. One antigen is a glycoprotein with an α-globulin electrophoretic mobility and a molecular weight of approximately 1.07–1.17 × 10⁵, while the other is a protein with faster mobility in the post-albumin region and a molecular weight of 2, 8–3, 0 × 10⁴. Both antigens are present in soluble form in the cell sap as well as insoluble components in the cell membranes but only one antigen, the glycoprotein, could be solubilized from the platelet membranes by sodium dodecyl sulphate and pyridine treatments.