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.     

Thrombocyte specific antigens. Detection of organ specific antigens by heterospecific anti-thrombocyte serum

Rabbit and chicken anti-human thrombocyte sera absorbed exhaustively with human plasma, fibrinogen, erythrocytes, leucocytes and liver powder, were used for the investigation of organ specific antigens of human thrombocytes.

The absorbed chicken anti-human thrombocyte serum revealed a very high agglutinating and fluorescence activity, which aided the characterization of two different types of organ-specific antigens of thrombocytes: antigens common to human and animal platelets, and antigens specific for human platelets only. The two types of antigens were found in all platelet fractions examined, the membrane, granule and soluble fractions.

     

Noncompetitive expansion of cytotoxic T lymphocytes specific for different antigens during bacterial infection

Listeria monocytogenes is an intracellular bacterium that elicits complex cytotoxic T-lymphocyte (CTL) responses in infected mice. The responses of CTL populations that differ in antigen specificity range in magnitude from large, dominant responses to small, subdominant responses. To test the hypothesis that dominant T-cell responses inhibit subdominant responses, we eliminated the two dominant epitopes of L. monocytogenes by anchor residue mutagenesis and measured the T-cell responses to the remaining subdominant epitopes. Surprisingly, the loss of dominant T-cell responses did not enhance subdominant responses. While mice immunized with bacteria lacking dominant epitopes developed L. monocytogenes-specific immunity, their ability to respond to dominant epitopes upon rechallenge with wild-type bacteria was markedly diminished. Recall responses in mice immunized with wild-type or epitope-deficient L. monocytogenes showed that antigen presentation during recall infection is sufficient for activating memory cells yet insufficient for optimal priming of naive T lymphocytes. Our findings suggest that T-cell priming to different epitopes during L. monocytogenes infection is not competitive. Rather, T-cell populations specific for different antigens but the same pathogen expand independently.     

Liver specific antigens: purification and characterization

Human liver has been homogenated and submitted to 150,000 g in order to investigate the supernatant for the presence of liver specific antigens. Gel-filtration on Sephadex G-100 revealed liver specific antigenicity in the first peak. Gel-filtration of the first peak on Sephadex G-200 revealed two peaks each exhibiting liver specific antigenicity. Immunochemical and physicochemical analysis made it possible to define the liver-specific antigen present in the first peak as a lipoprotein with the characteristics of a high molecular weight, low density protein. It is partly located in the membrane of liver cells. The liver specific protein of the second Sephadex G-200 peak has a molecular weight of about 190,000. It is located within the cytoplasm of hepatocytes.     

Leukaemia antigens: mixed leucocyte culture tests on twelve leukaemic patients with identical twins

Peripheral blood leucocytes from twelve leukaemic patients and their normal genetically identical twins were studied in one-way mixed leucocyte culture tests. In no case did normal twin cells stimulate the patient cells, nor did cells from nine patients with leukaemia (one acute lymphoblastic, four acute myeloblastic, two chronic myelogenous leukaemia in blast crisis and two lymphosarcoma-leukaemia) stimulate their normal twin cells. However, peripheral blood leucocytes from two patients with acute lymphoblastic and one with acute myeloblastic leukaemia significantly stimulated the lymphocytes of their normal twins. Cells from two of the latter three patients stimulated unrelated lymphocytes more than did cells from the corresponding normal twins. The results suggest the existence of new or modified antigens on leukaemia cells recognizable by cells from normal individuals who never had detectable leukaemia.     

Evidence that the low-incidence red cell antigens Rla and Lsa are identical

Testing of Ls(a+) and Rl(a+) red cells with numerous antisera containing antibodies to low-incidence antigens indicated that these antigens are identical. This conclusion was confirmed by adsorption and elution tests, and supported by immunoblotting of Ls(a+) and Rl(a+) cells with antibodies to glycophorin C and glycophorin D.     

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.     

Analysis of soluble antigens in guinea-pig epidermis. 3. The localization of the different tissue specific antigens in the epidermis

The distribution of five epidermis specific antigens in three fractions of guinea-pig epidermis (epidermal cells, intercellular substances and the superficial epidermis, or granular and horny layers) was investigated and compared with the antigens found in guinea-pig embryo and adult epidermis and in guinea-pig hairs. Four of the antigens were considered to be non-bacterial as they were demonstrated in embryo epidermis as well as in the adult tissue. These antigens appeared to be synthesized in the epidermal cells, after which they migrated to the superficial epidermal layers, where they were shown to accumulate and, in some cases, undergo fragmentation. Only one antigen was found in any quantity in the intercellular spaces, however although three of these antigens were found to be stable in 8 M urea, none of these could be demonstrated in guinea-pig hairs. The fifth antigen (Sp4), not found in the embryo tissue, could be of bacterial nature, as its presence, in small amounts, in the epidermal cells is probably due to contamination of this fraction by material from the superficial layers of the epidermis.     

Isolation of specific antigens fromAngiostrongylus cantonensis

The crude adult worm extract ofAngiostrongylus cantonensis was subjected to a series of affinity chromatography for selective removal of host antigens as well as cross-reactive components with other helminths. The purified fraction designated as AC(p) with molecular weights ranging from 10,000–42,000 was found to contain antigenic components specific toA. cantonensis as determined by immunoelectrophoresis and enzyme-linked immunosorbent assay.     

Detection of specific immunoglobulin M antibody to different flaviviruses by use of enzyme-labeled antigens.

An enzyme immunoassay was developed for the detection of human immunoglobulin M (IgM) antibody to different flavivirus antigens. The IgM antibody of human sera was selectively bound to anti-IgM antibody-coated solid-phase plates. Flavivirus IgM antibodies were then detected by use of various enzyme-labeled antigens. The flavivirus antigens (dengue type 2 virus, West Nile virus, and tick-borne encephalitis virus) were produced in suckling mice. The antigens were labeled with horseradish peroxidase by adding the activated enzyme at alkaline pH to sucrose-acetone-treated antigens. Addition of unlabeled mouse brain suspension of uninfected animals to the diluted enzyme-labeled antigens effectively reduced nonspecific binding to the solid phase. In patients with acute flavivirus infections, viral IgM antibody could be demonstrated with high sensitivity. Furthermore, the enzyme-labeled antigen-IgM test showed greater specificity than the hemagglutination inhibition test.     

Role of different B-cell subsets in the specific and polyclonal immune response to T-independent antigens type 2

Role of different B-cell subsets in the immune response to T-independent antigen type 2 (TI-2) was studied. BALB/c and C57BL/6 mice were immunized by polyvinylpyrrolidon (PVP), and the numbers of antibody- and Ig-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The number of cells producing non-specific Ig (nIFC) was calculated as the difference between the number of IFC and AFC; the number of nIFC induced by PVP was calculated as the difference between the number of nIFC in immune and control splenocytes. Immunization by PVP induced not only the AFC appearance, but also the increase in the number of the antigen-induced nIFC. The treatment of splenocytes by anti-CD5 antibodies and guinea pig complement reduced the increase in the numbers of newly formed AFC and nIFC to approximately 40% of control level. It means that CD5+ cells play an important role not only in the specific, but also in polyclonal immune response to non-self TI-2. To be sure that the decrease of AFC and nIFC numbers is due to depletion for CD5+ B-cells, but not CD5+ T-cells, splenocytes were separated to B-1 and B-2 subsets, and the numbers of AFC, IFC and nIFC were determined in each B-cell subpopulation separately. The overwhelming majority of newly formed AFC and nIFC was detected in B-1 subset. The numbers of AFC and nIFC in B-1 compartment was approximately 10-fold greater than in B-2 cells. A close parallelism between AFC and nIFC formation was observed. It is concluded that specific and polyclonal immune response to non-self TI-2-PVP-depends mainly on CD5+ B-1 subset.     

Virus specific antigens in mammalian cells infected with herpes simplex virus

Antisera to specific proteins in herpes simplex infected cells were produced by immunization of rabbits with infected rabbit kidney cells. These antisera were highly virus specific and produced up to twelve lines in immunodiffusion tests against infected cell extracts. Acrylamide electrophoresis and immunoelectrophoresis revealed up to ten virus specific proteins of varying size.     

Urinary excretion of placenta specific antigens in rats with normal and pathological pregnancy

Organ specific antisera were used to search for placental antigens in the urine of rats. The urine of male and virgin female rats was devoid of placental antigens. The antigens were detected in three of 150 urine specimens obtained from 30 presumably normal pregnant rats. When nephrotoxic serum or endotoxin were administered in order to intervene in the normal course of gestation, placental antigens were found in urine samples obtained during 30 of 40, and eight of 32 pregnancies, respectively. Demonstration of placenta specific antigens in the urine may be of aid in the diagnosis of pathological processes involving the placenta.     

Stage specific secreted and somatic antigens of Trichinella spiralis

Infective larvae, adult males and newborn larvae of Trichinella spiralis were cultured with [35S]methionine in vitro. Total secreted and total somatic (sodium deoxycholate-soluble) proteins were analyzed by electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Secreted proteins were relatively few in number and were different for each stage, whereas somatic proteins gave an unresolved smear in all cases. Immune precipitation with serum from infected mice revealed only one major antigen in secretions of all stages. In a similar investigation of the solubilised somatic antigens, the complexity of antigens ranged from none in infective larvae, through few in the adult, to many in the newborn larvae. The total concanavalin A-binding glycoproteins of each stage exhibited considerable individuality, and hence stage specificity, when resolved by two dimensional gel analysis. These results extend our knowledge of stage specific components of T. spiralis, and allow a rational approach towards the construction of diagnostic procedures.     

Intradermal skin test with diabetes specific antigens in patients with type 1 diabetes

Cell mediated immune response in vitro to a number of antigens has been reported in patients with Type 1 diabetes. The aim of the present study was to develop an in vivo intradermal (delayed type hypersensitivity) skin test using antigens known to be recognized by lymphocytes of patients with Type 1 diabetes and to compare, where possible, the in vivo response to the in vitro T cell proliferation to the same antigens. The skin test was performed in the following group of patients: 55 with recent onset Type 1 diabetes; 16 patients with Type 1 diabetes of longer duration; 10 patients with autoimmune thyroid disease and 20 patients with Latent Autoimmune Diabetes in Adults (LADA). Type 1 diabetes specific antigens for the skin test included glutamic acid decarboxilase (GAD65), insulin and beta casein, whereas diabetes non specific antigens included tetanus toxoid, diphteria, proteus, tubercolin, streptococcus, and glycerol as control. A multitest device consisting of heads delivering intradermally 10 microl of solution containing the antigens was applied to the forearms; the specific antigens were injected in one forearm whereas the non specific antigens were injected in the other forearm. Reading of the reaction, which was considered positive in the presence of a nodule of 2 mm diameter was performed 48 h after the multitest application. The in vitro T cell response to diabetes specific antigens used in the multitest was studied using conventional proliferation assays in patients with recent onset Type 1 diabetes and in age matched normal subjects. Only recent onset Type 1 diabetes patients showed an in vivo positive response to GAD65, such response being detectable in 10 patients (18%). Two patients reacted also to beta casein and insulin, all other patient groups resulted negative but 2 patients with longer duration of Type 1 diabetes. There was no apparent link between the in vivo skin test and in vitro T cell proliferation to GAD65. We conclude that in vivo cell mediated immune reaction to GAD65, insulin and beta casein can be visualized in a minority of patients with recent onset Type 1 diabetes. Further studies are required to determine specificity and whether altering the dose can improve the sensitivity of the test.     

A simple immunoelectrophoretic method for detecting specific antigens in mixtures of antigens

A fetal-specific antigen was detected in newborn serum by a modification of Mead's procedure of preparative immunoelectrophoresis.