Ig VH-dependent interaction between immunoglobulins and CD4.

CD4 interacts with the immunoglobulin (Ig)-VH domains by a virtue of its solvent-exposed C and C strands. These two strands also contribute to the full HIV-gp120 binding and participate significantly in binding to class II MHC molecules. In this paper we hypothesize that any high-affinity interaction between serum (or membrane-expressed) Ig and CD4 may have impact on early T cell activation events. The existing data provide evidence for different outcomes of a high affinity Ig/CD4 interaction on T cell proliferation and cytokine secretion: costimulation and inhibition. We will also discuss how a low affinity CD4/Ig interaction could play an important role in B cell stimulation initiated through surface Ig receptors, and how CD4 may be involved in shaping the B cell repertoire.

Human CD4-reactive antibodies from SLE patients induce reversible inhibition of polyclonal T lymphocyte proliferation.

We report on isolation of human polyclonal CD4-reactive antibodies of IgM and/or IgG isotypes from several SLE patients. These antibodies bound specifically to CD4-expressing cell lines and to rCD4 in ELISA and immunoblots. Saturation of CD4-binding sites occurred at antibody concentrations between 5 and 15 micrograms/ml. Anti-CD4 antibodies, in a dose-dependent manner, suppressed the proliferative responses of human peripheral blood mononuclear cells (PBMC) to superantigens (Staphylococcal enterotoxins A and B), anti-CD3 antibodies, and mitogens (PWM and Con A, but not PHA). They could also inhibit the proliferation of highly purified human T cells induced by immobilized anti-CD3 antibodies. To promote their effects on T cells, human anti-CD4 antibodies had to be present at lymphocyte cultures before or at the time of priming. There was no significant inhibition when antibodies were added more than 24 h following T cell activation. Substantial evidence that the immunosuppression induced by anti-CD4 antibodies was due to their direct effect on T cells was obtained. Down-regulatory effect of anti-CD4 antibodies could be significantly reversed by addition of exogenous IL-2 and by preincubation with soluble recombinant (r)CD4. Interestingly, at least one affinity-purified anti-CD4 antibody could costimulate the T cell proliferation induced by superantigens or anti-CD3 antibodies, especially when used at subsaturating concentrations (1-4 micrograms/ml) and when added subsequently to the initiation of cultures.

CD4-reactive antibodies in systemic lupus erythematosus.

An important place in the immune network is reserved for specific interactions between regulatory antibodies (Ab) and their ligands on T and B lymphocytes. Several lines of evidence indicate that the CD4 glycoprotein may be recognized by such Ab. High levels of CD4-reactive Ab occur in approximately 10-20% of HIV-infected patients. Moreover, between 20 and 30% SLE patients have Ab preferentially reactive with the CD4+ T cells. In relation to this, we have done studies aimed at demonstrating the existence and characteristics of Ab directly targeting CD4 in patients with SLE in comparison with rheumatoid arthritis and normal controls. Assessment of the CD4-reactive Ab by different approaches revealed a several-fold increase in serum concentration of anti-CD4 Ab restricted to a subset of SLE patients (n = 15/87, 17.2%). Enhanced binding was shown to occur specifically both on native CD4 (by immunofluorescence) and on recombinant CD4 (by ELISA and Western blot). Anti-CD4 Ab belonged to IgM and/or IgG isotypes. The overall binding of immunoglobulins to the CD4 molecule was not significantly contributed by DNA/anti-DNA and other circulating immune complexes, and there was no restriction in the usage of kappa and lambda light chains. Clinically, high CD4 reactivity occurred in SLE patients with active disease, as measured by the SLEDAI, and was associated with particular clinical manifestations, including neuropsychiatric disease and lymphopenia.

Human recombinant CD4 and CD4-derived synthetic peptides agglutinate immunoglobulin-coated latex particles. Evidence that residues 25-28 and 35-38 of human CD4 form two separate immunoglobulin binding sites.

It has been shown previously that amino acid residues 21-49 of the first extracellular domain of human CD4 form the core of an immunoglobulin (Ig) binding site. Synthetic peptides of human CD4 that encompass this region also bind Ig and, with higher affinity, antigen/antibody complexes. Synthetic peptides also enhance binding of both monomeric and aggregated Ig to monocytic U937 cells and Staphylococcus aureus Protein A. To better characterize the nature of the Ig binding site on CD4, we tested the ability of human recombinant CD4 (rCD4) to agglutinate polystyrene particles coated with Ig. Evidence is presented that soluble rCD4 and CD4 peptide p21-49 were capable of specific agglutination of polystyrene particles coated with polyclonal Ig of either human or sheep origin. Agglutination could be blocked by soluble human polyclonal IgG or F(ab')2 fragments. Both heparin and sulfated dextrans also inhibit agglutination, suggesting that charged residues on rCD4 played an important role in agglutination mediated by rCD4 or CD4 peptide. Similarly, aurintricarboxylic acid (ATA) also blocked agglutination of Ig-coated particles by rCD4. Agglutination mapping studies performed using truncated peptides revealed the existence of two discrete, closely related Ig binding sites (residues 25-28 and 35-38).

[Comparative value of skin tests and IgE antibodies in the serum in patients with chronic vasomotor rhinitis]. / Uporedne vrednosti koznog testiranja i IgE antitela u serumu obolelih od hronicnog vazomotornog rinitisa.

In order to assess values of skin testing and determination of pulp, as a credible indicator considering sensibilisation of an organism, 61 patients suffering from chronic vasomotor rhinitis were tested. By application of "prick" test, skin test results were positive in 30 patients and negative in 31. Presence of specific IgE antibodies in the serum was assessed in all patients by "ELISA" test. In 51 patients (83.6%) both kinds of findings corresponded. In 26 patients both skin parameters were negative while in 10 patients (16.40%) findings did not correspond. It was established that for Dermatophagodies pteronyssinus specific IgE antibodies are determined in serum when pap is 5 mm wide. Grasp pollen always caused strong local reactions, regularly more than 10 mm. Considerably high levels of IgE antibodies was also established while other pollens did not show such correspondence.

Idiotypes of pre-existing human anti-carcinoma anti-T and anti-Tn antibodies.

All humans normally possess antibodies, predominantly IgM, that react specifically with the Thomsen-Friedenreich (T) and the Tn antigens which are present in immunoreactive form on > 85% of all human carcinomas, but not in healthy and otherwise diseased tissues. We report here a serological study of idiotype expression and antigen reactivity of the anti-T and anti-Tn antibodies. Idiotypy was analyzed with rabbit antibodies raised against, and made specific for, affinity-purified polyclonal anti-T and anti-Tn antibodies from blood group A1B healthy adult donors. Anti-T and anti-Tn antibodies cross-reacted idiotypically in spite of their distinct epitope specificities. By adsorbing anti-T antibodies on insolubilized synthetic T carbohydrate we could firmly link idiotype expression with antigen reactivity. The relation of idiotype expression to the antigen-binding site of plant seed lectins was also studied; one originated from Arachis hypogaea [peanut agglutinin (PNA)], the other from Artocarpus integrifolia (Jacalin). PNA inhibited only anti-T antibodies. Jacalin inhibited both anti-T and anti-Tn antibodies in a dose-dependent manner. Neither idiotypic nor anti-idiotypic antibodies diminished the binding of lectins to T and Tn epitopes. The shared idiotypes on natural anti-T and anti-Tn antibodies permit consideration of application of their anti-idiotypes in treatment and/or prevention of human carcinoma.

[Immunologic characterization of autoreactivity to nuclear antigens in patients with systemic lupus erythematosus]. / Imunoloska karakterizacija autoreaktivnosti prema nuklearnim antigenima u bolesnika sa sistemskim eritemskim lupusom.

Abnormal immune reactivity, with a production of multiple autoantibodies specially against the components of a nucleoplasm is one of the hallmarks of systemic lupus erythematosus (SLE). Our investigations were conducted on 102 patients with SLE, classified according to the criteria of ARA, aiming to better characterize the overall incidence of anti-nuclear antibodies in SLE, to determine the type of immunofluorescent staining of the nuclei, and to characterize the fine specificity of such antibodies using modified ELISA procedure. Results of our investigation show that 95% of patients with SLE have detectable anti-nuclear antibodies. Predominant pattern of nuclear staining is homogeneous, followed by a speckled type, while the rim (peripheral) pattern is relatively infrequent. Anti-nuclear antibodies showed the highest reactivity against native DNA (70% of patients), which was followed by binding to SS-A, eRNP and SS-B antigens. Interestingly, using ELISA procedure we could observe the reactivity against Sm antigen only in 5% of SLE patients. In patients who showed homogeneous or rim pattern of nuclear staining the predominant type of reactivity was against native DNA, while in patients with speckled type most frequent binding to non-histone proteins was observed. The most frequently observed individual pattern of ANA reactivity was of polyreactive type.

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA.

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.