anti-H1 Antibodies Research Articles

Molecular Interactions, T‐Cell Subsets and a Role of the CD4/CD8:p56lck Complex in Human T‐Cell Activation

Several T-cell structures are capable of generating intracellular signals linked to T-cell proliferation. Crosslinking of CD2, CD4 and CD45 with Ti/CD3 to several of these antigens can augment the minimal signal induced by antigen binding to the Ti/CD3 complex. Importantly, some of these regulatory structures (CD4, CD8 and CD45) are also expressed on subsets of T cells with distinct activation requirements and functional programs (helper, suppressor, suppressor-inducer and cytotoxic function). The CD4+ CD45RA+ (2H4+) subset responds well to self-Ia, poorly to soluble antigen and possesses suppressor-inducer function. A reciprocal subset CD4+ CD45RA- (4B4+) is preferentially activated by soluble recall antigens and possesses helper function. Each of these subsets can be distinguished by virtue of the differential expression of CD45 antigens. Importantly, the anti-2H4 antibody which reacts with a specific region near the N-terminus of two CD45 isoforms can effectively block its function. Crosslinking of CD4 with the Ti/CD3 complex preferentially activated the CD4+ CD45+ RA- subset, while soluble antibodies to CD2 preferentially affected the CD45 CD45RA+ subset. Thus, CD3 and CD4 more effectively synergize in the activation process on the CD4+ CD45RA- subset, a result consistent with the ability of this subpopulation to respond to recall antigens. The regulatory role of the CD4, CD8 and CD45 antigens may be mediated by an interactive network of protein-tyrosine phosphorylation and dephosphorylation. We have shown the CD4 and CD8 antigens to be associated with the T cell-specific protein-tyrosine kinase (p56lck). p56lck is a member of a family of protein-tyrosine kinases with an established ability to activate and transform mammalian cells. The CD4/CD8:p56lck complex is catalytically active as shown by its ability to phosphorylate various members of the Ti/CD3 complex. By contrast, the CD45 antigens possess protein-tyrosine phosphatase activity within their intracellular domains and are postulated to function by virtue of a regulatory interaction with CD4/CD8:p56lck and its potential substrates. Thus, the differences in the response of the CD4+ CD45RA+/- subsets to various stimuli and the expansion of T-cell subsets with distinct immunoregulatory programs may be governed by a pathway of tyrosine-mediated events.

Histone H1 in the centromeric heterochromatin of Glyptotendipes barbipes larval polytene chromosomes

Immunofluorescent analysis with antibodies against histone H1 failed to detect H1 in the centromeric heterochromatin blocks of the polytene chromosomes of Glyptotendipes barbipes larvae. Centromeric regions were dissected microsurgically and acid-extracted. Electrophoresis in SDS and acid-urea gels revealed a band comigrating with H1 of calf thymus and of Gl. barbipes salivary gland nuclei. ELISA dot assay of the extracted material gave a positive reaction with anti-H1 monoclonal antibodies and with anti-H1 affinity-purified polyclonal antibodies. This shows that the centromeric heterochromatin contains histone H1 but packed in a way which prevents the H1 antigenic determinants from reacting in situ with the specific antibodies.

Amplification of suppressor inducer pathway with monoclonal antibody, anti-2H4, identifying a novel epitope of the common leukocyte antigen/T200 antigen

The 2H4 antigen, comprised of a 200 220 -kDa glycoprotein of the leukocyte common antigen (LCA) family, is expressed on a suppressor inducer, but not a helper inducer subset of T4 cells. Earlier studies have demonstrated that the T4+2H4+ subset of cells maximally responded to the AMLR and this molecule had an important role in generated suppressor signals in AMLR/ Con A-activated T cell systems. In the present study, we examined the effect of a series of monoclonal antibodies including anti-2H4 antibody on the initial activation of T4 cells in response to self-Ia antigens. We found that the addition of anti-2H4 antibody resulted in an augmentation of the proliferative response of T4 cells in AMLR, whereas other antibodies reactive with LCA/ T200 antigens lacked this ability. Furthermore, anti-2H4 antibody enhanced both IL-2 production and IL-2R expression in this AMLR system. This enhancing effect was inhibited by anti-T3 antibody. Moreover, the suppressor inducer function of AMLR T4 cells was enhanced with anti-2H4 antibody by increasing the number of 2H4+ cells with high antigen density. Taken together, these results suggest that the 2H4 antigen may serve as an accessory structure for enhancing the activation of the T4+2H4+ suppressor inducer subset at initiation of cell triggering.

Role of the 2H4 molecule in the activation of suppressor inducer function.

The monoclonal antibody anti-2H4 recognizes a 220-kDa and 200-kDa glycoprotein and subdivides T4+ cells into distinct subpopulations: the T4+2H4+ inducer of suppression and the T4+2H4- inducer of help. The T4+2H4+ subset has been shown to play a crucial role in the activation of T8+ suppressor cells. In the present study, we attempted to determine whether the 2H4 molecule itself may be involved in initial triggering of suppressor inducer function. The results show that the addition of anti-2H4 antibody to a mixture of B and T cells resulted in a marked suppression of pokeweed mitogen-driven Ig synthesis. When anti-2H4 was added to B cell cultures containing T4+2H4+ or T4+2H4- cells but lacking T8 cells, no suppression was generated. In addition to the requirement for T8 cells, no suppression was generated if T4+2H4+ cells were absent. These results suggest that the anti-2H4 antibody contributes to the activation of the T4+2H4+ lymphocyte subset, which in turn induces T8 cells to suppress B cell Ig synthesis. Biochemical analysis of the T4+2H4+ subset of lymphocytes indicated that the in vitro addition of anti-2H4 antibody resulted in an increased expression of the 220-kDa and 200-kDa structure on T4 cells. We conclude that perturbation of the 2H4 molecule may potentiate the activation of the suppressor inducer subset and that the T200 molecule may be directly involved in suppressor inducer function.

The H1 and H2 polypeptides associate to form the asialoglycoprotein receptor in human hepatoma cells.

Antibody-induced degradation and chemical cross-linking experiments have been carried out to assess the nature of the interaction between the two asialoglycoprotein-receptor polypeptides, H1 and H2, synthesized in HepG2 cells. Incubation of HepG2 cell monolayers with anti-H1 antibody caused a specific and equal loss of both H1 and H2 polypeptides. The same result was obtained with anti-H2 antibody. Control serum did not affect the level of H1 or H2 not did anti-H1 or anti-H2 antibodies affect the level of the transferrin receptor. The chemical cross-linking reagent, difluorodinitrobenzene, has been used to demonstrate that H1 can be cross-linked to H2 in HepG2 cell microsomal membranes. Dimer and trimer species with apparent molecular masses of 93 and 148 kD, respectively, were readily observed upon chemical cross-linking and some dimers and trimers were immunoreactive with both anti-H1 and anti-H2 antibodies. The putative trimer, possibly two H1 and one H2 molecules, is a minimum estimate of the true size of the asialoglycoprotein receptor in intact HepG2 cell, and it is possible that larger hetero-oligomeric forms of the receptor exist. The results of both types of experiments indicate that H1 and H2 form an oligomeric complex in HepG2 cells and thus, both polypeptides constitute the human asialoglycoprotein receptor.

Identification of the sequence required for expression of the 2H4 epitope on the human leukocyte common antigens.

The anti-2H4 antibody, which subdivides the T4+ population of human T lymphocytes into T4+, 2H4+ suppressor-inducer cells and T4+, 2H4- helper cells, recognizes an epitope on a subset of the human leukocyte common antigens (LCAs). LCAs are a family of cell surface glycoproteins generated from a single gene by the differential usage of three exons near the NH2-terminus. Using cDNA clones corresponding to four of the different forms of LCA molecules, extracellular domains of the LCA molecules were synthesized in vitro. Immunoprecipitation of these molecules with the anti-2H4 antibody demonstrated that exon A is required for the expression of the 2H4 epitope.

A defect of immunoregulatory T cell subsets in systemic lupus erythematosus patients demonstrated with anti-2H4 antibody.

The cell surface phenotype of peripheral blood lymphocytes (PBL) of systemic lupus erythematosus (SLE) patients was characterized with the anti-2H4 monoclonal antibody that defines the human suppressor inducer subset. The T4+2H4+ population of cells has been shown to be critical for the activation of T8+ suppressor cells. Patients with SLE has a markedly decreased percentage of T4+2H4+ cells (13 +/- 2%) in their PBL compared with normal controls (21 +/- 1%) (P less than 0.001). This reduction was greatest in patients with active SLE, especially those with renal disease. Serial analysis of patients with SLE and renal disease showed a correlation between percent positive circulating T4+2H4+ cells and disease activity. Moreover, there was a significant correlation between a low percentage of T4+2H4+ cells and decreased suppressor-inducer function in autologous mixed lymphocyte reaction-activated T4+ cells from SLE patients. Thus, a deficiency exists in SLE patients with active renal disease in the T4+2H4+ suppressor-inducer T cell subset.

Selective loss of the suppressor-inducer T-cell subset in progressive multiple sclerosis. Analysis with anti-2H4 monoclonal antibody.

The T4+ lymphocyte population includes a subset that induces suppressor T lymphocytes (T8+ cells) and can be distinguished by dual-color fluorescence analysis with anti-2H4 and anti-T4 monoclonal antibodies. To investigate the possible role of these cells in multiple sclerosis, we used anti-2H4 antibody to characterize peripheral-blood lymphocyte subsets in 63 patients with multiple sclerosis that was progressive, stable, or acute (relapsing-remitting). Twenty-three of 37 patients with progressive multiple sclerosis had a selective decrease in the number and percentage of peripheral-blood T cells that induce suppressor cells (T4+2H4+ cells), whereas only 3 of 16 patients with stable disease and 2 of 10 patients in the midst of an acute attack had a significant decrease. These selective decreases of circulating T4+2H4+ cells occurred in only 1 of 34 patient controls with other neurologic diseases and in 2 of 50 healthy controls (P less than 0.0001 by Fisher's exact test). The absolute number of T4+2H4+ cells and the percentage of reactivity in the populations studied were 187 +/- 28 per cubic millimeter and 8.3 +/- 1 percent in patients with progressive multiple sclerosis; 353 +/- 60 per cubic millimeter and 14.5 +/- 2 percent in patients with stable disease; 368 +/- 72 and 14.6 +/- 2.1 percent in patients with acute disease; 402 +/- 64 and 15.6 +/- 2 percent in controls with other neurologic diseases; and 519 +/- 44 and 19.7 +/- 1 percent in healthy controls. Functional studies using a pokeweed mitogen-driven IgG assay demonstrated a correlation between decreased numbers of T4+2H4+ cells and increased production of IgG in vitro. Family studies showed that the 2H4 antigen was not part of an inherited polymorphic antigenic determinant. Our results suggest that in progressive multiple sclerosis decreases in inducers of suppressor T cells may permit the activation of cells reactive with elements of the central nervous system.

Induction of suppression following autologous mixed lymphocyte reaction; role of a novel 2H4 antigen.

In this study, we have investigated the cellular and molecular basis for immunoregulatory function of T4+ cells after autologous mixed lymphocyte reaction (AMLR) activation. We demonstrated that the T4+ 2H4+ subset but not the T4+2H4- subset can proliferate maximally in response to autologous non-T cells. T4+ cells activated by AMLR exerted suppressor activity on pokeweed mitogen-driven IgG synthesis of autologous peripheral blood lymphocytes. The suppressor activity by AMLR-activated T4+ cells required the presence of fresh T8+ cells in the secondary culture, indicating that AMLR-activated T4+ cells functioned as a suppressor inducer rather than as a suppressor effector population. Following activation of T4+ cells in AMLR, it is the T4+2H4+ subset which induces suppression through the T8 population. Moreover, the treatment of AMLR-activated T4+2H4+ cells with anti-2H4 antibody, but not other antibodies, resulted in the abolishment of suppressor inducer function of such cells, suggesting that the 2H4 molecule itself may be involved in the suppressor inducer function. The 2H4 antigen on such cells was shown to be comprised of 220-kDa and 200-kDa glycoproteins. These results support the notion that the AMLR may play an important role in generating suppressor inducer signals and in down-regulating the immune response following self major histocompatibility complex recognition. More importantly, the present studies indicate that the 2H4 antigen on T4 cells serves not only as a phenotypic marker of suppressor inducer cells, but may have a functionally important role itself inducing suppression.

The role of the 2H4 molecule in the generation of suppressor function in Con A-activated T cells.

The molecular basis for the suppression generated in a concanavalin A (Con A)-activated T cell culture remains unknown. In this study, we have attempted to determine whether the 2H4 and 4B4 molecules on Con A-activated T cells play some role in the generation of suppression by such cells. We have shown that Con A-activated suppressor cells belong to the 2H4+ subset of T cells but not the 4B4+ (2H4-) subset. Con A-activated T cells exerted their optimal suppressor function on day 2 in culture, a time at which the expression of 2H4 on such cells was maximal and 4B4 was minimal. Furthermore, the stimulation of T cells with the higher concentration of Con A generated the stronger suppressor function. At the same time, both 2H4 expression and density were increased and 4B4 expression and density were decreased on such Con A-activated T cells. More importantly, the treatment of Con A-activated T cells with anti-2H4 antibody but not with anti-4B4, anti-TQ1, or anti-T4 antibodies can block the suppressor function of such cells. Taken together, the above results strongly suggest that the 2H4 molecule itself may be involved in the generation of suppressor function in Con A-activated T cells. The 2H4 antigen on such cells was shown to be comprised of 220,000 and 200,000 m.w. glycoproteins. Thus this study indicates that the 220,000 and 200,000 m.w. structure of the 2H4 molecule may itself play a crucial role in the generation of suppressor signals of Con A-activated cells.

The cellular basis for the induction of antigen‐specific T8 suppressor cells

The cellular basis for the generation of antigen-specific T8 suppressor cells with high doses of antigen has been studied. We separated the T4 subset of human T cells into T4+2H4+ and T4+2H4- subpopulations with a recently developed monoclonal anti-2H4 antibody. T8 cells could be consistently activated to suppress a primary anti-2,4-dinitrophenyl (DNP) antibody response in vitro with unfractionated T4 cells or with the T4+2H4+ subset but not the T4+2H4- subset. In contrast, the T4+2H4- subset functioned as the helper inducer for the anti-DNP antibody response. With keyhole limpet hemocyanin (KLH)-stimulated T4+2H4+ cells we could efficiently induce antigen-specific suppressor activity of fresh T8 cells. In contrast, the T4+2H4+ subset could not effect suppression in the absence of T8 cells. Our findings indicate that the T4+2H4+ subset of human T cells is the suppressor inducer of specific T8 cells in an antigen-specific DNP-KLH system.

The isolation and characterization of the human suppressor inducer T cell subset.

Immunization of mice with lower primate lymphoid cells has provided a useful strategy for raising monoclonal antibodies against functionally important surface determinants on human T lymphocytes. We have developed a monoclonal antibody, anti-2H4, which defines functionally unique human T cell subsets. This anti-2H4 antibody was reactive with approximately 42% of unfractionated T cells, 41% of T4+ inducer cells, and was reactive with approximately 54% of T8+ cytotoxic/suppressor population. Anti-2H4 was not reactive with human thymocytes, but reacted with subsets of peripheral blood B cells and null cells. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+2H4+ subset proliferate well to concanavalin A (Con A) stimulation, but poorly to soluble antigen stimulation, and provides poor help to B cells for PWM-induced Ig synthesis. The T4+2H4- subset, in contrast, proliferates poorly upon stimulation with Con A, but well on exposure to soluble antigen, and provides a good helper signal for PWM-induced Ig synthesis. What is, perhaps, most important, the T4+2H4+ subset functions as the inducer of the T8+ suppressor cells. Previous attempts to define the latter subset of cells has relied heavily on the use of specific autoantibodies present in the sera of patients with juvenile rheumatoid arthritis (JRA) and systemic lupus erythematosus (SLE). The present results suggest that anti-2H4 antibody defines the human suppressor induced subset of lymphocyte previously described as T4+JRA+. Last, the results reemphasize the previously documented remarkable structural conservation of certain T cell-specific determinants on lymphocytes of phylogenetically distant primates.

Antibodies to histones in systemic lupus erythematosus: localization of prominent autoantigens on histones H1 and H2B.

By the technique of immunoblotting we have assessed the ability of sera from 24 patients with systemic lupus erythematosus to bind nuclear proteins. Of the 11 patients who had antibodies to histones, 10 had antibodies to histone H1 and 9 of these also had antibodies to histone H2B. Antibodies to the other histones (H2A, H3, and H4) were less apparent. Five of the 11 patients (and two others in the remainder of the sample of 24) also had antibodies to a small number of nonhistone proteins that are probably components of ribonucleoprotein particles, but there was no obvious correlation between the presence of antihistone antibodies and the known antiribonucleoprotein activity of these sera. Separate determinants on H1 and H2B were demonstrated by immunoblotting with affinity-purified anti-H1 and anti-H2B antibodies derived from serum that showed both specificities. The localization of the determinants within the histone polypeptide chains was shown by immunoblotting with large fragments produced by specific proteolytic or chemical cleavage of the histones. The strongest determinant on H1 was located within the COOH-terminal half, with a weaker determinant being present within the NH2-terminal half; the H2B determinant(s) was located entirely within the NH2-terminal half of the molecule. The selectivity with which the antihistone antibodies in systemic lupus erythematosus are produced against the more exposed histones in the nucleosome (and perhaps against the most exposed regions of these histones) is consistent with the involvement of intact chromatin structures as immunogens in this disease.

Detection of antibodies to histones in human systemic lupus erythematosus and in murine lupus-like syndromes using micro-ELISA

An enzyme-linked immunosorbent microassay (micro-ELISA) was improved for the detection of anti-histone antibodies. Detection of these antibodies in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerodermitis sclerosis (SCL), showed a positive correlation between the presence of antinuclear antibodies detected by indirect immunofluorescence and the level of anti-histone antibodies (p less than or equal to 0.02) having anti-H1 (p less than or equal to 0.001) and anti-H2B (p less than or equal to 0.01) specificities. These antibodies were more frequently detected, and at a higher level, in human SLE than in RA and SCL. Anti-H1 and anti-H2B (and less frequently anti-H3) antibodies were also found in mice with lupus-like syndrome (MRL/1, PN and Swan). Their level rose with age and they were prevalent in the severe forms, especially in MRL/1 and PN.

Serological detection of homologies of H1o with H5 and H1 histones.

Immunospecifically purified anti-chicken H5 antibodies caused bright staining of mouse liver nuclei in an indirect immunofluorescence assay. Analysis with the fluorescence-activated cell sorter showed that 95% of the nuclei were in the brightest category. Histones extracted from mouse liver nuclei, analyzed by sodium dodecyl sulfate-gel electrophoresis, presented an extra band with the same mobility as chick erythrocyte H5. Electrophoretic blots on nitrocellulose paper were treated with anti-H5 or anti-H1 antibodies and iodinated protein A. The affinity purified anti-H5 bound to chicken H5 and mouse H1o fractions only. Anti-calf H1 bound to calf, chicken, and mouse H1 proteins and to mouse H1o as well. Antibodies to purified subfractions of rat thymus H1 showed binding to both H1 and H1o. The results support the suggestions that H1o bears significant structural homology with H5 and H1.