Heterogeneity of immunological abnormalities in ataxia-telangiectasia

Peripheral blood mononuclear cells from five patients with ataxia-telangiectasia were evaluated for their reactivity with a panel of monoclonal antibodies directed against T-cell subsets and for theirin vitro functions in a pokeweed mitogen-induced immunoglobulin biosynthesis assay. All the patients had significantly reduced proportions of cells identified by monoclonal antibodies to subpopulations of T lymphocytes with helper activity (OKT4 and 5/9) and produced low amounts or no IgA and IgGin vitro. Immunoglobulin biosynthesis was increased by the addition of normal x-irradiated peripheral blood mononuclear cells in one of three patients, suggesting a helper T-cell deficiency in this patient and intrinsic B-cell defects in the other two. Two patients had increased proportions of cells identified by a monoclonal antibody to a subpopulation of T lymphocytes which includes suppressor T cells (OKT8), and their cells were able to suppress immunoglobulin biosynthesis by peripheral blood mononuclear cells from normal donors. These findings indicate heterogeneous disturbances of immunoregulatory mechanisms in ataxia-telangiectasia.     

Characterization of a major virion envelope glycoprotein complex of murine cytomegalovirus and its immunological cross-reactivity with human cytomegalovirus

Three glycoproteins on the murine cytomegalovirus (MCMV) virion with apparent molecular weights of 150K (gp 150), 105K (gp 105), and 52K (gp52) were immunoprecipitated by two monoclonal antibodies (MAbs) 8G5.12A and 2E.12A. However, only 8G5.12A was able to neutralize MCMV infectivity in the presence of complement. The accessibility of these three glycoproteins to radiolabeling by surface-iodination reactions suggested that they were exposed on the surface of the virion. Western blot analysis of the three glycoproteins showed that gp150 shared antigenic determinants with gp105 and gp52. Briefly, the MAb 8G5.12A reacted with gp150 and gp105, whereas the MAb 2E8.12A reacted with gp150 and gp52. A third MAb 3H2.12A was also found to be reactive with gp150 and gp105 in Western blots, but was unable to immunoprecipitate these glycoproteins. Data from pluse-chase experiments suggested that all three virion glycoproteins were synthesized from a common 128K precursor, providing a partial explanation of their antigenic relatedness. Furthermore, we have demonstrated the presence of high-molecular-weight complexes formed by disulfide bonding between gp150, gp105, and gp52. Lastly, the MAb 8G5.12A was able to immunoprecipitate 84K and 99-110K glycoproteins from human CMV-infected WI-38 cells, demonstrating that conserved determinants exist between murine and human CMV envelope glycoproteins.     

Characterization of two monoclonal antibodies (UCL4D12 and UCL3D3) that discriminate between human mantle zone and marginal zone B cells.

Two new monoclonal antibodies (MoAbs), UCL3D3 and UCL4D12 were obtained following immunization with follicular lymphoma (UCL3D3) or low-grade primary B cell gastric lymphoma cells (UCL4D12). In normal splenic white pulp, tonsil and small intestinal Peyer's patches, UCL4D12 recognizes marginal zone B cells and a subpopulation of follicle centre cells, whereas mantle zone B cells are UCL4D12 negative. In contrast, UCL3D3 recognizes mantle zone B cells and follicular dendritic cells, but not marginal zone B cells or follicle centre B cells. Double-immunofluorescence studies showed that in the splenic white pulp, these antibodies stain reciprocally. The majority of UCL3D3+ cells are sIgM+ and sIgD+ whereas a higher proportion of UCL4D12+ cells express surface IgM (sIgM) but not surface IgD (sIgD). Less than 10% of splenic B cells express both 3D3 and 4D12 antigens. None of the cell lines tested expressed either antigen. Functional studies showed that both antigens play a role in B cell activation as the MoAbs increase the mitogenic effect of Staphylococcus aureus Cowan I on tonsil B cells. This effect was maximal at 72 h in culture. TPA activation was reduced, and no effect was observed with anti-immunoglobulin (anti mu) or CDw40 (G28.5). UCL3D3 and UCL4D12 did not show any stimulatory effect on their own. Biochemical studies show that both MoAbs recognize proteins of 80-90 kD under reducing conditions. These two MoAbs appear to recognize new B cell surface antigens which may be useful for identifying subpopulations of B cells.     

Functional and immunochemical cross-reactivity of V2-specific monoclonal antibodies from HIV-1-infected individuals

The recent analysis of the first successful RV144 vaccine trial revealed that a high titer of plasma anti-V2 antibodies (Abs) correlated with a decreased risk of HIV-1 infection in vaccine recipients. To understand the mechanism of immune correlates, we studied seven anti-V2 monoclonal Abs (mAbs) developed from HIV-1 infected individuals. The V2 mAbs target conserved epitopes, including the binding site for α4β7 integrin, and are broadly cross-reactive with various gp120 proteins. Preferential usage of the VH1-69 gene by V2 mAbs may depend on selection by the same antigenic structure. Six of seven V2 mAbs weakly neutralized four to eight of the 41 pseudoviruses tested and resistance to neutralization was correlated with longer V2 domains. The data suggest the presence of shared, conserved structural elements in the V2 loop, and these can be used in the design of vaccine immunogens inducing broadly reactive Abs with anti-viral activities.     

Monoclonal antibodies to cultured human glomerular mesangial cells. I. Reactivity with haematopoietic cells and normal kidney sections.

The aim of this study was to produce monoclonal antibodies to cultured human glomerular mesangial cells in order to obtain specific markers for these cells and to aid the study of their function. Using standard monoclonal antibody techniques, 29 hybridomas producing antibodies directed to cultured mesangial cells were obtained. Most of these antibodies were not reactive with normal or neoplastic haematopoietic cell lines by flow cytometry. Fourteen of the 29 culture supernatants bound to various components of normal human kidney sections stained by the alkaline phosphatase/anti-alkaline phosphatase (APAAP) method. Ten of these supernatants reacted with components within the glomerulus, with six binding to the mesangium. These studies suggest that (1) mesangial cells in culture may show significant de-differentiation, because most supernatants which reacted with mesangial cells in culture did not do so in tissue sections; (2) antibodies reactive with haematopoietic cells may not detect the majority of immunogenic surface antigens on cells in tissues; and (3) some of the antibodies which we have produced may prove to be useful markers for mesangial cells in glomerular disease.     

Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies.

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37 degrees C using non-reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides, the largest of which with an apparent molecular weight (MWap) of 40 kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18-24 h of incubation. Tryptic peptides of Tg, released at 1 h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti-Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWap of 10-25 kD and above. Four other MoAbs reacted with peptides of MWap of 25-43 kD and above, and the remaining four reacted with peptides of MWap > 43 kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation-dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.     

Conventional type 1 dendritic cells induce TH1, TH1-like follicular helper T cells and regulatory T cells after antigen boost via DEC205 receptor

Conventional dendritic cells (cDCs) are specialized in antigen presentation. In the mouse spleen, cDCs are classified in cDC1s and cDC2s, and express DEC205 and DCIR2 endocytic receptors, respectively. Monoclonal antibodies (mAbs) αDEC205 (αDEC) and αDCIR2 have been fused to different antigens to deliver them to cDC1s or cDC2s. We immunized mice with αDEC and αDCIR2 fused to an antigen using Poly(I:C) as adjuvant. The initial immune response was analyzed from days 3 to 6 after the immunization. We also studied the influence of a booster dose. Our results showed that antigen targeting to cDC1s promoted a pro-inflammatory T_H1 cell response. Antigen targeting to cDC2s induced T_FH cells, GCs, and plasma cell differentiation. After boost, antigen targeting to cDC1s improved the T_H1 cell response and induced T_H1-like T_FH cells that led to an increase in specific antibody titers and IgG class switch. Additionally, a population of regulatory T cells was also observed. Antigen targeting to cDC2s did not improve the specific antibody response after boost. Our results add new information on the immune response induced after the administration of a booster dose with αDEC and αDCIR2 fusion mAbs. These results may be useful for vaccine design using recombinant mAbs.     

A new mouse anti-mouse complement receptor type 2 and 1 (CR2/CR1) monoclonal antibody as a tool to study receptor involvement in chronic models of immune responses and disease

Although reagents are available to block mouse complement receptor type 2 and/or type 1 (CR2/CR1, CD21/CD35) function in acute or short term models of human disease, a mouse anti-rat antibody response limits their use in chronic models. We have addressed this problem by generating in Cr2−/− mice a mouse monoclonal antibody (mAb 4B2) to mouse CR2/CR1. The binding of murine mAb 4B2 to CR2/CR1 directly blocked C3dg (C3d) ligand binding. In vivo injection of mAb 4B2 induced substantial down regulation of CR2 and CR1 from the B cell surface, an effect that lasted six weeks after a single injection of 2 mg of mAb. The 4B2 mAb was studied in vivo for the capability to affect immunological responses to model antigens. Pre-injection of mAb 4B2 before immunization of C57BL/6 mice reduced the IgG1 antibody response to the T-dependent antigen sheep red blood cells (SRBC) to a level comparable to that found in Cr2−/− mice. We also used the collagen-induced arthritis (CIA) model, a CR2/CR1-dependent autoimmune disease model, and found that mice pre-injected with mAb 4B2 demonstrated substantially reduced levels of pathogenic IgG2a antibodies to both the bovine type II collagen (CII) used to induce arthritis and to endogenous mouse CII. Consistent with this result, mice pre-injected with mAb 4B2 demonstrated only very mild arthritis. This reduction in disease, together with published data in CII-immunized Cr2−/− mice, confirm both that the arthritis development depends on CR2/CR1 receptors and that mAb 4B2 can be used to induce biologically relevant receptor blockade. Thus mAb 4B2 is an excellent candidate for use in chronic murine models to determine how receptor blockage at different points modifies disease activity and autoantibody responses.     

Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis C virus glycoprotein E1.

Although both envelope glycoproteins of the hepatitis C virus, E1 and E2/NS1, show a high degree of sequence variation, the E1 protein includes a well conserved domain, which may be functionally important. We have analysed the human B cell response to a peptide fragment from amino acid residues 314-330 (EP3) covering the central conserved sequence of this domain. Anti-hepatitis C virus-positive blood donors were screened for anti-EP3 antibodies with an ELISA based on immobilized peptide. Thirty out of 92 (32%) RIBA-confirmed donors displayed a significant antibody response to EP3. From three of these blood donors we established four anti-EP3-producing heterohybridoma cell lines: Ul/F30 and Ul/F31 produced IgM-kappa, whereas Ul/F32 and Ul/F33 secreted the isotypes IgG1-lambda and IgG1-kappa, respectively. Epitope analysis with overlapping nonapeptides suggests the existence of different antigenic determinants within the EP3 fragment. Although both IgG antibodies Ul/F32 and Ul/F33 have dissociation constants to the peptide of approximately 10(-9) M, binding to recombinant E1 protein expressed in COS-7 cells was different. Only Ul/F33 detected envelope protein of approximately 24-35 kD in Western blot. This human MoAb will be useful for further investigations on the hepatitis C virus glycoprotein E1.     

Mononuclear-cell subsets in human idiopathic crescentic glomerulonephritis (ICGN): Analysis in tissue sections with monoclonal antibodies

Mononuclear inflammatory cells (MIC) were analyzed in renal biopsies from 16 patients with ICGN (7 with glomerular immune complex deposits, 3 with anti-GBM disease, and 6 without immune deposits) by the avidin-biotin-immunoperoxidase technique utilizing monoclonal antibodies to cell surface antigens: T11 (total T), T4 (inducer/helper T), T8 (suppressor/cytotoxic T), B1 (B cells), M1 (monocytes/granulocytes), and Leu 7 [natural killer (NK) cells]. Total MIC were significantly increased in both glomeruli and interstitial tissues of the patients. Interstitial MIC consisted mainly of lymphocytes (80%) and monocytes (19%), with small numbers of B and NK cells present. In contrast, MIC in renal glomeruli of patients with ICGN were composed of monocytes (65%) rather than T lymphocytes (34%). A majority of T lymphocytes found in renal tissues of patients and controls had the helper/inducer phenotype. Tissue T4/T8 ratios were not significantly different in the glomeruli and interstitium. Monocytes and T lymphocytes accumulating in renal tissues of patients with ICGN may mediate glomerular injury in all forms of human ICGN.Presented in part at the 15th Annual Meeting of the American Society of Nephrology, Chicago, Illinois, 1982.     

The specificity of antibodies induced by infection with rhinovirus type 2.

Mouse monoclonal antibodies (MAbs) against three distinct antigenic sites on rhinovirus type 2 have been obtained and the sites identified. We describe how these MAbs were used in a blocking test to detect antibodies in human sera directed against the same three defined sites. Sera from twelve volunteers were studied. All had been exposed to rhinovirus type 2 by intranasal inoculation, four had been uninfected, eight were infected of whom four developed a cold while four did not. Blocking antibodies were high and did not increase in the resistant volunteers, and were lower and increased in the infected volunteers. The antibodies were almost as sensitive as other antibody assays for detecting infection. The responses to all three sites were similar. Correlations between the results of all tests were calculated and the results are summarised. Tests were also devised to measure the Ig subclass of antibodies against the whole virus particle. The A1, G1, and G4 classes showed most frequent rises in response to infection. Correlations between these results and other antibody assays were found and are presented.     

Human hybridomas derived from CD5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies.

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B-CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT-sensitive heteromyeloma line (CB-F7). A fusion frequency of up to 10(-5) allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi-specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a 'switch-on' of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi-reactive antibody repertoire of CD5+ B cells.     

Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies

The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB⁺⁺), while activation of the alternative pathway of C (AP) was assessed in 10% Clq-depleted serum in the presence of 5 mm MgCl₂ in GVB⁺⁺. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG 1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG 1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')₂ fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP. whereas Fab'did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity.     

Modulation of murine complement receptor type 2 (CR2/CD21) ectodomain shedding by its cytoplasmic domain

Ectodomain shedding is a mechanism that regulates numerous functions of cell surface proteins. The extracellular domain of the human complement receptor 2 (CR2/CD21) is released by proteolytic cleavage as a soluble protein through a variety of stimuli including the thiol antioxidants N-acetylcysteine (NAC) and glutathione (GSH), and the oxidant pervanadate (PV). In addition, PV mimics B cell antigen receptor (BCR) signaling. Here, we show that murine CD21 is shed upon those stimuli and that the cytoplasmic domain is an important modulator for CD21-shedding. B cells expressing a mutant CD21 cytoplasmic domain with only three amino acids (KHR) showed increased CD21-shedding and required lower stimuli concentrations. At lower PV concentrations, wildtype CD21 was up-regulated on the cell surface, whereas at higher PV concentrations the ectodomain was shed. These findings further indicate that GSH and NAC utilize different pathways than PV to activate CD21-shedding. Altogether, as pre-activated B cells express higher CD21 levels than resting mature B cells or fully activated and antigen-experienced B cells, we suggest CD21-shedding to be a mechanism to fine-tune B cell activation.     

The expression of B cell surface receptors. III. The murine low-affinity IgE Fc receptor is not expressed on Ly 1 or 'Ly 1-like' B cells.

The distribution of IgE FcR (Fc epsilon R)-positive and -negative B cells was examined in normal adult mice. Using three-color flow cytometry, the expression of the Fc epsilon R was analyzed on various B-cell subsets present in the peritoneum and spleen. The results demonstrate that in the peritoneal cavity, the Fc epsilon R is not expressed on the large majority of Ly 1+ B cells and Ly 1-, Mac 1+ sister B cells. The receptor is present, however, on the small number of conventional B cells residing in the peritoneum. Although interleukin 4 (IL-4) can increase the levels of the Fc epsilon R on conventional B cells, incubation of Ly 1 and sister B cells with IL-4 did not result in the expression of the Fc epsilon R. When examining B cells present in the spleen, a small subset of B cells was consistently found to be Fc epsilon R-. These Fc epsilon R- cells were IgM-bright, IgD-dull and largely Ly 1- and Mac 1-negative. Staining of splenic tissue sections revealed that the Fc epsilon R- B cells were primarily localized to the marginal zones, whereas the Fc epsilon R+ B cells were found in the follicles. Taken together, the results indicate that the Fc epsilon R may be a useful marker in delineating the various B-cell subsets. In the peritoneum, the Fc epsilon R appears to discriminate conventional B cells from those of the Ly 1/sister lineage, and in the spleen it is likely to distinguish resting follicular B cells from Ly 1/sister and marginal zone B cells.     

Characterization of the B-cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite‐evasion immune‐mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69‐expressing peritoneal and splenic B cells. This early B‐cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin‐secreting splenic B cells and a rise in serum levels of N. caninum‐specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum‐infected mice. The B‐cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with γ‐irradiated parasites. Contrasting with the peripheral B‐cell expansion, a depletion of B‐lineage cells was observed in the bone‐marrow of the N. caninum‐infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite‐specific antibodies nevertheless impaired interferon‐γ (IFN‐γ) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non‐lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti‐N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite‐specific, non‐polyclonal, B‐cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN‐γ in the host protective immune mechanisms against this parasite.     

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope.

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.