Glória László

Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.

Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.

Differential expression of GL7 activation antigen on bone marrow B cell subpopulations and peripheral B cells

GL7 was originally described as a 35-kDa late activation antigen on mouse T and B cells. GL7 expression has also been demonstrated on thymocytes, germinal center B cells and some neuronal cell types. Flow-cytometry and immunohistochemistry were used to follow changes in the expression of GL7 during B cell development, amongst B cell subpopulations and various anatomical locations. GL7 is expressed as early as the pro-B cell stage and increases up to the pre-B-I stadium. Expression remains high on pre-B-II and on immature B cells, although slightly decreases during maturation. GL7 is almost completely downregulated when IgD appears on the cell surface. On the periphery only a few B cells are positive and these cells are almost exclusively found in the sIgD- germinal center areas of lymph nodes and spleen. The staining pattern of GL7 is very similar to that of PNA in the lymph nodes but in the bone marrow we have found both B220+PNA+GL7- and B220+PNA+GL7+ populations, showing that GL7 and the antigen recognized by PNA are different. After in vitro stimulation, the GL7(hi) B cell population has also been found to be IgD negative. Functional comparison between in vitro activated and MACS sorted GL7(hi) and GL7(lo/-) spleen B cells of immunized mice showed significantly higher specific and total antibody production as well as antigen presenting capacity in the GL7(hi) population.

Some new faces of membrane microdomains: A complex confocal fluorescence, differential polarization, and FCS imaging study on live immune cells

Lipid rafts are cholesterol‐ and glycosphingolipid‐rich plasma membrane microdomains, which control signal transduction, cellular contacts, pathogen recognition, and internalization processes. Their stability/lifetime, heterogeneity remained still controversial, mostly due to the high diversity of raft markers and cellular models. The correspondence of the rafts of living cells to liquid ordered (Lo) domains of model membranes and the effect of modulating rafts on the structural dynamics of their bulk membrane environment are also yet unresolved questions. Spatial overlap of various lipid and protein raft markers on live cells was studied by confocal laser scanning microscopy, while fluorescence polarization of DiIC18(3) and Bodipy‐phosphatidylcholine was imaged with differential polarization CLSM (DP‐CLSM). Mobility of the diI probe under different conditions was assessed by fluorescence correlation spectroscopic (FCS). GM1 gangliosides highly colocalized with GPI‐linked protein markers of rafts and a new anti‐cholesterol antibody (AC8) in various immune cells. On the same cells, albeit not fully excluded from rafts, diI colocalized much less with raft markers of both lipid and protein nature, suggesting the Lo membrane regions are not equivalents to lipid rafts. The DP‐CLSM technique was capable of imaging probe orientation and heterogeneity of polarization in the plasma membrane of live cells, reflecting differences in lipid order/packing. This property—in accordance with diI mobility assessed by FCS—was sensitive to modulation of rafts either through their lipids or proteins. Our complex imaging analysis demonstrated that two lipid probes—GM1 and a new anti‐cholesterol antibody—equivocally label the membrane rafts on a variety of cell types, while some raft‐associated proteins (MHC‐II, CD48, CD59, or CD90) do not colocalize with each other. This indicates the compositional heterogeneity of rafts. Usefulness of the DP‐CLSM technique in imaging immune cell surface, in terms of lipid order/packing heterogeneities, was also shown together with its sensitivity to monitor biological modulation of lipid rafts.

Novel anti-cholesterol monoclonal immunoglobulin G antibodies as probes and potential modulators of membrane raft-dependent immune functions.

Natural autoantibodies against cholesterol are present in the sera of all healthy individuals; their function, production, and regulation, however, are still unclear. Here, we managed to produce two monoclonal anti-cholesterol antibodies (ACHAs) by immunizing mice with cholesterol-rich liposomes. The new ACHAs were specific to cholesterol and to some structurally closely related 3β-hydroxyl sterols, and they reacted with human lipoproteins VLDL, LDL, and HDL. They bound, usually with low avidity, to live human or murine lymphocyte and monocyte-macrophage cell lines, which was enhanced substantially by a moderate papain digestion of the cell surface, removing some protruding extracellular protein domains. Cell-bound ACHAs strongly colocalized with markers of cholesterol-rich lipid rafts and caveolae at the cell surface and intracellularly with markers of the endoplasmic reticulum and Golgi complex. These data suggest that these IgG ACHAs may serve as probes of clustered cholesterol (e.g., different lipid rafts) in live cells and thus may also have immunomodulatory potential.

FcRn Overexpression in Transgenic Mice Results in Augmented APC Activity and Robust Immune Response with Increased Diversity of Induced Antibodies

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.

Structural characteristics influencing the carrier function of synthetic branched polypeptides based on poly[Lys-(DL-Ala)3)]backbone

Effective carrier function of selected representatives of new branched polypeptides covalently coupled with the synthetic monovalent hapten, oxazolone was studied. The effectiveness of oxazolone-synthetic polypeptide conjugates in inducing oxazolone-as well as carrier-specific antibody responses in inbred mice was compared to that of bovine serum albumin (BSA)- and KLH-oxazolone conjugates. The synthetic polypeptides, poly[Lys-(D-Leui-DL-Alam)] (D-LAK), LAK and FAK, as well as the common poly[Lys-(DL-Alam)](AK) core covalently coupled to oxazolone (Ox) induced a T cell-dependent antibody response when repeatedly administered with or without Freunds adjuvant in mice. This was evidenced by: the increasing titer of oxazolone-specific IgG during the course of the memory response; the appearance of all IgG subclasses; the effective oxazolone-specific priming by the conjugates; and the induction of an intense oxazolone- and carrier-specific DTH reaction. Although the oxazolone-specific antibody response was 10-100 times lower than that induced by KLH- or BSA-oxazolone conjugates, it was accompanied by a lower level or no detectable carrier-specific antibody response despite an effective carrier-specific T cell-mediated response. Significant differences were observed between the effectiveness of synthetic polypeptides used as carrier: highest oxazolone-specific antibody titers were observed using the AK, LAK and FAK conjugates. The intensity and specificity of the DTH reaction and antibody response induced by the carrier-oxazolone conjugates suggested that the distinct effectiveness of L- and D-amino acid-containing conjugates (LAK vs D-LAK and FAK vs D-FAK) was dependent on altered B cell recognition of the haptenic group. Circular dichroism (CD) spectra indicating different local orientation of oxazolone, when coupled to L or D side chain-terminating amino acids, support this suggestion.

A dynamic network of estrogen receptors in murine lymphocytes: fine-tuning the immune response

The actual level of circulating estrogen (17β‐estradiol, E2) has a serious impact on regulation of diverse immune cell functions, where their classical cytoplasmic receptors, ERα and ERβ, act as nuclear transcriptional regulators of multiple target genes. There is growing evidence, however, for rapid, “non‐nuclear” regulatory effects of E2 on lymphocytes. Such effects are likely mediated by putative membrane‐associated receptor(s) (mER), but the mechanistic details and the involved signaling pathways still remained largely unknown because of their complexity. Here, we show that in lymphocytes, mERs can signalize themselves, and upon ligation, they are able to coordinate translocation of other E2Rs to the PM. Our data firmly imply existence of a complex, dynamic network of at least seven ER forms in murine lymphocytes: cytoplasmic and membrane‐linked forms of ERα, ERβ, or GPR30 and a mER that can receive extracellular E2 signals. The latter mERs are likely palmitoylated, as they are enriched in lipid‐raft microdomains, and their E2 binding is also cholesterol dependent. The data also support that ligation of mERs can induce rapid regulatory signals to lymphocytes and then internalize and let the E2 liberate in lysosomes. In addition, they can dynamically control the cell‐surface linkage of other cytoplasmic ERs. As demonstrated by the differential effects of mER or cytoplasmic ER ligation on the proliferation of activated T and B lymphocytes, such a dynamic E2R network can be considered as a tool to manage accommodation/fine‐tuning of lymphocytes to rapidly changing hormone levels.

The influence of Igh-1 genes on the class and subclass distribution of oxazolone-specific antibodies

Previous studies have demonstrated that the level of the oxazolone-specific antibody response induced by contact sensitization is under the control of H-2 and Igh-1-linked genes. The aim of the present study was to clarify the role of H-2 and Igh-1 genes in the regulation of antibody affinity and isotype composition of oxazolone-specific antibodies. Analysis of the antibody response to oxazolone has revealed different ratios of IgG2a and IgG2b antibodies in mice carrying the Igh-1b allele and in strains carrying alleles a, c, and e. The characteristic ratio of IgG2a and IgG2b isotypes persisted during the whole period of the primary and secondary antibody response of CBA and CBA-IgbIgh-C congenic mice. The Igh-1-linked genes influenced the isotype distribution and not the affinity of oxazolone-specific antibodies induced by contact sensitization.

Collaboration of TCR-, CD4- and CD28-mediated signalling in antigen-specific MHC class II-restricted T-cells

A previously developed experimental system was applied to obtain qualitative and quantitative data on the contribution of TCR-, CD4- and CD28-mediated signalling in the activation of an antigen specific T-cell hybridoma. All the three signal transducing receptors were stimulated by their natural ligands, and intermediate and late responses of an I-Ed restricted, CD4 +, influenza HA specific murine T-hybridoma (IP-12-7) were monitored by measuring the concentration of intracellular calcium [Ca2+]i and secreted IL-2. This type of analysis of T-cell activation revealed: (i) calcium mobilization induced by peptide loaded APC requires rapid conjugate formation; (ii) a direct correlation between the magnitude of the intermediate and the late responses was observed as a consequence of differential TCR ligation modulated by peptide dose or by the presence CD4; (iii) considering the APC/peptide and T/APC ratios, the concentration dependence of the intermediate and late responses was similar in both assays but a substantial difference in the sensitivity of the two methods was observed; (iv) CD4 mediated signalling has a co-stimulatory effect predominantly at suboptimal in vitro conditions; and (v) sustained increase of [Ca2+]i as well as the production of high concentrations of IL-2 is highly dependent on the CD28-B7 interaction. These results demonstrate that distinct peptide doses and the presence or absence of CD4 result in quantitative changes in T-cell responses, while the degree of CD28 mediated signalling has a qualitative affect on the outcome of T-cell activation, revealed by complete or partial inhibition of IL-2 secretion as a result of limited CD28-B7 interaction as well as by alteration in the duration and time kinetics of the calcium response.

New cholesterol-specific antibodies remodel HIV-1 target cells’ surface and inhibit their in vitro virus production

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.