Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.     

Immunoselection of human lymphocytes producing xenoreactive natural antibodies against Galocl,3Gal terminal residues

Abstract: In the pig-to-human xenograft combination, human xenoreactive natural antibodies (XNA) bind to carbohydrate moieties, especially those with Galα1,3Gal terminal residues, expressed on the surface of most cells. The aim of this study was to select the cells that produce XNA by functionally characterizing a subset of cells for future studies on immunosuppression of XNA formation. We especially addressed the question whether XNA could be produced by CD5+ B lymphocytes, a subset of cells producing antibodies of high connectivity and polyreactivity, possibly involved in autoimmune processes. For that purpose we used porcine thyroglobulin (PTg), which expresses several Galα,3Gal terminal residues, for the immunoselection of XNA-producing cells. On FACS analysis, biotinylated PTg appeared to react with 5% of the B lymphocytes but the proportion of CD5+ B lymphocytes was not enriched in the PTg-reactive population. Similarly, magnetic beads coated with PTg were used to sort lymphocytes with Ig receptors recognizing PTg. Two and one-half percent of cells reacted, mainly B lymphocytes (89%), but they were not enriched for CD5+ B lymphocytes. When PTg-reactive lymphocytes were cultivated in presence of irradiated T cells and stimulated with PWM, the synthesis of Galα1,3Gal reactive antibodies, mainly of the IgG class, was demonstrated. The results of this study suggest that a high percentage of B lymphocytes react with Galα1,3Gal terminal residues. Such XNA-producing cells are not particularly common in the CD5+ subset.     

天然抗角蛋白单克隆抗体3B4的噬菌体呈现的scFv的构建及表达

目的:构建并表达噬菌体呈现的天然抗角蛋白单克隆抗体(mAb)3B4的单链抗体(scFv),并测定其活性。方法:分别以pMD18TmAb3B4VH和pMD18TmAb3B4VL为模板进行PCR,扩增mAb3B4的VH和VL基因,然后将其依次插入噬菌体表达载体pscMH中。经DNA序列测定正确后,转化大肠杆菌,用辅助噬菌体VCSM13超感染诱导表达scFv;用ELISA检测其与亲本mAb3B4的抗原结合活性的改变。结果:对扩增的mAb3B4的VH和VL基因进行测序,结果表明VH基因与原序列完全一致,VL基因在骨架区FR1有1个碱基发生突变。用VCSM13超感染后能检测到scFv的表达,其与亲本一样具有多反应性,但抗原结合模式不完全相同。结论:成功地构建并表达了天然抗角蛋白mAb3B4噬菌体呈现的单链抗体,表达的scFv与mAb3B4的抗原结合活性有一定的差异,为探讨天然自身抗体的抗原结合模式奠定了基础。     

Induced antigen-binding polyreactivity in human serum IgA

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.     

Polyreactivity of human monoclonal antibodies: Human IgM anti-rhesus monoclonal antibodies are frequently polyreactive

The aim of this study was to characterize human anti-Rhesus monoclonal antibodies cross-reacting with tissue antigens. Of the 155 monoclonal alloantibodies tested, 49 also reacted with intracellular antigens, as demonstrated by immunofluorescence assay on cryostat sections of animal and human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 49 cross-reacting Mabs, 37 were IgM). The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells.     

Murine lupus strains differentially model unique facets of human lupus serology

Systemic lupus erythematosus (SLE) is a polygenic autoimmune disease characterized by the production of anti-nuclear autoantibodies that lead to subsequent end organ damage. Previous array-based studies in patients with SLE have shown that high immunoglobulin (Ig)G anti-nuclear autoantibody reactivity was associated with severe renal lupus, whereas IgM polyreactivity was associated with less severe disease. To ascertain how different murine lupus strains recapitulate these different autoantibody profiles seen in patients, serum from New Zealand black (NZB)/NZ white (W) F(1), Murphy Roths large (MRL)/lpr, NZ mixed (M)2410 and BXSB strains were compared using a comprehensive array-based screen. The array results were verified using enzyme-linked immunosorbent assays (ELISAs). Serum from MRL/lpr mice exhibited high levels of IgG anti-nuclear antibodies as well as anti-glomerular antibodies and variable levels of antibodies to myosin, Matrigel and thyroglobulin. Elevated anti-nuclear IgG antibodies were associated with severe nephritis in this strain. In contrast, NZM2410 mice exhibited lower IgG autoantibody levels with less severe nephritis but a significantly higher polyreactive IgM autoantibody profile. ELISA analysis confirmed these results. The NZB/NZW F(1) and BXSB strains exhibited an intermediate serological profile. Hence, just as in patients with SLE, whereas strong IgG reactivity to nuclear antigens is associated with severe renal disease, a polyreactive IgM seroprofile is also less ominous in murine lupus.     

Restricted immunoglobulin variable region (Ig V) gene expression accompanies secondary rearrangements of light chain Ig V genes in mouse plasmacytomas

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.     

B-cell receptor epitope recognition correlates with the clinical course of chronic lymphocytic leukemia

BACKGROUND:

B‐cell receptors (BCRs) and their recognition of specific epitopes may play a pivotal role in the development and progression of chronic lymphocytic leukemia (CLL). In this study, the authors set up a model system to explore epitope reactivity and its clinical relevance in CLL.

METHODS:

Epitope‐mimicking peptides were selected from phage display libraries on 6 CLL BCRs from randomly chosen patients. The binding of the 6 index epitope mimics was evaluated in a set of 100 unrelated CLL samples. Epitope recognition patterns were correlated with the clinical course of the disease.

RESULTS:

Surprisingly, all CLL samples recognized 1 or several index epitopes, and some revealed marked polyreactivity. Patients with CLL who expressed BCRs that reacted with ≥5 epitope mimics had a significantly worse clinical course than less polyreactive patients (median time to first treatment, 24 months vs 102 months). This effect was independent of otherwise known prognostic markers.

CONCLUSIONS:

The authors introduced a system with which to model epitope reactivity of CLL BCRs without previous knowledge of potential antigens. The findings indicated that a polyreactive epitope recognition pattern may be a determinant of an aggressive clinical course in this disease. This further emphasizes the functional and prognostic relevance of BCR epitope recognition in CLL. Cancer 2011. © 2010 American Cancer Society.     

Limited specificity of xenoantibodies in diabetic patients transplanted with fetal porcine islet cell clusters. Main antibody reactivity against α-linked galactose-containing epitopes

Abstract: The immunological specificity of antibodies formed as a result of xenotransplantation of fetal porcine islet-like cell clusters to diabetic patients was characterized. High titer increases were recorded against porcine cells, solubilized membrane fractions of porcine cells, and purified MHC class I antigens. However, titer increases were also noted against ssDNA and dsDNA and against pig thyroglobulin but not against actin, myoglobin, or haptenated BSA. Antibody titers against tetanus toxoid were unaffected. The reactivity against porcine RBC could be completely blocked by absorption with pig thyroglobulin. Since pig thyroglobulin contains the galαl-3gal antigen, the reactivity against RBC was most probably mainly due to antibodies against this oligosaccharide epitope. The reactivity against porcine mononuclear cells was only partially absorbed by pig thyroglobulin, indicating a heterogeneity of the clonal response. These conclusions were substantiated by data showing that the antigenic determinants on pig thyroglobulin were completely destroyed by treatment with α- but not with β-galactosidase. Further studies showed that immune reactivity against pig RBCs, platelets, islet-cells, endothelial cells and pig MHC class I molecules, caused by xenoimmunization, was almost completely blocked by either absorption of antibodies on a column of Sepharose beads coated with galα1-3gal or by pretreatment of the antigen fractions with α-galactosidase. Western blot experiments revealed that both the natural and xenoimmune antibodies reacted with a large number of different glycoproteins. There was no difference in heterogeneity of the response when comparisons were made between pre-and posttransplantation sera, nor was there any difference of patterns caused by IgM or IgG antibodies. Absorption studies revealed that this epitope was present in a large number of different glycoproteins, a conclusion verified by staining with the eluate from a thyroglobulin-immunosorbent column or with the eluate from the galα1-3gal-coupled Sepharose column. Our findings demonstrate that the xenoimmune response is mainly directed against oligosaccharide residues and that there is a limited clonal heterogeneity of antibodies in xenografted patients. There was no direct evidence of reactivity against any porcine proteins nor of specific immunization against porcine MHC peptides. The data form a basis for a future understanding of means to cope with and prevent the rejection of xenogeneic islet cells in man. We also suggest, based on the specificity found in xenoimmunized patients sera, that a major part of naturally occurring antibodies might be specifically reactive with carbohydrate antigens.