A quantitative ELISA procedure for the measurement of membrane-bound platelet-associated IgG (PAIgG)

A quantitative ELISA assay for the measurement of in vivo bound platelet-associated IgG (PAIgG) using intact patient platelets is presented. The assay requires quantitation and standardization of the number of platelets bound to microtiter plate wells and an absorbance curve using quantitated IgG standards. Platelet-bound IgG was measured using an F(ab')2 peroxidase labeled anti-human IgG and o-phenylenediamine dihydrochloride (OPD) as the substrate. Using this assay, PAIgG for normal individuals was 2.8 +/- 1.6 fg/platelet (mean +/- 1 SD; n = 30). Increased levels were found in 28 of 30 patients with clinical autoimmune thrombocytopenia (ATP) with a range of 7.0-80 fg/platelet. Normal PAIgG levels were found in 26 of 30 patients with nonimmune thrombocytopenia. In the sample population studied, the PAIgG assay showed a sensitivity of 93%, specificity of 90%, a positive predictive value of 0.90, and a negative predictive value of 0.93. The procedure is highly reproducible (CV = 6.8%) and useful in evaluating patients with suspected immune mediated thrombocytopenia.     

Characterization of host CD4+ T lymphocytes in mice neonatally tolerized to alloantigens

BALB/c mice injected at birth with semi-allogeneic F1 spleen cells become tolerant to alloantigens as shown by their CTL unresponsiveness to the corresponding alloantigen and the persistence of donor F1 cells into the BALB/c host. Moreover, these mice develop a transient systemic lupus erythematosis-like autoimmune syndrome characterized by splenomegaly, glomerulonephritis, thrombocytopenia and abnormal serological findings, such as several autoantibodies and IgG1 hypergammaglobulinemia. Recent studies done in our laboratory have shown that donor F1 B cells persisting in the host are responsible for the production of autoantibodies and must be activated in vivo by the host CD4⁺ T lymphocytes in a MHC class II-restricted fashion. In the present work, we have focused our attention on the ability of splenic CD4⁺ T cells recovered at different periods from BALB/c mice injected at birth with (CBA/Ca × BALB. Igh^b) Fl spleen cells to interact with and activate F1 semi-allogeneic spleen cells in vitro. We show that (i) only CD4⁺ T cells from 2- and 3-week-old tolerant BALB/c mice preferentially produce IL-4 and IL-5 in response to a F1 semi-allogeneic in vitro stimulation, (ii) CD4⁺ T cells purified from 3-week-old tolerant BALB/c mice are able to induce in vitro IgG and IgM production by F1 B cells. Taken together, these results strongly suggest that host CD4+ T cells, belonging to the TH2 subset progressively lose their reactivity towards the F1 semi-allogeneic persistent B cells, reaching a state of unresponsiveness that correlates with the disappearance of serum autoantibodies and autoimmune pathology.     

Autoimmune syndrome after induction of neonatal tolerance to alloantigens: analysis of the role of donor T cells in the induction of autoimmunity.

The injection of (C57BL/6 x BALB/c) F1 spleen cells into BALB/c newborn mice leads to activation of persisting F1 donor B cells and development of a lupus-like syndrome in tolerized BALB/c mice. This syndrome is characterized by hypergammaglobulinaemia, high levels of anti-DNA and anti-Sm antibodies, circulating immune complexes and deposits of immunoglobulin in renal glomeruli. The role of donor T cells in this model was investigated by injecting the newborn mice with F1 cells depleted in different T cell subsets by using specific monoclonal antibodies (MoAbs). Tolerance, as shown by an absence of H-2b-specific CTL alloreactivity and persistence of immunoglobulin bearing the donor allotype were observed in mice injected with F1 cells previously depleted in the CD4+ and/or CD8+ T cell subsets as well as in those which received Thy-1+-depleted F1 spleen cells. In these mice, a typical autoimmune syndrome was found, including splenomegaly and lymphadenopathy, anti-ssDNA and anti-aortic myosin IgG antibodies and renal deposition of immunoglobulin. However, some quantitative changes were seen: the levels of anti-aortic myosin antibodies were lower in mice tolerized with CD4+-depleted F1 cells than in those receiving untreated F1 cells. Conversely, higher levels of these autoantibodies were observed in mice tolerized with CD8+-depleted F1 cells. These results suggest that mature donor T cells are not necessary neither for the establishment of neonatal tolerance to alloantigens nor for the activation of F1 donor B cells in the production of the autoimmune syndrome in tolerant mice, but they may contribute in the regulation of the expression of autoreactive B cell clones.     

Self-limited autoimmune disease related to transient donor B cell activation in mice neonatally injected with semi-allogeneic F1 cells.

BALB/c mice injected at birth with 10(8) semi-allogeneic (C57BL/6 x BALB.IgHb)F1 spleen cells develop a lupus-like syndrome in which autoantibodies bear exclusively the donor allotype. We have analyzed the evolution of donor B cell chimerism and the autoimmune manifestations during the first year of life in these mice. Anti-DNA, -histone, and -cardiolipin IgG antibodies as well as circulating immune complexes appeared in the second week of life, reached the highest values around the sixth week, and then progressively dropped to normal values after the sixth month in most mice. The kinetics of the evolution of the autoimmune manifestations, as well as the kinetics of serum donor Ig allotype, were parallel to the kinetics of donor B cell chimerism, which was particularly prominent in the spleens in early weeks of life, and progressively decreased after remission of the autoimmune syndrome. Membrane-proliferative glomerulonephritis, which was followed as the more representative histological abnormality in this model, was particularly evident after 10 weeks of life, but disappeared by the end of the follow-up. Interestingly, when mice with a self-limited disease were re-injected with 10(8) F1 spleen cells i.v., a flare in the serological manifestations was observed. In these re-injected mice a predominance of anti-DNA, IgG1 antibodies bearing exclusively the donor allotype was also observed, as in the early weeks of life.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-idiotype antibodies neutralize in vivo the blistering effect of Pemphigus foliaceus IgG

Idiotypes are molecular clues used to explore the specificity and diversity of immune response. In the present study, anti-idiotype antibodies were used to neutralize the pathogenic effects induced by the injection of pemphigus immunoglobulin(Ig)G into BALB/c mice. To achieve our goal, antidesmoglein 1 IgG was obtained from a patient with pemphigus foliaceus with high titer of antiepithelial antibodies. The IgG was isolated by ion exchange chromatography, then digested by pepsin. F(ab')2 fragments were purified in Sephacryl S-300 and injected in rabbits to produce anti-idiotype antibodies. The rabbit sera reacted with the pemphigus F(ab')2 fragments. Eleven pemphigus foliaceus sera were recognized by the anti-idiotype serum at the light or heavy chains whereas bullous pemphigoid and normal IgG were negative. Neonatal BALB/c mice injected with pemphigus IgG developed intraepidermal blisters, mimicking the clinical and immunopathological features of the pemphigus. In contrast, the animals treated with anti-idiotype antibodies and pemphigus IgG did not develop blisters. Thus, anti-idiotype antibodies neutralize in vivo the pathogenic effects of pemphigus IgG.     

Persistence of anti-donor allohelper T cells after neonatal induction of allotolerance in mice

BALB/c mice rendered tolerant to A/J alloantigens by neonatal injection of 10(8) (A/J X BALB/c)F1 spleen cells develop an autoimmune disease associated with a polyclonal activation of donor B cells. To study the mechanisms leading to donor B cell activation in tolerant mice, we prepared mixed lymphocyte cultures (MLC) between splenic T cells from neonatally injected mice and donor-type (A/J X BALB/c)F1 or third-party (C57BL/6 X BALB/c)F1 B cells. T cells from tolerized mice were unable to generate cytotoxic T lymphocytes, to proliferate or to secrete interleukin (IL)2 after stimulation with donor alloantigens in MLC. These T cell responses were present after MLC with third-party antigens, but were of lower intensity than those generated by control BALB/c T cells. In contrast, T cells from tolerized mice stimulated immunoglobulin production by donor-type (A/J X BALB/c)F1 B cells much more powerfully than T cells from control BALB/c mice. The stimulation of donor-type (A/J X BALB/c)F1 B cells was polyclonal, as attested by the levels of anti-hapten and anti-DNA antibodies in the MLC supernatants. IgM was the dominant isotype secreted in vitro, but IgG1 and IgG3 were also produced in significant amounts. Lysis experiments indicated that the T cells responsible for F1 B cell stimulation in MLC were CD4+ host T cells. These T helper cells were alloreactive since they did not stimulate syngeneic BALB/c B cells, and their effect on donor B cells was specifically blocked by anti-donor Ia monoclonal antibodies. Addition of anti-IL 4 monoclonal antibody to MLC between T cells from tolerant mice and (A/J X BALB/c)F1 B cells almost completely abolished the production of IgG1, but not that of IgM or IgG3. Taken together, these findings indicate that neonatal injection of alloantigens in BALB/c mice induces a state of dissociated tolerance, with unresponsiveness of anti-donor T cells secreting IL 2 on the one hand, and persistence of T cells responsible for B cell help and IL 4 secretion on the other hand.     

Expression of anti-DNA clonotypes and the role of helper T-lymphocytes during the autoimmune response in mice tolerant to alloantigens

BALB/c mice neonatally injected with semiallogeneic (C57BL/6 x BALB/c) F1 spleen cells become tolerant to C57BL/6 alloantigens and exhibit chimaerism due to persistence of F1 lymphocytes. Such mouse chimaeras develop an autoimmune (lupus-like) disease characterised by hypergammaglobulinaemia with production of autoantibodies against DNA, Sm antigen and other self-antigens characteristic of SLE in addition to circulating immune complexes and glomerular deposition of immunoglobulins. We have studied the autoimmune response by analysing the isoelectric focusing (IEF)+ patterns (spectrotypes) of anti-ss and anti-dsDNA antibodies produced by these animals. The results show that the anti-DNA response is remarkably restricted, only a very small number of lymphoid cell clones responding in the majority of animals. The behaviour of these clones has been followed during the development of the autoimmune response by analysis of their individual IEF patterns (clonotypes). The first appearance of clones secreting anti-DNA autoantibodies was observed in 3-4 week old mice. Changes in spectrotype occurred during the course of the response but they remained restricted to a very small number of clones in almost all the animals studied. Changes in clonotype consistent with somatic mutation in committed, anti-DNA-secreting clones were also observed. Helper T-lymphocytes of host origin are shown to be required for the development of an autoimmune response.     

Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies.

BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.     

Selective expansion of idiotype sharing T and B cells in cyclosporin A-mediated autoimmunity.

CBA/N mice submitted to autologous bone marrow reconstitution after lethal irradiation and simultaneous Cyclosporin A (CsA) treatment develop a chronic graft-versus-host disease with autoimmune characteristics. When compared to normal controls, diseased mice show an overrepresentation of V beta 8-expressing T cells (65-80% of all CD3+ lymphocytes), together with a marked increase in the titres of serum Ig that specifically bind to F(ab')2 fragments of anti-V beta 8 F23.1 antibodies. Such 'V beta 8-like' Ig V regions are abundantly represented among the IgG2b and mAbs of an unselected collection of hybridomas derived from these mice. These mAbs are not multireactive Ig as they fail to bind to a panel of various antigens and antibodies, but often show simultaneous reactivity with anti-idiotypic mAbs to F23.1 and auto-binding. These molecules may provide the structural basis of V-region specific complementarities, driving the expansion of restricted T and B cell repertoires associated with pathological autoimmunity.     

The use of immunodeficient male (CBA/N x BALB/c) F1 mice to produce monoclonal antibodies directed to proteins of Leptospira interrogans rather than to immunodominant lipopolysaccharides.

A method, using an immunodeficient mouse strain, for the production of monoclonal antibodies directed exclusively against the proteins in an antigen mixture also containing immunodominant LPS, is described. Male (CBA/N x BALB/c) F1 mice were immunized with an outer envelope antigen mixture from Leptospira interrogans strain Wijnberg containing both lipopolysaccharides and proteins. The immune response in these mice was shown to be predominantly directed against protein antigens. Hybridoma cell lines were generated by fusing spleen cells from a (CBA/N x BALB/c) F1 mouse with BALB/c Sp2/0 plasmacytoma cells. Hybridoma cell lines producing monoclonal antibodies reacting with the outer envelope preparation were identified by ELISA. All epitopes recognized by the monoclonal antibodies are sensitive to proteinase K degradation and resistant to oxidation by periodate indicating that they are located on proteins. All epitopes are located on a 35 kDa protein and specific for the pathogenic L. interrogans species.     

Circulating levels of chromatin fragments are inversely correlated with anti-dsDNA antibody levels in human and murine systemic lupus erythematosus

Anti-dsDNA antibodies represent a central pathogenic factor in Lupus nephritis. Together with nucleosomes they deposit as immune complexes in the mesangial matrix and along basement membranes within the glomeruli. The origin of the nucleosomes and when they appear e.g. in circulation is not known. Serum samples from autoimmune (NZBxNZW)F1 mice, healthy BALB/c mice, patients with SLE, RA and normal healthy individuals were analyzed for presence and amount of circulating anti-dsDNA antibodies and nucleosomal DNA. Here we use a quantitative PCR to measure circulating DNA in sera. We demonstrate an inverse correlation between anti-dsDNA antibodies and the DNA concentration in the circulation in both murine and human serum samples. High titer of anti-DNA antibodies in human sera correlated with reduced levels of circulating chromatin, and in lupus prone mice with deposition within glomeruli. The inverse correlation between DNA concentration and anti-dsDNA antibodies may reflect antibody-dependent deposition of immune complexes during the development of lupus nephritis in autoimmune lupus prone mice. The measurement of circulating DNA in SLE sera by using qPCR may indicate and detect the development of lupus nephritis at an early stage.     

Relationship between the synthesis of autoimmune antibodies and the formation of clusters of B,T and APC cells during the syngeneic mixed lymphocyte reaction in BALB/c and NZB mice: A technique for isolation of the spleen autoimmune compartment of non-immunized pathogen-free mice

Cells from the spleens of non-immunized mice were cultured in horizontal tubes, rotating very slowly around their long axis. Under these conditions, the flux speed gradient of the cell suspension near the tube walls greatly increased the chances of cells coming into contact with one another. Mixed clusters of B, T and APC cells were soon found adhering firmly to the walls of the tube; cluster formation leveled off after about 3 h. The clustered cells were easily separated from those remaining in suspension and constituted a particular cell compartment comprising a maximum of 20–30 % of the total.B cells from this compartment, cultured in complete medium for 48 h, almost exclusively produced IgM antibodies. Antibodies reacting with self antigens were so numerous in the culture medium that it is likely all IgM were self antibodies.That the clusters obtained under these conditions constituted a compartment of autoimmune cells is supported by previous work which showed that 20–30 % of spleen cells secrete IgM antibodies almost exclusively.Cluster formation as a function of age was compared in NZB mice which are used as a model of lupus erythematosus, and in BALB/c mice which never manifest self-immune pathology. The number of cells found in clusters per whole spleen increased exponentially with age in NZB mice and linearly in BALB/c mice. The production of autoimmune antibodies as a function of age also increased exponentially for NZB mice and linearly in BALB/c mice, which provides further striking support for the hypothesis that the clusters formed constitute the autoimmune comportment.     

Relationship between the synthesis of autoimmune antibodies and the formation of clusters of B, T and APC cells during the syngeneic mixed lymphocyte reaction in BALB/c and NZB mice: a technique for isolation of the spleen autoimmune compartment of non-immunized pathogen-free mice

Cells from the spleens of non-immunized mice were cultured in horizontal tubes, rotating very slowly around their long axis. Under these conditions, the flux speed gradient of the cell suspension near the tube walls greatly increased the chances of cells coming into contact with one another. Mixed clusters of B, T and APC cells were soon found adhering firmly to the walls of the tube; cluster formation leveled off after about 3 h. The clustered cells were easily separated from those remaining in suspension and constituted a particular cell compartment comprising a maximum of 20-30% of the total. B cells from this compartment, cultured in complete medium for 48 h, almost exclusively produced IgM antibodies. Antibodies reacting with self antigens were so numerous in the culture medium that it is likely all IgM were self antibodies. That the clusters obtained under these conditions constituted a compartment of autoimmune cells is supported by previous work which showed that 20-30% of spleen cells secrete IgM antibodies almost exclusively. Cluster formation as a function of age was compared in NZB mice which are used as a model of lupus erythematosus, and in BALB/c mice which never manifest self-immune pathology. The number of cells found in clusters per whole spleen increased exponentially with age in NZB mice and linearly in BALB/c mice. The production of autoimmune antibodies as a function of age also increased exponentially for NZB mice and linearly in BALB/c mice, which provides further striking support for the hypothesis that the clusters formed constitute the autoimmune comportment.     

Capping and Co-Capping of Membrane Immunoglobulin and Lipid-Conjugated Immunoglobulin Inserted in the Cell Membrane of B Lymphocytes

Human and mouse immunoglobulins (Ig) or F(ab')2 fragments of rabbit Ig were conjugated to lipids and the conjugates inserted into the membrane of mouse spleen cells. It was found that nearly all B cells, but not T cells, became decorated with lipid immunoglobulin. Both endogenous Ig receptors and inserted Ig capped after the addition of cross-linking F(ab')2 antibodies and in both cases capping required energy. Capping of endogenous mouse Ig led to co-capping of inserted human Ig, but the reverse was not true.     

Selection of anti-F protein B-cell repertoires in normal mice

The hypothesis that self-tolerance to F protein antigen exclusively concerns T cells was tested by determining the frequencies of B lymphocytes producing anti-F antibodies in bone marrow (BM), spleen and peritoneal exudate (PEC) cells from normal, immune or tolerant animals, and in responder and non-responder mouse strains. Using an ELISA spot assay and lipopolysaccharide stimulation, we found that anti-F frequencies were highest in BM and “naturally activated” large spleen cells, followed by resting spleen and PEC cells. Anti-F specificities were also induced among “natural” Ig-secreting cells of normal individuals. Specific immunization of responder mice doubled the splenic frequencies, while tolerization had no effect. Similar results were obtained in BALB/c and A/J mice, while C57BL/6 contained fewer anti-F B cells in spleen, but not in BM. These results support the notion that self-tolerance to F antigen can primarily be ascribed to T cells, but they also show F-antigen-specific selection of B-cell repertoires.     

Idiotypes of monoclonal anti-DNA antibodies produced in autoimmune B/W mice are expressed in normal mice.

An anti-idiotypic antiserum was prepared in a rabbit immunized against a pool of six monoclonal anti-DNA antibodies generated in B/W mice. This antiserum detected idiotypic determinants in four of the six monoclonal anti-DNA antibodies but also in the serum of several non autoimmune strains (BALB/c, NZB X BALB/c) F1 hybrids & CBA/LH). The antiserum also reacted, but only to a weak degree, with B/W mouse sera. These results indicate that some idiotypes of anti-DNA antibodies produced by autoimmune B/W mice are present in normal mouse sera.     

Anti-LFA-1 (CD11a) monoclonal antibody interferes with neonatal induction of tolerance to alloantigens

The injection of (C57BL/6 × BALB/c)F1 spleen cells into newborn BALB/c mice results in the induction of a specific cytotoxic T lymphocyte (CTL) tolerance to the alloantigens. On the contrary, alloreactive CD4⁺ T cells persist in the host and are still able to activate autoreactive F1 B cells to produce autoantibodies. This state of “split tolerance” is closely associated with the development of a lupus-like autoimmune syndrome. The LFA-1 integrin plays a relevant role in homing, intercellular adhesion and tranduction of co-stimulatory signals in leukocytes. Because of the beneficial effects of anti-LFA-1 monoclonal antibodies (mAb) treatment in various models of organ transplantation and autoimmune disease, we have investigated if such a treatment could interfere with the induction of neonatal tolerance or the development of the autoimmune syndrome in F1 cell-injected newborn mice. For this purpose, BALB/c mice neonatally injected with F1 cells were treated from day 1 up to day 15 with a non-cytotoxic anti-LFA-1 (CD11a) mAb. Anti-LFA-1 mAb treatment interfered with the persistence of a stable chimerism and with the establishment of CTL tolerance, as shown by rejection of allogeneic skin grafts and F1 B cells, and by a normal in vitro CTL activity against the corresponding alloantigens. As a consequence, these mice did not develop the characteristic autoimmune features seen in close association with an effective induction of CTL tolerance to alloantigens. These results stress the importance of the interactions between LFA-1 and its ligands during the neonatal induction of tolerance to alloantigens.     

Selection of anti-F protein B-cell repertoires in normal mice

The hypothesis that self-tolerance to F protein antigen exclusively concerns T cells was tested by determining the frequencies of B lymphocytes producing anti-F antibodies in bone marrow (BM), spleen and peritoneal exudate (PEC) cells from normal, immune or tolerant animals, and in responder and non-responder mouse strains. Using an ELISA spot assay and lipopolysaccharide stimulation, we found that anti-F frequencies were highest in BM and "naturally activated" large spleen cells, followed by resting spleen and PEC cells. Anti-F specificities were also induced among "natural" Ig-secreting cells of normal individuals. Specific immunization of responder mice doubled the splenic frequencies, while tolerization had no effect. Similar results were obtained in BALB/c and A/J mice, while C57BL/6 contained fewer anti-F B cells in spleen, but not in BM. These results support the notion that self-tolerance to F antigen can primarily be ascribed to T cells, but they also show F-antigen-specific selection of B-cell repertoires.     

Termination of Immunologic Tolerance with Tolerated Antigen Complexed with Specific Antibody

Female NTH albino and BALB/c mice were tolerized with chicken egg lysozyme or capsular polysaccharide of Diplococcus pneumoniae type III (SIII). Isolated spleen cells from tolerant mice were incubated in vitro with antigen-antibody complexes formed with the tolerated antigen and specific xenogeneic, allogeneic and syngeneic immune serum. The spleen cells were washed and returned to the individual donor mice and their immune response to the tolerated antigen was assayed. In all cases one or more concentrations of specific antibody when complexed with the tolerated antigen was able to terminate the tolerant state. The successful use of specific antibodies raised in a syngeneic strain of mice (BALB/c) to terminate immunologic tolerance in other BALB/c mice with demonstrated T and B cell tolerance, precludes T cell recognition of a foreign antibody as a mechanism to explain these results.     

Idiotypic interactions between normal human polyspecific IgG and natural IgM antibodies

Pooled normal human polyspecific IgG (IVIg) contain anti-idiotypes against a variety of autoantibodies from patients with autoimmune diseases and IgG autoantibodies present in IVIg. The present study indicates that IVIg may also react through idiotypic/anti-idiotypic interactions with human natural IgM antibodies. Sixty-four percent of IgM secreted by B lymphoid cell lines derived from B cells of healthy elderly donors and 18% of IgM secreted by cloned EBV-transformed cord B cells that were tested, bound through their variable region to F(ab')2 fragments of IVIg. The binding to 2,4,6-trinitrophenyl (TNP) of a polyreactive IgM with anti-TNP specificity, was inhibited by F(ab')2 fragments from IVIg, indicating the presence in IVIg of anti-idiotypes that may interfere with the antibody-combining site of polyreactive IgM antibodies. The ability of IgM antibodies to interact with idiotypes on IVIg was not related to the degree of polyreactivity of natural antibodies. Our observations further document that IVIg contain antibody specificities against Ig from normal individuals and suggest that IgG originating from the physiologically expressed repertoire may modulate the expression of the potential B cell repertoire. The results may be relevant to the suppressive effect of IVIg in autoimmune diseases.