Delineation of spontaneous erythrocyte autoantibody responses of NZB and other strains of mice.

Four anti-erythrocyte autoantibody responses (anti-X, anti-HB, anti-HOL, and anti-I) that occur spontaneously in mice have been characterized with regard to antigenic specificities, predominant immunoglobulin class, and pathogenetic importance. Each autoantibody response exhibits specificity for an independent erythrocyte membrane autoantigen (X, HB, HOL, or I) or a soluble analogue (SEA-X or SEA-HB) present in the plasma. The anti-X response, unique to NZB mice, is directed to a normally exposed murine erythrocyte autoantigen, whereas the anti-HB response is directed to a cryptic erythrocyte autoantigen exposed by limited enzymatic cleavage of the membrane. The anti-I response also is directed to a cryptic but distinct autoantigen, and anti-HOL autoantibodies react with an erythrocyte autoantigen located at the cytoplasmic surface of the membrane. Analysis of the predominant immunoglobulin class of each of the autoantibodies has demonstrated that anti-HB and anti-I antibodies are predominantly of IgM class, whereas anti-X and anti-HOL antibodies are IgG immunoblobulins. Only anti-X and anti-HB autoantibodies are recovered from Coombs' positive erythrocytes from NZB mice and erythrocytes with surface C3 are detected only in NZB mice greater than 9 months of age. These data suggest that only the anti-X and anti-HB responses are pathogenetically implicated in the autoimmune hemolytic anemia of NZB mice.     

Specific antigen-binding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice.

The receptor characteristics as well as incidence of antigen-binding lymphocytes (ABL) or B and T cell classes with membrane receptors specific for the exposed (X) and cryptic (HB) murine erythrocyte autoantigens were examined in NZB and nine control strains of mice. Whereas only NZB and NZB hybrid mice synthesize anti-X autoantibody pathogenetically implicated in the genetically determined autoimmune hemolytic anemia, the NZB as well as control strains synthesize the ubiquitous anti-HB anti-erythrocyte autoantibody. By utilizing immunocytoadherence assays, maximum numbers of specific ABL of both B and T lymphocyte classes were optimally demonstrated at erythrocyte:lymphocyte ratios of 20:1 and after lymphocyte fixation at 56 degrees C for 20 min. Surface membrane receptor specificity was established by inhibition with semi-purified soluble X or HB autoantigen. Inhibition of immunocyto-adherence with class specific antisera to mouse immuno-globulins demonstrated that the receptors on both B and T cells were of IgM class. Specific receptors regenerated in vitro after trypsinization which excluded the role of cytophilic antibody in the immunocytoadherence reactions. B lymphocyte ABL reactive with the X autoantigen were demonstrable in NZB, NZB hybrid, and control mice. Only in NZB and NZB hybrid mice, strains that uniformly synthesize anti-X autoantibody, were X ABL of T lymphocyte class demonstrated. The presence and incidence of T lymphocyte X ABL is compatible with the expression of a single dominant gene carried by the NAB strain. The incidence of B lymphocyte X ABL increased with age, suggesting proliferation of this cell population. HB ABL of both B and T lymphocyte classes were observed in all strains, concordant with the ubiquitous presence of humoral anti-HB autoantibodies. Differentiation of precursor B cells are evaluated by PFC assay of cells secreting specific autoantibodies. Anti-X PFC were observed only in NZB and NZB hybrid mice; and the observed frequency suggested that less than 3.5% of the specific ABL were differentiated for the secretion of anti-X autoantibody. Anti-HB PFC were observed in all strains and represented as high as 11.8% of specific ABL. Genetic determination of the anti-X anti-erythrocyte autoantibody response does not prescribe the presence of precursors of the antibody-forming cell, but rather appears to influence regulation of the differentiation of these cells. These data suggest that circumvention of immunologic tolerance to this specific erythrocyte autoantigen may occur at the level of the T lymphocyte; or alternatively, that T lymphocytes as well as B lymphocytes, are induced to proliferate and differentiate in the NZB strain.     

Autoantibodies against bromelainized mouse erythrocyte: strain distribution of serum idiotype expression and relative peritoneal cell activity

Previously, we demonstrated that the naturally occurring mouse autoantibodies directed against bromelainized mouse red blood cells (BrMRBC) comprised a family of structurally related molecules bearing a common idiotypic determinant (CP) based on structural and idiotypic analysis of a series of anti-BrMRBC monoclonal autoantibodies derived from a fusion of peritoneal cells (PerC) with plasmacytomas. In the present studies, we have evaluated the quantitative expression of circulating CP idiotype related to autoantibodies against BrMRBC in relation to specific PerC anti-BrMRBC plaque-forming activity in an individual mouse of different strains. The data presented here show no direct relationship between serum CP idiotype expression and PerC anti-BrMRBC plaque-forming activity in an individual mouse of all strains tested. However, the circulating CP idiotype content is higher in strains, viz., CBA/J, NZB, C3H, BXSB, and Biozzi high responder (H) mice which exhibit a high perC autoantibody secretory activity against BrMRBC. The strains such as BALB/c, DBA2, SJL/J, CBA/N, and Biozzi low responder (L) express little or no circulating CP idiotype with a corresponding small or no PerC anti-BrMRBC activity. Furthermore, the PerC "auto"-immune phenomenon is markedly expressed in the normal CBA/J strain since these mice show a higher percentage ratio of CP idiotype over serum IgM (2.68%) as well as highest PerC anti-BrMRBC plaque-forming activity (11,319 +/- 18,029 plaques per million viable cells) compared to other normal and autoimmune strains tested. Nevertheless, the highest circulating serum CP idiotype (49.4 micrograms/ml) is observed in the autoimmune NZB mouse. The immunodeficient CBA/N mice fail to express detectable levels of CP idiotype in their serum. The experiments conducted in genetically selected outbred Biozzi (H and L) strain have revealed remarkable differences in serum CP idiotype expression as well as PerC anti-BrMRBC plaque-forming activity in these two lines. The expression of mouse PerC "auto"-immune phenomenon and quantitative circulation of CP idiotype in the serum seem to be related to regulatory mechanisms as for sheep erythrocytes and other natural antigens earlier demonstrated to be under polygenic regulation in Biozzi (H and L) mice.     

A pathogenic monoclonal antibody, G8, is characteristic of antierythrocyte autoantibodies from Coombs'-positive NZB mice.

With age, NZB mice develop anti-RBC autoantibodies resulting in the development of autoimmune hemolytic anemia. We now have evidence that this spontaneous autoantibody response consists of antibodies that are similar in specificity and Id expression to a pathogenic autoantibody (G8) that was cloned from an autoimmune NZB mouse. Similar to autoantibodies eluted from Coombs'-positive mouse E (MRBC), the G8 mAb recognizes native (unmodified) MRBC but not RBC from other species. Interestingly, G8 and four additional mAb bind with a higher titer to bromelain-treated MRBC than to native MRBC. Nucleotide sequence analysis reveals, however, that unlike "natural" antibodies that react solely with bromelain-MRBC, G8 is encoded by a J558 VH gene and a V kappa 12,13 L-chain gene. Thus G8 is clearly distinct from antibodies to bromelain-MRBC which are encoded by unrelated V genes. Instead, the sequence of the G8 VH chain was found to be nearly identical to that of an anti-DNA mAb derived from an MRL-lpr/lpr mouse. The results suggest Coombs'-positive autoantibodies from NZB mice are not derived from "natural" antibodies, but rather, consist of a restricted set of autoantibodies expressing the G8 IdX.     

Genetic control of glycoprotein 70 autoantigen production and its influence on immune complex levels and nephritis in murine lupus

The F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop an autoimmune disease that serves as a model for human systemic lupus erythematosus. Autoimmunity in (NZB x NZW)F1 mice includes the production of autoantibodies to the endogenous retroviral envelope glycoprotein, gp70, and gp70-anti-gp70 immune complexes (gp70 IC) have been implicated in the development of lupus nephritis in these animals. We used backcross and intercross combinations of C57BL/6 (B6; low gp70 levels) and NZB mice (high gp70 levels) to examine the contribution of serum gp70 Ag levels to the development of gp70 IC and nephritis. Analysis of (B6.H2z x NZB)F1 x NZB backcross mice and (NZB x B6)F2 mice showed a much stronger association of gp70 IC with kidney disease compared with IgG anti-chromatin autoantibodies in both populations of mice. Serum levels of gp70 correlated with production of gp70 IC in mice producing autoantibodies, although the overall effect on nephritis appeared to be small. Genetic mapping revealed three NZB-derived regions on chromosomes 2, 4, and 13 that were strongly linked with increased gp70 levels, and together, accounted for over 80% of the variance for this trait. However, additional linkage analyses of these crosses showed that loci controlling autoantibody production rather than gp70 levels were most important in the development of nephritogenic immune complexes. Together, these studies characterize a set of lupus-susceptibility loci distinct from those that control autoantibody production and provide new insight into the components involved in the strong association of gp70 IC with murine lupus nephritis.     

IgG1 and IgG2a production by autoimmune B cells treated in vitro with IL-4 and IFN-gamma

Autoimmune MRL-lpr/lpr and NZB/W mice spontaneously secrete large quantities of pathogenic IgG1 and IgG2a autoantibodies. NZB mice also produce autoantibodies but these tend to be of the IgM H chain class. This work examines whether differences in the isotype of autoantibody produced by lupus-prone mice reflects differences in the sensitivity of autoreactive B cells to lymphokine-mediated IgG secretion. Twenty-five percent of normal BALB/c B cells produced IgG1 when stimulated in vitro with IL-4 plus LPS. This was comparable with the effect of IL-4 on small resting B cells from MRL-lpr/lpr and NZB/W mice. In contrast, less than 8% of the resting B cells from NZB mice produced IgG1 under these conditions. LPS plus IFN-gamma induced 5% of BALB/c and NZB/W but only 1% of NZB B cells to secrete IgG2a. Because lymphocytes from both young and old NZB mice showed diminished IgG1 and IgG2a secretion after lymphokine treatment, B cells from this strain appeared to be intrinsically resistant to the effects of IL-4 and IFN-gamma. In contrast, a disproportionately large proportion (22%) of B cells from adult MRL-lpr/lpr mice produced IgG2a when treated with IFN-gamma in vitro. Only B cells from MRL-lpr/lpr mice with active disease responded with such high levels of IgG2a production: cells from animals that had not yet developed clinical disease produced normal levels of IgG2a. Within each strain, B cells producing antibodies against autoantigens such as DNA, bromelain-treated mouse RBC and Sm responded to treatment with IL-4 and IFN-gamma in a manner indistinguishable from B cells producing antibodies against conventional Ag such as TNP and ARS.     

Studies of congenitally immunologically mutant New Zealand mice. IX. Age-related microenvironmental effects on autoantibody production in NZB and NZB.Xid mice studied by transplantation

The mechanism of polyclonal expansion of B cells and subsequent autoantibody production in New Zealand mice remains a critical question. We have been studying the requirements for autoantibody production both in NZB mice as well as NZB mice congenic with the Xid gene of CBA/N mice. In this study, we have attempted to alter the immunologic phenotype of NZB.Xid mice by transfer of cells from young and old NZB mice. There was little difficulty in restoring normal levels of serum IgM, IgG3, splenic Lyb-5 cells, and response to DNP-Ficoll in young NZB.Xid mice that were injected with young NZB bone marrow cells. Although such animals had an almost immediate change in their immune profile to values characteristic of NZB mice, they required, much like unmanipulated NZB mice, a latency period of an additional 6 mo before autoantibodies were detected. In contrast, adult NZB.Xid mice, who likewise developed an immune profile similar to NZB after transfer of bone marrow cells from young NZB mice, began to express autoantibodies immediately without any latency period. NZB.Xid mice who were recipients of adult NZB bone marrow cells did not show sustained autoantibody production, reflecting the limited state of B cell precursors in adult NZB mice. Thus, the age of both donor cells and the age of recipient mice are critical factors for determining the latency period and the age at which autoantibodies will appear. Similarly we attempted to alter the production of autoantibodies in NZB mice that were irradiated and injected with bone marrow cells from NZB.Xid animals. NZB mice had a major amelioration of disease when they received cell transfers from young NZB.Xid mice. This amelioration, which included the acquisition of the immune profile of NZB.Xid animals, was not seen in adult NZB mice that were recipient of young NZB cells. We suggest that although Lyb-5 cells may be the effective mechanism for autoantibody production, there are other interacting influences that may selectively turn on or turn off autoantibodies and that are required and are responsible for the latency period.     

Evidence that autoimmunity in NZB mice is caused by a defect in immune specificity rather than a generalized defect in tolerance of self-antigens

NZB mice which were already producing anti-erythrocyte autoantibodies were not able to respond to their own liver F antigen, thus providing evidence that their autoimmunity is not caused by a generalized breakdown in self-tolerance mechanisms. The specificity of autoantibodies produced in the spontaneous hemolytic anemia was different from that of antierythrocyte antibodies induced in normal mice and in young NZB mice by injections of rat erythrocytes. This indicates that the B-cell clones which can be triggered by heterologous antigen are different from those responsible for the NZB disease; the latter clones may not exist in normal mice.     

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.     

Expression and regulation of a recurrent anti-erythrocyte autoantibody idiotype in spleen cells from neonatal and adult BALB/c mice.

BALB/c spleen cells depleted of CD8+ T cells generate an autoantibody response to mouse RBC (MRBC) when cultured 5 days in the presence of syngeneic RBC. More than 80% of the cells secreting anti-MRBC antibody are blocked by an antiidiotypic mAb that recognizes the G8 Id. This G8 Id was originally identified in an autoimmune NZB mouse derived anti-MRBC mAb and later characterized as a dominant Id in NZB anti-MRBC autoantibodies. Furthermore, the CD8+ regulatory T cells that control this autoimmune response in BALB/c mice are specifically eliminated by cytotoxic treatment with the G8 mAb + C, suggesting that the regulatory cells recognize the G8 Id. Spleen cells from neonatal BALB/c mice, which lack those regulatory cells can generate an in vitro antibody response to MRBC without depletion of CD8+ cells. More than 80% of these AFC were also found to express the G8 Id. We propose that Id determinants on autoantibodies that are produced neonatally induce Id-specific regulatory cells that maintain peripheral tolerance to self-RBC throughout the life of normal animals.     

Restricted IgH V gene usage in the response to the ese epitope on "Hi" sheep red blood cells.

We had previously shown that the in vitro antibody response to a single epitope (ese; extra sheep E Ag) present on some sheep E but absent from others could be monitored by assay of the plaque-forming cell response on both Lo3 and Hi SRBC. We had shown also that the response was seen only in certain strains of mice and that the gene(s) controlling the response mapped to the IgH V region of the IgH chain complex. An additional feature of the response is that it is only seen in vitro and is absent and, we hypothesize, is suppressed in vivo. The strain distribution of the response to the ese determinant suggested that the response may only use one V gene (or a small set of closely related V genes) that would be present in the responder strains and absent from the nonresponder strains. To test this hypothesis, we made hybridomas with specificity for the ese determinant and for the shared determinants. cDNA from these hybridomas were sequenced. All four anti-ese hybridomas were almost identical in V region sequence, but varied considerably in D and J segment usage, thus confirming the hypothesis that the ese response would be limited at the V segment. The four anti-ese hybridomas used two Vh J558 genes that differed only by one, or possibly two, nucleotide(s). Importantly, these genes are quite different from most other published J558 sequences. The sequence is very similar to an unexpressed sequence from a C57Bl/6 perinatal mouse and slightly less similar to two other Vhb sequences. It was quite similar to two sequences from autoantibodies, one an anti-DNA hybridoma antibody, BXW-14, isolated from an NZB x NZWF1 mouse, and the other, an NZB hybridoma, G8, with specificity for a mouse E Ag. We speculate that the Ig encoded by the V ese gene react with an autoantigen, that the B cells persist in the animal, but that the secretion of Ig is somehow suppressed.     

Interclonal and intraclonal diversity among anti-DNA antibodies from an (NZB x NZW)F1 mouse

The immunologic basis for the generation of autoantibodies that are characteristic of systemic autoimmunity in mice and humans remains obscure. Experiments directed toward the analysis of serum antibody and the cell populations that combine to generate antibody in autoimmune mice have led to the proposition that autoantibody production, including anti-DNA, results from the nonselective, polyclonal activation of B cells. The present results from the molecular analyses of anti-DNA autoantibodies from an individual (NZB x NZW)F1 autoimmune mouse, however, are inconsistent with a clonally nonselective model for autoantibody production and are most consistent with a clonally selective, Ag-driven model for anti-DNA autoantibody production. These results demonstrate that Ig V region structures contributed by germ-line V region genes; recombinational diversity, including unusual DH gene usage and DH-DH recombination; and somatic mutation during B cell clonal expansion are all important for generating antibody and presumably B cell Ig receptor specificity for nucleic acids including native, duplex DNA.     

Lupus prone (SWR x NZB)F1 mice produce potentially nephritogenic autoantibodies inherited from the normal SWR parent

The incidence of nephritis in autoimmune NZB mice is low, but when they are crossed with normal SWR mice, almost 100% of the female F1 hybrids (SNF1) develop lethal glomerulonephritis. To define the contribution of the normal SWR strain to the development of nephritis, we analyzed 65 monoclonal anti-DNA autoantibodies derived from SNF1 mice and compared them with those obtained from the NZB parent. The majority of the SNF1-derived anti-DNA antibodies were IgG and cationic in charge. By contrast, 77% of the NZB-derived antibodies were IgM. Moreover, all three NZB-derived IgG anti-DNA antibodies were anionic. The cationic property of the SNF1-derived IgG autoantibodies was not restricted to any particular antigenic specificity pattern or IgG subclass, nor was there a preference for the allotype of either parent. However, we identified a set of highly cationic (pI at 8.2 to 8.8 pH) IgG2b anti-DNA antibodies from SNF1 hybrids that had the SWR allotype. Isoelectric focusing of intact antibodies and isolated heavy and light chains showed that the highly cationic charge of these antibodies was determined by the variable regions of their heavy chains. Because IgG anti-DNA antibodies with cationic charge are especially pathogenic, those antibodies bearing the allotype of the normal SWR parent may account for the high incidence of severe nephritis in the F1 hybrids. The results indicate that pathogenic autoantibodies, which are encoded by genes of the nonautoimmune SWR parent, are expressed in the SNF1 mice due to some cellular and genetic regulatory influence of the NZB parent.     

Effects of MHC and gender on lupus-like autoimmunity in Nba2 congenic mice

The lupus-like disease that develops in hybrids of NZB and NZW mice is genetically complex, involving both MHC- and non-MHC-encoded genes. Studies in this model have indicated that the H2d/z MHC type, compared with H2d/d or H2z/z, is critical for disease development. C57BL/6 (B6) mice (H2b/b) congenic for NZB autoimmunity 2 (Nba2), a NZB-derived susceptibility locus on distal chromosome 1, produce autoantibodies to nuclear Ags, but do not develop kidney disease. Crossing B6.Nba2 to NZW results in H2b/z F1 offspring that develop severe lupus nephritis. Despite the importance of H2z in past studies, we found no enhancement of autoantibody production or nephritis in H2b/z vs H2b/b B6.Nba2 mice, and inheritance of H2z/z markedly suppressed autoantibody production. (B6.Nba2 x NZW)F1 mice, compared with MHC-matched B6.Nba2 mice, produced higher levels of IgG autoantibodies to chromatin, but not to dsDNA. Although progressive renal damage with proteinuria only occurred in F1 mice, kidneys of some B6.Nba2 mice showed similar extensive IgG and C3 deposition. We also studied male and female B6.Nba2 and F1 mice with different MHC combinations to determine whether increased susceptibility to lupus among females was also expressed within the context of the Nba2 locus. Regardless of MHC or the presence of NZW genes, females produced higher levels of antinuclear autoantibodies, and female F1 mice developed severe proteinuria with higher frequencies. Together, these studies help to clarify particular genetic and sex-specific influences on the pathogenesis of lupus nephritis.     

Role of the chemokine stromal cell-derived factor 1 in autoantibody production and nephritis in murine lupus

In normal mice, stromal cell-derived factor 1 (SDF-1/CXCL12) promotes the migration, proliferation, and survival of peritoneal B1a (PerB1a) lymphocytes. Because these cells express a self-reactive repertoire and are expanded in New Zealand Black/New Zealand White (NZB/W) mice, we tested their response to SDF-1 in such mice. PerB1a lymphocytes from NZB/W mice were exceedingly sensitive to SDF-1. This greater sensitivity was due to the NZB genetic background, it was not observed for other B lymphocyte subpopulations, and it was modulated by IL-10. SDF-1 was produced constitutively in the peritoneal cavity and in the spleen. It was also produced by podocytes in the glomeruli of NZB/W mice with nephritis. The administration of antagonists of either SDF-1 or IL-10 early in life prevented the development of autoantibodies, nephritis, and death in NZB/W mice. Initiation of anti-SDF-1 mAb treatment later in life, in mice with established nephritis, inhibited autoantibody production, abolished proteinuria and Ig deposition, and reversed morphological changes in the kidneys. This treatment also counteracted B1a lymphocyte expansion and T lymphocyte activation. Therefore, PerB1a lymphocytes are abnormally sensitive to the combined action of SDF-1 and IL-10 in NZB/W mice, and SDF-1 is key in the development of autoimmunity in this murine model of lupus.     

The presence of autoantibodies specific for NZB serum factors in adult NZB mice and the establishment of monoclonal autoantibodies against these humoral factors

Humoral factors in serum of young NZB mice enhance maturation of B-lymphocyte precursors in vitro. A blot ELISA assay identified autoantibodies against the serum factors. NZB-SFs (designated NZB-SF alpha, pI 3.5-4.0, and NZB-SF beta, pI 7.8) were purified by sequential steps. Both had a molecular weight (MW) of approximately 23,000 in SDS-PAGE. NZB mice develop autoantibodies against NZB-SFs by 2 months of age; titers increased progressively with age. Non-autoimmune-prone mice did not produce autoantibodies against NZB-SFs. We then developed two hybridoma clones, IIC1C1 and IIC1M4, which produce monoclonal autoantibodies against NZB-SF alpha and NZB-SF beta, respectively. Both IgM autoantibodies could be affinity purified with a column of CNBr-Sepharose 4B gel conjugated with anti-mouse IgM antibody. Neither IIC1C1 nor IIC1M4 abolished bioactivity of recombinant mouse IL-1 alpha, human IL-1, mouse, rat, or human IL-2, mouse IL-3, or colony-stimulating factor. Neither antibody reacted to recombinant mouse IL-1 alpha, IL-4, TNF alpha, or IFN gamma in blot ELISA assays. Monoclonal autoantibodies IIC1C1 and IIC1M4 were used to purify NZB-SFs. SDS-PAGE of the affinity-purified NZB-SFs revealed bands of 23 and 60 kDa, and proteins extracted from the bands were reactive to our monoclonal autoantibodies.     

Haemagglutinating activity of Campylobacter pylori

Abstract Thirty-one isolates of Campylobacter pylori , screened for their ability to agglutinate a panel of erythrocyte species, could be divided into two phenotypic groups on the basis of their ability to agglutinate human A and O erythrocytes, a property which correlated strongly with their ability to agglutinate horse and cat erythrocytes. Isolates which agglutinated human red blood cells exhibited a broad-spectrum haemagglutination profile on other red blood cells including dog, goat, guinea-pig, ox, rat and sheep erythrocytes. Agglutination dog, guinea-pig, horse and human erythrocytes by C. pylori was mannose-resistant. Haemagglutination was not inhibited by other saccharides tested nor by two glycoproteins or serine. The bacterial ligand was protease- and heat-sensitive. Neither protease nor neuraminidase treatment of erythrocytes prevented agglutination.     

Regulation of self-recognition by T-cell hybrids

Somatic cell hybrids were prepared between BW 5147, an AKR T lymphoma, and purified T cells from three sources: spleen cells exposed to sheep red blood cells, lymph node cells from mice sensitized to ovalbumin, and spleen cells of mice injected with azobenzenearsonate-IgG. Hybrid lines expressed constitutive markers of both parents which include H-2 antigens and the isoenzymes glucose phosphate isomerase and isocitrate dehydrogenase. Furthermore, they expressed both parental alleles of Thy 1, a differentiation antigen. Many of the hybrid lines formed rosettes with mouse erythrocytes. T-cell hybrids did not bind human or chicken red blood cells, though they did rosette with sheep erythrocytes to the same extent as with mouse red cells. We interpret the latter reaction as due to recognition of shared antigens by the murine T cells. This form of self-recognition is influenced by culture conditions and is expressed optimally by cells in late logarithmic phase of growth.     

Proliferation of anti-DNA-producing NZB B cells in a non-autoimmune environment

This study demonstrates that purified NZB B cells, but not other NZB spleen cell populations, are capable of transferring anti-DNA antibody production into unirradiated H-2-compatible xid recipients. The number of autoantibody-producing B cells and the concentration of anti-DNA antibody found in the recipients correlated directly with the number of NZB B cells transferred. In addition, the number of anti-DNA-secreting lymphocytes found in the xid hosts increased exponentially with time post cell transfer. Several lines of evidence suggest that this phenomenon reflected the rapid proliferation of donor NZB B cells in the xid environment. Significantly, such proliferation was characteristic of donor cells that produced autoantibodies, but not of splenic B cells as a whole. These results suggest that stimulated NZB B cells can both induce and perpetuate autoantibody production in a normally non-autoimmune environment and in the absence of autoimmune helper cells.     

Histone autoantigens in murine lupus. Definition of a major epitope within an accessible region of chromatin.

In SLE and in the (NZB x NZW)F1 murine model of this disease, IgG autoantibodies are frequently produced to DNA and histones. In the present study, we define a linear epitope on histone H2B that is recognized by (NZB x NZW)F1 mice. IgG antibodies from anti-H2B positive (but not anti-H2B negative) mice bound strongly to a peptide containing the first 15 N-terminal amino acids, a region that is exposed in chromatin. Competitive inhibition studies showed that the binding of autoantibodies to H2B in ELISA as well as the binding to soluble H2B was substantially blocked by this peptide. Studies with smaller peptides mapped the epitope to residues 3-12. Individual mice recognized different residues within this region, and a sequence search did not reveal proteins other than H2B that could elicit this spectrum of antibodies. Interestingly, these autoantibody specificities were not a component of those induced in preautoimmune mice by immunization with H2B/RNA complexes or with H2B peptide 1-30 containing the autoantigenic sequence. These findings argue that recognition of a specific N-terminal region of self histone contributes to the anti-H2B autoantibody response in lupus. Autoreactive B cells with specificity for this sequence seem to develop only after the autoimmune process has been initiated.