Linda Spatz - Profile on Academia.edu (original) (raw)
Papers by Linda Spatz
Mapping the epitope in Epstein Barr Virus nuclear antigen-1 that elicits cross-reactivity with dsDNA (101.43)
Journal of Immunology, Apr 1, 2011
A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)
Journal of Immunology, May 1, 2014
One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Bar... more One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. Anti-EBNA-1 antibody obtained from mice from all three strains was observed to cross-react with dsDNA. However, the frequency of mice producing cross-reactive antibody was greater and the amount of cross-reactive antibody was higher in BAFF than wild type mice. This suggests that a viral protein such as EBNA-1 may trigger the development of antibodies that cross-react with dsDNA and that genetic susceptibility to lupus may enhance the development of cross-reactive antibodies upon viral exposure.
Antibodies to a peptide derived from EBNA-1 may be a potential biomarker for SLE
Journal of Immunology, May 1, 2018
The Epstein Barr virus (EBV) has been associated with the autoimmune disease Systemic lupus eryth... more The Epstein Barr virus (EBV) has been associated with the autoimmune disease Systemic lupus erythematosus (SLE). We previously demonstrated that mice injected with EBNA-1, a major nuclear protein of EBV, develop anti-dsDNA antibodies that cross-react with EBNA-1. We generated two monoclonal antibodies, 3D4 and 16D2, from EBNA-1 injected mice, and showed that they bind to both EBNA-1 and dsDNA. We also identified a peptide, PFM-15, in EBNA-1 (PQPGPLRESIVCYFM) that is recognized by 3D4 and 16D2. We hypothesize that this peptide can act as a molecular mimic of dsDNA and elicit antibodies to EBNA-1 that cross-react with dsDNA in some individuals who develop lupus. In the present study, we screened the sera of 17 lupus patients and 19 healthy individuals to determine if they have antibodies to PFM-15. We observed a highly significant increase (p<.05) in the number of SLE patients that have antibodies to PFM-15 compared to the number of healthy individuals with these antibodies. Furthermore, statistical analysis revealed that the anti-PFM-15 test has an 80% positive predictive value for SLE suggesting potential to be used diagnostically to detect a subset of patients with lupus. We also observed that 75% of the lupus patients tested, who had antibodies to PFM-15, also had antibodies to dsDNA and a moderately strong correlation was observed between the level of antibodies to PFM and dsDNA in these patients. This is consistent with our observation that antibodies to PFM-15 cross-react with dsDNA. These studies suggest that antibodies to PFM-15 may be a useful biomarker for a subset of patients exposed to EBV who develop lupus. Future studies with a much larger cohort of patients and controls will be needed to confirm this.
Antibodies that bind to an EBV derived peptide mimic of dsDNA, have pathogenic potential
Journal of Immunology, May 1, 2018
Anti-DNA producing B cells from mice transgenic for a gamma-2b anti-DNA heavy chain express Vk1 light chains and are not allelically excluded
A Second Heavy Chain Permits Survival of High Affinity Autoreactive B Cells
Autoimmunity, Feb 1, 2004
Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibo... more Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibodies with this specificity may be generated in many individuals, only patients with SLE fail to regulate them effectively. We have demonstrated previously that in non-autoimmune mice transgenic for the heavy chain of the R4A-gamma2b anti-DNA antibody, the existence of high affinity, IgG2b dsDNA binding B cells is tightly correlated with the co-expression of endogenous IgM heavy chain. These cells are anergic. In contrast, low affinity IgG2b dsDNA binding B cells do not express an endogenous heavy chain and represent a population of immunocompetent autoreactive B cells. In order to determine whether the presence of a second heavy chain permits the high affinity autoreactive B cells to escape deletion, the R4A-gamma2b mouse was mated to a strain with a targeted deletion of the transmembrane portion of the mu heavy chain, muMT mice, to produce R4A-gamma2b/muKO mice. Serum titers of anti-DNA antibodies were negligible in both R4A-gamma2b and R4A-gamma2b/muKO mice. In R4A-gamma2b/muKO mice, however, LPS was able to activate a DNA-reactive population although an LPS inducible DNA-reactive population. Light chain gene usage in transgene expressing B cells from R4A-gamma2b/muKO mice was similar to that of the previously defined low affinity anti-DNA B cells that escape tolerance. These data suggest a requirement for a second heavy chain for the survival of this anergic B cell subset.
Lack of allelic exclusion permits autoreactive B cells to escape deletion
The Journal of Immunology
The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA ... more The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA Ab. Approximately 80% of B cells expressing the transgene display allelic exclusion. Although the transgenic mice have little to no detectable serum anti-DNA activity, splenic B cells can be stimulated in vitro with LPS to secrete anti-DNA Ab. Hybridomas derived from LPS-stimulated splenic B cells were analyzed for expression of the transgene and for DNA binding. All nine transgene-encoded anti-DNA-producing lines were found to express an endogenous IgM heavy chain. Of 19 randomly selected lines producing a transgene-encoded non-DNA binding Ab, none expressed a second heavy chain. The tight correlation between lack of allelic exclusion and anti-dsDNA specificity provides strong support for the hypothesis that a major function of allelic exclusion is to prevent the maintenance of a pool of potentially activatable autoreactive cells.
Mice immunized with EBNA-1 protein produce antibodies that cross-react with double stranded DNA (129.5)
The Journal of Immunology
Systemic Lupus Erythematosus (SLE) is characterized by the production of antibodies to nuclear an... more Systemic Lupus Erythematosus (SLE) is characterized by the production of antibodies to nuclear antigens, in particular antibodies to double stranded DNA (dsDNA). The etiology of these autoantibodies is unknown but one theory suggests that viruses may play a role. One virus that has been shown to have an association with SLE is the Epstein Barr Virus (EBV). We previously demonstrated that mice immunized with an EBNA-1 expression vector or with purified EBNA-1 protein develop antibodies to dsDNA as well as to the viral antigen. We have recently demonstrated that these antibodies are pathogenic and deposit in the kidney and induce moderate proteinuria. We hypothesized that since EBNA-1 is a dsDNA binding protein, it could complex with chromosomal dsDNA in order to render it immunogenic and elicit an anti-dsDNA response. To test this hypothesis we generated recombinant EBNA-1 protein that lacks the 3 known chromosomal binding sites and immunized mice with it. We observed that mice immun...
A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)
The Journal of Immunology
One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Bar... more One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. An...
Anti-DNA producing B cells from mice transgenic for a gamma-2b anti-DNA heavy chain express Vk1 light chains and are not allelically excluded
Antibodies to an Epstein Barr Virus protein that cross-react with dsDNA have pathogenic potential
Molecular Immunology, 2021
Pathogens such as the Epstein Barr virus (EBV) have long been implicated in the etiology of syste... more Pathogens such as the Epstein Barr virus (EBV) have long been implicated in the etiology of systemic lupus erythematosus (SLE). The Epstein Barr virus nuclear antigen I (EBNA-1) has been shown to play a role in the development of anti-nuclear antibodies characteristic of SLE. One mechanism by which EBV may play a role in SLE is molecular mimicry. We previously generated two monoclonal antibodies (mAbs) to EBNA-1 and demonstrated that they cross-react with double-stranded DNA (dsDNA). In the present study, we demonstrate that these mAbs have pathogenic potential. We show that they can bind to isolated rat glomeruli and that binding can be greatly diminished by pretreatment of glomeruli with DNase I, suggesting that these mAbs bind dsDNA in the kidney. We also demonstrate that these antibodies can deposit in the kidney when injected into mice and can induce proteinuria and elicit histopathological alterations consistent with glomerulonephritis. Finally, we show that these antibodies can cross-react with laminin and collagen IV in the extracellular matrix suggesting that direct binding to the glomerular basement membrane or mesangial matrix may also contribute to the antibody deposition in the kidney. In summary, our results indicate that EBNA-1 can elicit antibodies that cross-react with dsDNA, that can deposit in the kidney, and induce kidney damage. These results are significant because they support the role of a viral protein in SLE and lupus nephritis.
Proceedings of the National Academy of Sciences, 1997
Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit i... more Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit in the kidneys of lupus patients to cause glomerulonephritis. Recent data suggest that a significant proportion of anti-DNA antibodies may cross-react with renal antigens and be sequestered in the kidney by virtue of this cross-reactivity. If this is true, antigenic competition for pathogenic antibodies might prevent their deposition in kidneys and the ensuing tissue damage. To generate surrogate antigens that could be used for this purpose, we have used peptide display phage libraries to identify peptides that react with R4A, a pathogenic mouse monoclonal anti-DNA antibody that deposits in glomeruli. We have demonstrated that the peptides bind in or near the double-stranded DNA binding site. Furthermore, the peptides are bound preferentially by the R4A antibody as compared with two closely related antibodies derived from it, one of which deposits in renal tubules and one of which display...
Mapping the epitope in Epstein Barr Virus nuclear antigen-1 that elicits cross-reactivity with dsDNA
The Journal of Immunology, Apr 1, 2011
PLOS ONE, 2015
Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid... more Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the fivefold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe-reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of V κ and V H genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein's antigenic sites.
PLoS ONE, 2011
Background: Several genetic and environmental factors have been linked to Systemic Lupus Erythema... more Background: Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1. Methodology/Principal Findings: In this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated mononclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs-3D4, generated in this laboratory, and 0211, a commercial MAb-revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm. Conclusions/Significance: In conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.
Journal of Clinical Investigation, 2000
A Second Heavy Chain Permits Survival of High Affinity Autoreactive B Cells
Autoimmunity, 2004
Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibo... more Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibodies with this specificity may be generated in many individuals, only patients with SLE fail to regulate them effectively. We have demonstrated previously that in non-autoimmune mice transgenic for the heavy chain of the R4A-gamma2b anti-DNA antibody, the existence of high affinity, IgG2b dsDNA binding B cells is tightly correlated with the co-expression of endogenous IgM heavy chain. These cells are anergic. In contrast, low affinity IgG2b dsDNA binding B cells do not express an endogenous heavy chain and represent a population of immunocompetent autoreactive B cells. In order to determine whether the presence of a second heavy chain permits the high affinity autoreactive B cells to escape deletion, the R4A-gamma2b mouse was mated to a strain with a targeted deletion of the transmembrane portion of the mu heavy chain, muMT mice, to produce R4A-gamma2b/muKO mice. Serum titers of anti-DNA antibodies were negligible in both R4A-gamma2b and R4A-gamma2b/muKO mice. In R4A-gamma2b/muKO mice, however, LPS was able to activate a DNA-reactive population although an LPS inducible DNA-reactive population. Light chain gene usage in transgene expressing B cells from R4A-gamma2b/muKO mice was similar to that of the previously defined low affinity anti-DNA B cells that escape tolerance. These data suggest a requirement for a second heavy chain for the survival of this anergic B cell subset.
Cellular Immunology, 1986
Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-... more Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-associated glycoprotein (MAG) antibodies were studied to determine whether secretion of anti-MAG IgM antibodies by B cells was autonomous, or whether the monoclonal B cells were responsive to T cells. Secretion of anti-MAG IgM by isolated B cells was stimulated by the addition of increasing numbers of pokeweed mitogen (PWM)-activated autologous OKT4+ helper T cells in all four patients. Secretion of anti-MAG IgM by peripheral blood lymphocytes was dependent on the ratio of OKT,+ T helper cells to OKTo+ T suppressor/cytotoxic cells. In three patients with an OKT4+ to OKTs+ T-cell ratio of 2: 1, PWM activation stimulated secretion of anti-MAG IgM; in one patient with an OKT4+ to OKTs+ ratio of 1:2, activation by PWM suppressed anti-MAG IgM secretion. These studies suggest that the monoclonal B cells that secrete anti-MAG IgM are responsive to regulatory T cells. 0 1986 Academic FWS, IIIC.
Immunity, inflammation and disease, 2016
The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Eryt... more The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen-I (EBNA-1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA-1, 3D4, and demonstrated that it cross-reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA-1. The aim of the present study was to characterize another antibody to EBNA-1 that cross-reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA-1 that is recognized by these cross-reactive antibodies. We generated an IgM MAb to EBNA-1, 16D2, from EBNA-1 injected mice and demonstrated by ELISA that it cross-reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA-1 recognized by these MAbs, we examined their bin...
Biological Psychiatry, 2010
Background: It has been postulated that brain inflammatory processes associated with autoimmune d... more Background: It has been postulated that brain inflammatory processes associated with autoimmune diseases may be causative factors in emotional disorders. Accordingly, we examined emotional behaviors in autoimmune-prone cytokine B-cell-activating factor (BAFF) transgenic mice, a model of systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. Methods: Male BAFF transgenic mice were examined on a series of standard laboratory assays of emotionality. Mice were also tested for brain inflammation, stress-induced c-Fos expression, hippocampal progenitor cell proliferation, and hippocampal neurogenesis-dependent and neurogenesis-independent long-term potentiation (LTP). Results: Our study revealed that older BAFF transgenic mice exhibit an anxiety-like phenotype associated with brain inflammation. Furthermore, anxious mice display an abnormal neuronal activation within the limbic system in response to mild anxiogenic stimuli. Proliferation of newly formed neurons in the subgranular zone of adult hippocampus was significantly decreased in anxious BAFF transgenic mice that also showed impaired neurogenesis-dependent and neurogenesis-independent dentate gyrus LTP. Our results suggest that anxiety associated with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome can be linked to brain inflammation, impaired neurogenesis, and hippocampal plasticity. BAFF transgenic mice can be used in future studies to test compounds of therapeutic value for the treatment of mood disorders associated with autoimmune diseases.
Mapping the epitope in Epstein Barr Virus nuclear antigen-1 that elicits cross-reactivity with dsDNA (101.43)
Journal of Immunology, Apr 1, 2011
A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)
Journal of Immunology, May 1, 2014
One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Bar... more One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. Anti-EBNA-1 antibody obtained from mice from all three strains was observed to cross-react with dsDNA. However, the frequency of mice producing cross-reactive antibody was greater and the amount of cross-reactive antibody was higher in BAFF than wild type mice. This suggests that a viral protein such as EBNA-1 may trigger the development of antibodies that cross-react with dsDNA and that genetic susceptibility to lupus may enhance the development of cross-reactive antibodies upon viral exposure.
Antibodies to a peptide derived from EBNA-1 may be a potential biomarker for SLE
Journal of Immunology, May 1, 2018
The Epstein Barr virus (EBV) has been associated with the autoimmune disease Systemic lupus eryth... more The Epstein Barr virus (EBV) has been associated with the autoimmune disease Systemic lupus erythematosus (SLE). We previously demonstrated that mice injected with EBNA-1, a major nuclear protein of EBV, develop anti-dsDNA antibodies that cross-react with EBNA-1. We generated two monoclonal antibodies, 3D4 and 16D2, from EBNA-1 injected mice, and showed that they bind to both EBNA-1 and dsDNA. We also identified a peptide, PFM-15, in EBNA-1 (PQPGPLRESIVCYFM) that is recognized by 3D4 and 16D2. We hypothesize that this peptide can act as a molecular mimic of dsDNA and elicit antibodies to EBNA-1 that cross-react with dsDNA in some individuals who develop lupus. In the present study, we screened the sera of 17 lupus patients and 19 healthy individuals to determine if they have antibodies to PFM-15. We observed a highly significant increase (p<.05) in the number of SLE patients that have antibodies to PFM-15 compared to the number of healthy individuals with these antibodies. Furthermore, statistical analysis revealed that the anti-PFM-15 test has an 80% positive predictive value for SLE suggesting potential to be used diagnostically to detect a subset of patients with lupus. We also observed that 75% of the lupus patients tested, who had antibodies to PFM-15, also had antibodies to dsDNA and a moderately strong correlation was observed between the level of antibodies to PFM and dsDNA in these patients. This is consistent with our observation that antibodies to PFM-15 cross-react with dsDNA. These studies suggest that antibodies to PFM-15 may be a useful biomarker for a subset of patients exposed to EBV who develop lupus. Future studies with a much larger cohort of patients and controls will be needed to confirm this.
Antibodies that bind to an EBV derived peptide mimic of dsDNA, have pathogenic potential
Journal of Immunology, May 1, 2018
Anti-DNA producing B cells from mice transgenic for a gamma-2b anti-DNA heavy chain express Vk1 light chains and are not allelically excluded
A Second Heavy Chain Permits Survival of High Affinity Autoreactive B Cells
Autoimmunity, Feb 1, 2004
Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibo... more Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibodies with this specificity may be generated in many individuals, only patients with SLE fail to regulate them effectively. We have demonstrated previously that in non-autoimmune mice transgenic for the heavy chain of the R4A-gamma2b anti-DNA antibody, the existence of high affinity, IgG2b dsDNA binding B cells is tightly correlated with the co-expression of endogenous IgM heavy chain. These cells are anergic. In contrast, low affinity IgG2b dsDNA binding B cells do not express an endogenous heavy chain and represent a population of immunocompetent autoreactive B cells. In order to determine whether the presence of a second heavy chain permits the high affinity autoreactive B cells to escape deletion, the R4A-gamma2b mouse was mated to a strain with a targeted deletion of the transmembrane portion of the mu heavy chain, muMT mice, to produce R4A-gamma2b/muKO mice. Serum titers of anti-DNA antibodies were negligible in both R4A-gamma2b and R4A-gamma2b/muKO mice. In R4A-gamma2b/muKO mice, however, LPS was able to activate a DNA-reactive population although an LPS inducible DNA-reactive population. Light chain gene usage in transgene expressing B cells from R4A-gamma2b/muKO mice was similar to that of the previously defined low affinity anti-DNA B cells that escape tolerance. These data suggest a requirement for a second heavy chain for the survival of this anergic B cell subset.
Lack of allelic exclusion permits autoreactive B cells to escape deletion
The Journal of Immunology
The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA ... more The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA Ab. Approximately 80% of B cells expressing the transgene display allelic exclusion. Although the transgenic mice have little to no detectable serum anti-DNA activity, splenic B cells can be stimulated in vitro with LPS to secrete anti-DNA Ab. Hybridomas derived from LPS-stimulated splenic B cells were analyzed for expression of the transgene and for DNA binding. All nine transgene-encoded anti-DNA-producing lines were found to express an endogenous IgM heavy chain. Of 19 randomly selected lines producing a transgene-encoded non-DNA binding Ab, none expressed a second heavy chain. The tight correlation between lack of allelic exclusion and anti-dsDNA specificity provides strong support for the hypothesis that a major function of allelic exclusion is to prevent the maintenance of a pool of potentially activatable autoreactive cells.
Mice immunized with EBNA-1 protein produce antibodies that cross-react with double stranded DNA (129.5)
The Journal of Immunology
Systemic Lupus Erythematosus (SLE) is characterized by the production of antibodies to nuclear an... more Systemic Lupus Erythematosus (SLE) is characterized by the production of antibodies to nuclear antigens, in particular antibodies to double stranded DNA (dsDNA). The etiology of these autoantibodies is unknown but one theory suggests that viruses may play a role. One virus that has been shown to have an association with SLE is the Epstein Barr Virus (EBV). We previously demonstrated that mice immunized with an EBNA-1 expression vector or with purified EBNA-1 protein develop antibodies to dsDNA as well as to the viral antigen. We have recently demonstrated that these antibodies are pathogenic and deposit in the kidney and induce moderate proteinuria. We hypothesized that since EBNA-1 is a dsDNA binding protein, it could complex with chromosomal dsDNA in order to render it immunogenic and elicit an anti-dsDNA response. To test this hypothesis we generated recombinant EBNA-1 protein that lacks the 3 known chromosomal binding sites and immunized mice with it. We observed that mice immun...
A higher frequency of anti-EBNA-1 antibodies that cross-react with dsDNA are elicited in BAFF mice than wild type mice (BA4P.224)
The Journal of Immunology
One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Bar... more One environmental factor strongly linked to Systemic Lupus Erythematosus (SLE) is the Epstein Barr Virus (EBV). Injecting mice with the major nuclear protein of EBV, EBNA-1, causes some to develop anti-EBNA-1 antibodies that cross-react with double stranded DNA (dsDNA). We hypothesized that autoimmune mice would regulate the development of cross-reactive autoantibodies more poorly than healthy mice. The goal of our study was to examine the potential of using biotin-labeled-EBNA-1 bound to streptavidin-linked magnetic beads (B-EBNA-s-A Dynabeads) to isolate anti-EBNA-1 antibody from sera and to use this method to examine if autoimmune BAFF transgenic mice are more likely to develop antibodies to EBNA-1 that cross-react with dsDNA than wild-type mice. We successfully isolated anti-EBNA-1 antibodies from polyspecific sera obtained from BALB/c, C57BL/6, and BAFF mice injected with EBNA-1, using B-EBNA-s-A-Dynabeads. Eluted antibody was tested by ELISA for binding to EBNA-1 and dsDNA. An...
Anti-DNA producing B cells from mice transgenic for a gamma-2b anti-DNA heavy chain express Vk1 light chains and are not allelically excluded
Antibodies to an Epstein Barr Virus protein that cross-react with dsDNA have pathogenic potential
Molecular Immunology, 2021
Pathogens such as the Epstein Barr virus (EBV) have long been implicated in the etiology of syste... more Pathogens such as the Epstein Barr virus (EBV) have long been implicated in the etiology of systemic lupus erythematosus (SLE). The Epstein Barr virus nuclear antigen I (EBNA-1) has been shown to play a role in the development of anti-nuclear antibodies characteristic of SLE. One mechanism by which EBV may play a role in SLE is molecular mimicry. We previously generated two monoclonal antibodies (mAbs) to EBNA-1 and demonstrated that they cross-react with double-stranded DNA (dsDNA). In the present study, we demonstrate that these mAbs have pathogenic potential. We show that they can bind to isolated rat glomeruli and that binding can be greatly diminished by pretreatment of glomeruli with DNase I, suggesting that these mAbs bind dsDNA in the kidney. We also demonstrate that these antibodies can deposit in the kidney when injected into mice and can induce proteinuria and elicit histopathological alterations consistent with glomerulonephritis. Finally, we show that these antibodies can cross-react with laminin and collagen IV in the extracellular matrix suggesting that direct binding to the glomerular basement membrane or mesangial matrix may also contribute to the antibody deposition in the kidney. In summary, our results indicate that EBNA-1 can elicit antibodies that cross-react with dsDNA, that can deposit in the kidney, and induce kidney damage. These results are significant because they support the role of a viral protein in SLE and lupus nephritis.
Proceedings of the National Academy of Sciences, 1997
Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit i... more Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit in the kidneys of lupus patients to cause glomerulonephritis. Recent data suggest that a significant proportion of anti-DNA antibodies may cross-react with renal antigens and be sequestered in the kidney by virtue of this cross-reactivity. If this is true, antigenic competition for pathogenic antibodies might prevent their deposition in kidneys and the ensuing tissue damage. To generate surrogate antigens that could be used for this purpose, we have used peptide display phage libraries to identify peptides that react with R4A, a pathogenic mouse monoclonal anti-DNA antibody that deposits in glomeruli. We have demonstrated that the peptides bind in or near the double-stranded DNA binding site. Furthermore, the peptides are bound preferentially by the R4A antibody as compared with two closely related antibodies derived from it, one of which deposits in renal tubules and one of which display...
Mapping the epitope in Epstein Barr Virus nuclear antigen-1 that elicits cross-reactivity with dsDNA
The Journal of Immunology, Apr 1, 2011
PLOS ONE, 2015
Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid... more Protein P7 is a component of the cystovirus viral polymerase complex. In the unpackaged procapsid, the protein is situated in close proximity to the viral directed RNA polymerase, P2. Cryo-electron microscopy difference maps from the species ϕ6 procapsid have demonstrated that P7 and P2 likely interact prior to viral RNA packaging. The location of P7 in the post-packaged nucleocapsid (NC) remains unknown. P7 may translocate closer to the fivefold axis of a filled procapsid but this has not been directly visualized. We propose that monoclonal antibodies (Mabs) can be selected that serve as probe-reagents for viral assembly and structure. A set of Mabs have been isolated that recognize and bind to the ϕ6 P7. The antibody set contains five unique Mabs, four of which recognize a linear epitope and one which recognizes a conformational epitope. The four unique Mabs that recognize a linear epitope display restricted utilization of V κ and V H genes. The restricted genetic range among 4 of the 5 antibodies implies that the antibody repertoire is limited. The limitation could be the consequence of a paucity of exposed antigenic sites on the ϕ6 P7 surface. It is further demonstrated that within ϕ6 nucleocapsids that are primed for early-phase transcription, P7 is partially accessible to the Mabs, indicating that the nucleocapsid shell (protein P8) has undergone partial disassembly exposing the protein's antigenic sites.
PLoS ONE, 2011
Background: Several genetic and environmental factors have been linked to Systemic Lupus Erythema... more Background: Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1. Methodology/Principal Findings: In this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated mononclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs-3D4, generated in this laboratory, and 0211, a commercial MAb-revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm. Conclusions/Significance: In conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.
Journal of Clinical Investigation, 2000
A Second Heavy Chain Permits Survival of High Affinity Autoreactive B Cells
Autoimmunity, 2004
Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibo... more Anti-DNA antibody is the serological hallmark of systemic lupus erythematosus (SLE). While antibodies with this specificity may be generated in many individuals, only patients with SLE fail to regulate them effectively. We have demonstrated previously that in non-autoimmune mice transgenic for the heavy chain of the R4A-gamma2b anti-DNA antibody, the existence of high affinity, IgG2b dsDNA binding B cells is tightly correlated with the co-expression of endogenous IgM heavy chain. These cells are anergic. In contrast, low affinity IgG2b dsDNA binding B cells do not express an endogenous heavy chain and represent a population of immunocompetent autoreactive B cells. In order to determine whether the presence of a second heavy chain permits the high affinity autoreactive B cells to escape deletion, the R4A-gamma2b mouse was mated to a strain with a targeted deletion of the transmembrane portion of the mu heavy chain, muMT mice, to produce R4A-gamma2b/muKO mice. Serum titers of anti-DNA antibodies were negligible in both R4A-gamma2b and R4A-gamma2b/muKO mice. In R4A-gamma2b/muKO mice, however, LPS was able to activate a DNA-reactive population although an LPS inducible DNA-reactive population. Light chain gene usage in transgene expressing B cells from R4A-gamma2b/muKO mice was similar to that of the previously defined low affinity anti-DNA B cells that escape tolerance. These data suggest a requirement for a second heavy chain for the survival of this anergic B cell subset.
Cellular Immunology, 1986
Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-... more Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-associated glycoprotein (MAG) antibodies were studied to determine whether secretion of anti-MAG IgM antibodies by B cells was autonomous, or whether the monoclonal B cells were responsive to T cells. Secretion of anti-MAG IgM by isolated B cells was stimulated by the addition of increasing numbers of pokeweed mitogen (PWM)-activated autologous OKT4+ helper T cells in all four patients. Secretion of anti-MAG IgM by peripheral blood lymphocytes was dependent on the ratio of OKT,+ T helper cells to OKTo+ T suppressor/cytotoxic cells. In three patients with an OKT4+ to OKTs+ T-cell ratio of 2: 1, PWM activation stimulated secretion of anti-MAG IgM; in one patient with an OKT4+ to OKTs+ ratio of 1:2, activation by PWM suppressed anti-MAG IgM secretion. These studies suggest that the monoclonal B cells that secrete anti-MAG IgM are responsive to regulatory T cells. 0 1986 Academic FWS, IIIC.
Immunity, inflammation and disease, 2016
The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Eryt... more The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen-I (EBNA-1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA-1, 3D4, and demonstrated that it cross-reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA-1. The aim of the present study was to characterize another antibody to EBNA-1 that cross-reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA-1 that is recognized by these cross-reactive antibodies. We generated an IgM MAb to EBNA-1, 16D2, from EBNA-1 injected mice and demonstrated by ELISA that it cross-reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA-1 recognized by these MAbs, we examined their bin...
Biological Psychiatry, 2010
Background: It has been postulated that brain inflammatory processes associated with autoimmune d... more Background: It has been postulated that brain inflammatory processes associated with autoimmune diseases may be causative factors in emotional disorders. Accordingly, we examined emotional behaviors in autoimmune-prone cytokine B-cell-activating factor (BAFF) transgenic mice, a model of systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome. Methods: Male BAFF transgenic mice were examined on a series of standard laboratory assays of emotionality. Mice were also tested for brain inflammation, stress-induced c-Fos expression, hippocampal progenitor cell proliferation, and hippocampal neurogenesis-dependent and neurogenesis-independent long-term potentiation (LTP). Results: Our study revealed that older BAFF transgenic mice exhibit an anxiety-like phenotype associated with brain inflammation. Furthermore, anxious mice display an abnormal neuronal activation within the limbic system in response to mild anxiogenic stimuli. Proliferation of newly formed neurons in the subgranular zone of adult hippocampus was significantly decreased in anxious BAFF transgenic mice that also showed impaired neurogenesis-dependent and neurogenesis-independent dentate gyrus LTP. Our results suggest that anxiety associated with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome can be linked to brain inflammation, impaired neurogenesis, and hippocampal plasticity. BAFF transgenic mice can be used in future studies to test compounds of therapeutic value for the treatment of mood disorders associated with autoimmune diseases.