Salar Khan - Academia.edu (original) (raw)

Papers by Salar Khan

Research paper thumbnail of A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence

PLOS Pathogens

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed... more Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activit...

Research paper thumbnail of Correction for Khan et al., “Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry”

Research paper thumbnail of Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry

Journal of Virology

Protection against acquiring human immunodeficiency virus (HIV) infection may not require a vacci... more Protection against acquiring human immunodeficiency virus (HIV) infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective, and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication both in vitro and in vivo. To expand the available array of multispecific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMAb targeting two epitopes on the major HIV coreceptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98 to 100% of a 109-virus panel, as well as additivity ...

Research paper thumbnail of Elicitation of Neutralizing Antibodies Targeting the V2 Apex of the HIV Envelope Trimer in a Wild-Type Animal Model

Cell reports, Jan 3, 2017

Recent efforts toward HIV vaccine development include the design of immunogens that can engage B ... more Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare "glycan hole" at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.

Research paper thumbnail of Apoptotic cells: a source of autoantigens for antiphospholipid antibodies

The Journal of Immunology, Apr 1, 2007

Research paper thumbnail of HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation

Scientific reports, Jan 16, 2016

The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) va... more The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs "knock out" trimers. Following two inoculations, the CD4bs-directed mAbs efficiently recognized the eliciting immunogen in their affinity-maturing state but did not recognize CD4bs-defective probes. We reverted these mAbs to their most likely inferred germline (igL) state, leaving the HCDR3 unaltered, to establish correlates of in vitro affinity to in vivo activation. Most igL-reverted mAbs bound the eliciting gp140 immunogen, indicating that CD4bs-directed B cells possessing reasonable affinity existed in the naïve repertoire. We detected relatively high affinities fo...

Research paper thumbnail of Rhesus Macaque B-Cell Responses to an HIV-1 Trimer Vaccine Revealed by Unbiased Longitudinal Repertoire Analysis

mBio, Jan 3, 2015

Next-generation sequencing (NGS) has been used to investigate the diversity and maturation of bro... more Next-generation sequencing (NGS) has been used to investigate the diversity and maturation of broadly neutralizing antibodies (bNAbs) in HIV-1-infected individuals. However, the application of NGS to the preclinical assessment of human vaccines, particularly the monitoring of vaccine-induced B-cell responses in a nonhuman primate (NHP) model, has not been reported. Here, we present a longitudinal NGS analysis of memory B-cell responses to an HIV-1 trimer vaccine in a macaque that has been extensively studied by single B-cell sorting and antibody characterization. We first established an NHP antibodyomics pipeline using the available 454 pyrosequencing data from this macaque and developed a protocol to sequence the NHP antibody repertoire in an unbiased manner. Using these methods, we then analyzed memory B-cell repertoires at four time points of NHP immunization and traced the lineages of seven CD4-binding site (CD4bs)-directed monoclonal antibodies previously isolated from this mac...

Research paper thumbnail of Editing and escape from editing in anti-DNA B cells

Proceedings of the National Academy of Sciences, 2008

Research paper thumbnail of Antibodies that bind complex glycosaminoglycans accumulate in the Golgi

Proceedings of the National Academy of Sciences, 2013

Research paper thumbnail of Divergent members of a single autoreactive B cell clone retain specificity for apoptotic blebs

Molecular Immunology, 2007

Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches... more Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches of an autoreactive B cell clone. However, it is not known whether a different autospecificity predates anti-dsDNA and whether separate offshoots of an expanding B cell clone retain or evolve alternative specificities. We compared 3H9, an anti-dsDNA IgG, to 4H8 and 1A11, antibodies produced by hybridomas representing an alternative branch of the 3H9 B cell clone. All three IgG bound chromatin in ELISA and apoptotic cells in confocal microscopy, yet only 3H9 bound dsDNA, as measured by plasmon resonance. Moreover, we demonstrate that despite the unique specificity of 3H9 for dsDNA, all three clone members exhibited indistinguishable binding to chromatin. The binding to chromatin and apoptotic cells was unaffected by N-linked glycosylation in L chain CDR1, a modification that results from a replacement of serine 26 with asparagine in 4H8 and 1A11. These data provide the first evidence that specificity for nucleosome epitopes on apoptotic cells provides the initial positive stimulus for somatic variants that comprise a B cell clone, including those that subsequently acquire specificity for dsDNA. Conversely, selection of autoreactive B cells for binding to apoptotic cells leads to clonal expansion, antibody diversification, and the development of linked sets of anti-nuclear autoantibodies.

Research paper thumbnail of Intra-Golgi Formation of IgM-Glycosaminoglycan Complexes Promotes Ig Deposition

The Journal of Immunology, 2011

Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. H... more Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. However, it is not known whether intracellular Ag-Ab interactions also contribute to the formation of extracellular immune complexes. In this study, we report that certain murine B cell hybridomas accumulate intracellular IgM and release large, spherical IgM complexes. The complexes (termed "spherons") reach 2 mm in diameter, detach from the cell surface, and settle out of solution. The spherons contain IgM multimers that incorporate the J chain and resist degradation by endoglycosidase H, arguing for IgM passage through the Golgi. Treatment of cells with inhibitors of proteoglycan synthesis, or incubation of spherons with chondroitinase ABC, degrades spherons, indicating that spheron formation and growth depend on interactions between IgM and glycosaminoglycans. This inference is supported by direct binding of IgM to heparin and hyaluronic acid. We conclude that, as a consequence of IgM binding to glycosaminoglycans, multivalent IgM-glycan complexes form in transit of IgM to the cell surface. Intra-Golgi formation of immune complexes could represent a new pathogenic mechanism for immune complex deposition disorders.

Research paper thumbnail of Histone Deimination As a Response to Inflammatory Stimuli in Neutrophils

The Journal of Immunology, 2008

Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl ar... more Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stimulate autoimmunity. To examine conditions leading to histone deimination in neutrophils, we used Abs that detect citrullines in the N terminus of histone H3. Deimination was investigated in human neutrophils and HL-60 cells differentiated into granulocytes. We observed rapid and robust H3 deimination in HL-60 cells exposed to LPS, TNF, lipoteichoic acid, f-MLP, or hydrogen peroxide, which are stimuli that activate neutrophils. Importantly, we also observed H3 deimination in human neutrophils exposed to these stimuli. Citrullinated histones were identified as components of extracellular chromatin traps (NETs) produced by degranulating neutrophils. In contrast, apoptosis proceeded without detectable H3 deimination in HL-60 cells exposed to staurosporine or camptothecin. We conclude that histone deimination in neutrophils is induced in response to inflammatory stimuli and not by treatments that induce apoptosis. Our results further suggest that deiminated histone H3, a covalently modified form of a prominent nuclear autoantigen, is released to the extracellular space as part of the neutrophil response to infections. The possible association of a modified autoantigen with microbial components could, in predisposed individuals, increase the risk of autoimmunity.

Research paper thumbnail of Intra-Golgi Formation of IgM–Glycosaminoglycan Complexes Promotes Ig Deposition

The Journal of …, 2011

Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. H... more Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. However, it is not known whether intracellular Ag–Ab interactions also contribute to the formation of extracellular immune complexes. In this study, we report that ...

Research paper thumbnail of Editing and escape from editing in anti-DNA B cells

Proceedings of the …, 2008

Tolerance to dsDNA is achieved through editing of Ig receptors that react with dsDNA. Nevertheles... more Tolerance to dsDNA is achieved through editing of Ig receptors that react with dsDNA. Nevertheless, some B cells with anti-dsDNA receptors escape editing and migrate to the spleen. Certain anti-dsDNA B cells that are recovered as hybridomas from the spleens of ...

Research paper thumbnail of Divergent members of a single autoreactive B cell clone retain specificity for apoptotic blebs

Molecular …, 2007

Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches... more Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches of an autoreactive B cell clone. However, it is not known whether a different autospecificity predates anti-dsDNA and whether separate offshoots of an expanding B cell clone retain or evolve alternative specificities. We compared 3H9, an anti-dsDNA IgG, to 4H8 and 1A11, antibodies produced by hybridomas representing an alternative branch of the 3H9 B cell clone. All three IgG bound chromatin in ELISA and apoptotic cells in confocal microscopy, yet only 3H9 bound dsDNA, as measured by plasmon resonance. Moreover, we demonstrate that despite the unique specificity of 3H9 for dsDNA, all three clone members exhibited indistinguishable binding to chromatin. The binding to chromatin and apoptotic cells was unaffected by N-linked glycosylation in L chain CDR1, a modification that results from a replacement of serine 26 with asparagine in 4H8 and 1A11. These data provide the first evidence that specificity for nucleosome epitopes on apoptotic cells provides the initial positive stimulus for somatic variants that comprise a B cell clone, including those that subsequently acquire specificity for dsDNA. Conversely, selection of autoreactive B cells for binding to apoptotic cells leads to clonal expansion, antibody diversification, and the development of linked sets of anti-nuclear autoantibodies.

Research paper thumbnail of Histone deimination as a response to inflammatory stimuli in neutrophils

The Journal of Immunology, 2008

Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl ar... more Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stimulate autoimmunity. To examine ...

Research paper thumbnail of Felty's syndrome autoantibodies bind to deiminated histones and neutrophil extracellular chromatin traps

Arthritis & Rheumatism, 2012

Objective. To test the hypothesis that autoantigen modifications by peptidylarginine deiminase ty... more Objective. To test the hypothesis that autoantigen modifications by peptidylarginine deiminase type 4 (PAD-4) increase immunoreactivity. Methods. We assembled sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty's syndrome (FS), and antineutrophil cytoplasmic antibody-associated vasculitides (AAVs), as well as sera from control subjects without autoimmune diseases. The sera were tested for binding to activated neutrophils, deiminated histones, and neutrophil extracellular chromatin traps (NETs). IgG binding to lipopolysaccharide-activated neutrophils was assessed with confocal microscopy, and binding to in vitro-deiminated histones was measured using enzyme-linked immunosorbent assay (ELISA) and Western blotting. In addition, we quantitated histone deimination in freshly isolated neutrophils from the blood of patients and control subjects. Results. Increased IgG reactivity with activated neutrophils, particularly binding to NETs, was paralleled by preferential binding to deiminated histones over nondeiminated histones by ELISA in a majority of sera from FS patients but only in a minority of sera from SLE and RA patients. Immunoblotting revealed autoantibody preference for deiminated histones H3, H4, and H2A in most FS patients and in a subset of SLE and RA patients. In patients with AAVs, serum IgG preferentially bound nondeiminated histones over deiminated histones. Increased levels of deiminated histones were detected in neutrophils from RA patients. Conclusion. Circulating autoantibodies in FS are preferentially directed against PAD-4-deiminated histones and bind to activated neutrophils and NETs. Thus, increased reactivity with modified autoantigens in FS implies a direct contribution of neutrophil activation and the production of NET-associated nuclear autoantigens in the initiation or progression of FS. Autoimmune disorders such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) may progress slowly and follow a chronic path with gradual worsening of disease manifestations (1,2). In other individuals, sudden flares of more intense disease manifestations may interrupt lengthy periods of symptom quiescence. One notable example of worsening in a chronically progressing disorder is provided by Felty's syndrome (FS), a variant of RA that is defined by arthritis involving axial joints, enlargement of the spleen, and a decline in neutrophil numbers (3). The decrease in neutrophil numbers is thought to be due to excessive activation of mature neutrophils and their clearance in the patient's spleen. FS occur in 1-3% of RA patients, usually after 10-15 years of fairly typical symptoms (3). An alternative viewpoint is that FS does not arise as chronic progression of RA, but instead may be closely

Research paper thumbnail of Regulation of extracellular chromatin release from neutrophils

Journal of Innate Immunity, 2009

Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mecha... more Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mechanism entails the release of relaxed chromatin from the cell. Microbes are trapped by the extracellular chromatin and exposed to high local concentrations of bactericidal compounds. We examine the regulation of chromatin release by testing the contribution of microtubules and the actin cytoskeleton to the deployment of neutrophil extracellular traps (NETs). Incubation of human neutrophils with nocodazole, a tubulin polymerization inhibitor, or cytochalasin D, an inhibitor of actin filamentation, severely diminished the ability of neutrophils to respond to LPS by releasing chromatin from the cells. In addition, pretreatment of neutrophils with M1/70, a monoclonal antibody to the Mac-1 integrin adhesion receptor, drastically reduced the deployment of chromatin into NETs. Analysis of histone deimination, the conversion of arginine to citrulline in 3 of the 4 core histones by peptidylarginine deiminase 4, revealed that the treatments inhibiting NET formation also reduced histone deimination. Our data indicate that NET formation requires functional tubulin and actin filaments and responds to engagement of Mac-1 integrins. Because histone deimination coincides with the release of NETs, we propose that these events represent overlapping mechanisms of neutrophil responses to infections.

Research paper thumbnail of A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence

PLOS Pathogens

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed... more Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activit...

Research paper thumbnail of Correction for Khan et al., “Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry”

Research paper thumbnail of Targeting the HIV-1 Spike and Coreceptor with Bi- and Trispecific Antibodies for Single-Component Broad Inhibition of Entry

Journal of Virology

Protection against acquiring human immunodeficiency virus (HIV) infection may not require a vacci... more Protection against acquiring human immunodeficiency virus (HIV) infection may not require a vaccine in the conventional sense, because broadly neutralizing antibodies (bNAbs) alone prevent HIV infection in relevant animal challenge models. Additionally, bNAbs as therapeutics can effectively suppress HIV replication in infected humans and in animal models. Combinations of bNAbs are generally even more effective, and bNAb-derived multivalent antibody-like molecules also inhibit HIV replication both in vitro and in vivo. To expand the available array of multispecific HIV inhibitors, we designed single-component molecules that incorporate two (bispecific) or three (trispecific) bNAbs that recognize HIV Env exclusively, a bispecific CrossMAb targeting two epitopes on the major HIV coreceptor, CCR5, and bi- and trispecifics that cross-target both Env and CCR5. These newly designed molecules displayed exceptional breadth, neutralizing 98 to 100% of a 109-virus panel, as well as additivity ...

Research paper thumbnail of Elicitation of Neutralizing Antibodies Targeting the V2 Apex of the HIV Envelope Trimer in a Wild-Type Animal Model

Cell reports, Jan 3, 2017

Recent efforts toward HIV vaccine development include the design of immunogens that can engage B ... more Recent efforts toward HIV vaccine development include the design of immunogens that can engage B cell receptors with the potential to affinity mature into broadly neutralizing antibodies (bnAbs). V2-apex bnAbs, which bind a protein-glycan region on HIV envelope glycoprotein (Env) trimer, are among the most broad and potent described. We show here that a rare "glycan hole" at the V2 apex is enriched in HIV isolates neutralized by inferred precursors of prototype V2-apex bnAbs. To investigate whether this feature could focus neutralizing responses onto the apex bnAb region, we immunized wild-type rabbits with soluble trimers adapted from these Envs. Potent autologous tier 2 neutralizing responses targeting basic residues in strand C of the V2 region, which forms the core epitope for V2-apex bnAbs, were observed. Neutralizing monoclonal antibodies (mAbs) derived from these animals display features promising for subsequent broadening of the response.

Research paper thumbnail of Apoptotic cells: a source of autoantigens for antiphospholipid antibodies

The Journal of Immunology, Apr 1, 2007

Research paper thumbnail of HIV-1 Vaccine-elicited Antibodies Reverted to Their Inferred Naive Germline Reveal Associations between Binding Affinity and in vivo Activation

Scientific reports, Jan 16, 2016

The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) va... more The elicitation of HIV-1 broadly neutralizing antibodies following envelope glycoprotein (Env) vaccination is exceedingly difficult. Suboptimal engagement of naïve B cells is suggested to limit these low frequency events, especially at the conserved CD4bs. Here, we analyzed CD4bs-directed monoclonal antibodies (mAbs) elicited by YU2 gp140-foldon trimers in a non-human primate by selective sorting using CD4bs "knock out" trimers. Following two inoculations, the CD4bs-directed mAbs efficiently recognized the eliciting immunogen in their affinity-maturing state but did not recognize CD4bs-defective probes. We reverted these mAbs to their most likely inferred germline (igL) state, leaving the HCDR3 unaltered, to establish correlates of in vitro affinity to in vivo activation. Most igL-reverted mAbs bound the eliciting gp140 immunogen, indicating that CD4bs-directed B cells possessing reasonable affinity existed in the naïve repertoire. We detected relatively high affinities fo...

Research paper thumbnail of Rhesus Macaque B-Cell Responses to an HIV-1 Trimer Vaccine Revealed by Unbiased Longitudinal Repertoire Analysis

mBio, Jan 3, 2015

Next-generation sequencing (NGS) has been used to investigate the diversity and maturation of bro... more Next-generation sequencing (NGS) has been used to investigate the diversity and maturation of broadly neutralizing antibodies (bNAbs) in HIV-1-infected individuals. However, the application of NGS to the preclinical assessment of human vaccines, particularly the monitoring of vaccine-induced B-cell responses in a nonhuman primate (NHP) model, has not been reported. Here, we present a longitudinal NGS analysis of memory B-cell responses to an HIV-1 trimer vaccine in a macaque that has been extensively studied by single B-cell sorting and antibody characterization. We first established an NHP antibodyomics pipeline using the available 454 pyrosequencing data from this macaque and developed a protocol to sequence the NHP antibody repertoire in an unbiased manner. Using these methods, we then analyzed memory B-cell repertoires at four time points of NHP immunization and traced the lineages of seven CD4-binding site (CD4bs)-directed monoclonal antibodies previously isolated from this mac...

Research paper thumbnail of Editing and escape from editing in anti-DNA B cells

Proceedings of the National Academy of Sciences, 2008

Research paper thumbnail of Antibodies that bind complex glycosaminoglycans accumulate in the Golgi

Proceedings of the National Academy of Sciences, 2013

Research paper thumbnail of Divergent members of a single autoreactive B cell clone retain specificity for apoptotic blebs

Molecular Immunology, 2007

Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches... more Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches of an autoreactive B cell clone. However, it is not known whether a different autospecificity predates anti-dsDNA and whether separate offshoots of an expanding B cell clone retain or evolve alternative specificities. We compared 3H9, an anti-dsDNA IgG, to 4H8 and 1A11, antibodies produced by hybridomas representing an alternative branch of the 3H9 B cell clone. All three IgG bound chromatin in ELISA and apoptotic cells in confocal microscopy, yet only 3H9 bound dsDNA, as measured by plasmon resonance. Moreover, we demonstrate that despite the unique specificity of 3H9 for dsDNA, all three clone members exhibited indistinguishable binding to chromatin. The binding to chromatin and apoptotic cells was unaffected by N-linked glycosylation in L chain CDR1, a modification that results from a replacement of serine 26 with asparagine in 4H8 and 1A11. These data provide the first evidence that specificity for nucleosome epitopes on apoptotic cells provides the initial positive stimulus for somatic variants that comprise a B cell clone, including those that subsequently acquire specificity for dsDNA. Conversely, selection of autoreactive B cells for binding to apoptotic cells leads to clonal expansion, antibody diversification, and the development of linked sets of anti-nuclear autoantibodies.

Research paper thumbnail of Intra-Golgi Formation of IgM-Glycosaminoglycan Complexes Promotes Ig Deposition

The Journal of Immunology, 2011

Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. H... more Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. However, it is not known whether intracellular Ag-Ab interactions also contribute to the formation of extracellular immune complexes. In this study, we report that certain murine B cell hybridomas accumulate intracellular IgM and release large, spherical IgM complexes. The complexes (termed "spherons") reach 2 mm in diameter, detach from the cell surface, and settle out of solution. The spherons contain IgM multimers that incorporate the J chain and resist degradation by endoglycosidase H, arguing for IgM passage through the Golgi. Treatment of cells with inhibitors of proteoglycan synthesis, or incubation of spherons with chondroitinase ABC, degrades spherons, indicating that spheron formation and growth depend on interactions between IgM and glycosaminoglycans. This inference is supported by direct binding of IgM to heparin and hyaluronic acid. We conclude that, as a consequence of IgM binding to glycosaminoglycans, multivalent IgM-glycan complexes form in transit of IgM to the cell surface. Intra-Golgi formation of immune complexes could represent a new pathogenic mechanism for immune complex deposition disorders.

Research paper thumbnail of Histone Deimination As a Response to Inflammatory Stimuli in Neutrophils

The Journal of Immunology, 2008

Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl ar... more Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stimulate autoimmunity. To examine conditions leading to histone deimination in neutrophils, we used Abs that detect citrullines in the N terminus of histone H3. Deimination was investigated in human neutrophils and HL-60 cells differentiated into granulocytes. We observed rapid and robust H3 deimination in HL-60 cells exposed to LPS, TNF, lipoteichoic acid, f-MLP, or hydrogen peroxide, which are stimuli that activate neutrophils. Importantly, we also observed H3 deimination in human neutrophils exposed to these stimuli. Citrullinated histones were identified as components of extracellular chromatin traps (NETs) produced by degranulating neutrophils. In contrast, apoptosis proceeded without detectable H3 deimination in HL-60 cells exposed to staurosporine or camptothecin. We conclude that histone deimination in neutrophils is induced in response to inflammatory stimuli and not by treatments that induce apoptosis. Our results further suggest that deiminated histone H3, a covalently modified form of a prominent nuclear autoantigen, is released to the extracellular space as part of the neutrophil response to infections. The possible association of a modified autoantigen with microbial components could, in predisposed individuals, increase the risk of autoimmunity.

Research paper thumbnail of Intra-Golgi Formation of IgM–Glycosaminoglycan Complexes Promotes Ig Deposition

The Journal of …, 2011

Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. H... more Immune complexes arise from interactions between secreted Ab and Ags in the surrounding milieu. However, it is not known whether intracellular Ag–Ab interactions also contribute to the formation of extracellular immune complexes. In this study, we report that ...

Research paper thumbnail of Editing and escape from editing in anti-DNA B cells

Proceedings of the …, 2008

Tolerance to dsDNA is achieved through editing of Ig receptors that react with dsDNA. Nevertheles... more Tolerance to dsDNA is achieved through editing of Ig receptors that react with dsDNA. Nevertheless, some B cells with anti-dsDNA receptors escape editing and migrate to the spleen. Certain anti-dsDNA B cells that are recovered as hybridomas from the spleens of ...

Research paper thumbnail of Divergent members of a single autoreactive B cell clone retain specificity for apoptotic blebs

Molecular …, 2007

Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches... more Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches of an autoreactive B cell clone. However, it is not known whether a different autospecificity predates anti-dsDNA and whether separate offshoots of an expanding B cell clone retain or evolve alternative specificities. We compared 3H9, an anti-dsDNA IgG, to 4H8 and 1A11, antibodies produced by hybridomas representing an alternative branch of the 3H9 B cell clone. All three IgG bound chromatin in ELISA and apoptotic cells in confocal microscopy, yet only 3H9 bound dsDNA, as measured by plasmon resonance. Moreover, we demonstrate that despite the unique specificity of 3H9 for dsDNA, all three clone members exhibited indistinguishable binding to chromatin. The binding to chromatin and apoptotic cells was unaffected by N-linked glycosylation in L chain CDR1, a modification that results from a replacement of serine 26 with asparagine in 4H8 and 1A11. These data provide the first evidence that specificity for nucleosome epitopes on apoptotic cells provides the initial positive stimulus for somatic variants that comprise a B cell clone, including those that subsequently acquire specificity for dsDNA. Conversely, selection of autoreactive B cells for binding to apoptotic cells leads to clonal expansion, antibody diversification, and the development of linked sets of anti-nuclear autoantibodies.

Research paper thumbnail of Histone deimination as a response to inflammatory stimuli in neutrophils

The Journal of Immunology, 2008

Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl ar... more Posttranslational modifications, such as the deimination of arginine to citrulline by peptidyl arginine deiminase (PAD4), change protein structure and function. For autoantigens, covalent modifications represent a mechanism to sidestep tolerance and stimulate autoimmunity. To examine ...

Research paper thumbnail of Felty's syndrome autoantibodies bind to deiminated histones and neutrophil extracellular chromatin traps

Arthritis & Rheumatism, 2012

Objective. To test the hypothesis that autoantigen modifications by peptidylarginine deiminase ty... more Objective. To test the hypothesis that autoantigen modifications by peptidylarginine deiminase type 4 (PAD-4) increase immunoreactivity. Methods. We assembled sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty's syndrome (FS), and antineutrophil cytoplasmic antibody-associated vasculitides (AAVs), as well as sera from control subjects without autoimmune diseases. The sera were tested for binding to activated neutrophils, deiminated histones, and neutrophil extracellular chromatin traps (NETs). IgG binding to lipopolysaccharide-activated neutrophils was assessed with confocal microscopy, and binding to in vitro-deiminated histones was measured using enzyme-linked immunosorbent assay (ELISA) and Western blotting. In addition, we quantitated histone deimination in freshly isolated neutrophils from the blood of patients and control subjects. Results. Increased IgG reactivity with activated neutrophils, particularly binding to NETs, was paralleled by preferential binding to deiminated histones over nondeiminated histones by ELISA in a majority of sera from FS patients but only in a minority of sera from SLE and RA patients. Immunoblotting revealed autoantibody preference for deiminated histones H3, H4, and H2A in most FS patients and in a subset of SLE and RA patients. In patients with AAVs, serum IgG preferentially bound nondeiminated histones over deiminated histones. Increased levels of deiminated histones were detected in neutrophils from RA patients. Conclusion. Circulating autoantibodies in FS are preferentially directed against PAD-4-deiminated histones and bind to activated neutrophils and NETs. Thus, increased reactivity with modified autoantigens in FS implies a direct contribution of neutrophil activation and the production of NET-associated nuclear autoantigens in the initiation or progression of FS. Autoimmune disorders such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) may progress slowly and follow a chronic path with gradual worsening of disease manifestations (1,2). In other individuals, sudden flares of more intense disease manifestations may interrupt lengthy periods of symptom quiescence. One notable example of worsening in a chronically progressing disorder is provided by Felty's syndrome (FS), a variant of RA that is defined by arthritis involving axial joints, enlargement of the spleen, and a decline in neutrophil numbers (3). The decrease in neutrophil numbers is thought to be due to excessive activation of mature neutrophils and their clearance in the patient's spleen. FS occur in 1-3% of RA patients, usually after 10-15 years of fairly typical symptoms (3). An alternative viewpoint is that FS does not arise as chronic progression of RA, but instead may be closely

Research paper thumbnail of Regulation of extracellular chromatin release from neutrophils

Journal of Innate Immunity, 2009

Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mecha... more Neutrophils use intricate mechanisms for capturing and killing invading microorganisms. One mechanism entails the release of relaxed chromatin from the cell. Microbes are trapped by the extracellular chromatin and exposed to high local concentrations of bactericidal compounds. We examine the regulation of chromatin release by testing the contribution of microtubules and the actin cytoskeleton to the deployment of neutrophil extracellular traps (NETs). Incubation of human neutrophils with nocodazole, a tubulin polymerization inhibitor, or cytochalasin D, an inhibitor of actin filamentation, severely diminished the ability of neutrophils to respond to LPS by releasing chromatin from the cells. In addition, pretreatment of neutrophils with M1/70, a monoclonal antibody to the Mac-1 integrin adhesion receptor, drastically reduced the deployment of chromatin into NETs. Analysis of histone deimination, the conversion of arginine to citrulline in 3 of the 4 core histones by peptidylarginine deiminase 4, revealed that the treatments inhibiting NET formation also reduced histone deimination. Our data indicate that NET formation requires functional tubulin and actin filaments and responds to engagement of Mac-1 integrins. Because histone deimination coincides with the release of NETs, we propose that these events represent overlapping mechanisms of neutrophil responses to infections.