Thiago Detanico - Academia.edu (original) (raw)
Papers by Thiago Detanico
<p>(A) Adoptive transfer and immunization scheme. Primary and secondary injections were as ... more <p>(A) Adoptive transfer and immunization scheme. Primary and secondary injections were as specified in the figure. Naïve mice were B6AF1 mice that received CA30 cells, but no primary or secondary injection. (B) Mean titration curves (above) and concentrations (below) of serum IgG anti-Id at day 21. Each point represents an experimental mouse (n = 5/group). Results representative of 2 independent experiments with n = 5 mice per group.</p
<p>(A) Adoptive transfer scheme designed to test early CA30 T cell proliferation after B ce... more <p>(A) Adoptive transfer scheme designed to test early CA30 T cell proliferation after B cell depletion. (B) B220+ cell numbers at day 5. (C) CA30 T cell numbers at day 5. Data are representative of two independent experiments. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test with n = 5 mice per group. Data are representative of 2 independent experiments using n ≥ 5 mice per group.</p
<p>(A) Adoptive transfer scheme. (B) Representative FACS plots of CFSE and CD45.1 staining ... more <p>(A) Adoptive transfer scheme. (B) Representative FACS plots of CFSE and CD45.1 staining in the CD4+, MHC II-, CD19-, CD8α-, F4/80- gate and representative CFSE histograms for CD4+ CD45.1+ (CA30) cells. (C) Graph of percentage or total numbers (D) of CD4+ CD45.1+ (CA30) cells with SEM in each cell cycle division gate as defined by the FlowJo CFSE proliferation algorithm. Data presented are from a single experiment with mice treated with deaggregated (n = 7), immune complex (n = 7), or heat aggregated (n = 4) mAb 36–71. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test (* = p<0.05, ** = p<0.005). Data are representative of 3 independent experiments using n ≥ 4 mice per group.</p
<p>(A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies d... more <p>(A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies directed against the Vκ of mAb 36–71 were quantified using DELFIA as described in Materials and Methods. Connected lines denote a single mouse. Data are representative of two independent experiments with n = 5 mice per group. (C) Table summarizing results from 2 experiments with n = 5 mice per group with positive titers at each time point by treatment. Both experiments had 3 mice with positive titers at d42 and 5 mice with positive titers at d63 in both the heat-aggregated and immune complex groups.</p
As visões atuais do processo inflamatório articular, demonstram que a articulação é um local de r... more As visões atuais do processo inflamatório articular, demonstram que a articulação é um local de resposta imune, apresentando células apresentadoras de antígeno (APCs), células T e B, e intensa produção de citocinas. Em diversas artropatias, o perfil de citocinas exerce um papel fundamental. Na artrite reumatóide, a neutralização local de TNF-alfa induz uma melhora sintomática nestes indivíduos, enquanto em artrites infecciosas, produção INF-gama no sinovio é correlacionado com a melhora clínica. Proteínas de choque de calor (HSPs) são historicamente relacionadas com autoimunidade, porém poucos trabalhos descrevem como as HSPs correlacionam-se com a autoimunidade. Estudos recentes afirmam que a HSP70 autóloga induz a produção de citocinas pró-inflamatórias. O objetivo deste trabalho foi de montar um sistema para o estudo da imunomodulação em pacientes com artrite utilizando a HSP70 de micobactéria. O material foi coletado por punção articular, e após realizada separação de células por Ficoll-Histopaque. As células foram postas em cultura com fitohemaglutinina, HSP70 de Mycobacterium tuberculosis e após 48horas as células foram analisadas por citometria de fluxo para HLA de classe I e II. O sobrenadante foi analisado para as seguintes citocinas: INF-gama, IL-10, IL-12 e TGF-beta. HSP70 micobacteriana induziu a produção de IL-10 com a sem produção de INF-gama, foi visto também que a proteína bacteriana aumenta a expressão de HLA I e II nos linfócitos. Os resultados preliminares sugerem que a HSP70 micobacteriana pode modular o perfil de citocinas, pela produção de IL-10 pelas APCs que inibe a produção de INF gama pelos linfócitos. Estamos atualmente testando esta hipótese.
PloS one, 2017
A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic pr... more A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36-71. We found that heat-aggregated or immune complexes (IC) of mAb 36-71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36-71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between ...
Frontiers in Immunology, 2016
In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by s... more In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase, we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in antiviral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses and found that mutations producing Arg codons in antiviral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with Ag. In many cases, mutations producing codons for these alternative amino acids in antiviral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which two-thirds of random mutations generate codons for these key residues. Finally, by directly analyzing X-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via SHM occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences.
Rev Bras Reumatol, Oct 1, 2002
Journal of immunology (Baltimore, Md. : 1950), Jan 18, 2015
We previously reported that selective ablation of certain γδ T cell subsets, rather than removal ... more We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations we...
Journal of Autoimmunity, 2015
Unfortunately some text was missing from the Acknowledgments section We thank Desiree Straign for... more Unfortunately some text was missing from the Acknowledgments section We thank Desiree Straign for help with the Southern blots; Katja Aviszus, James B. St. Clair, Greg Kirchenbaum and Amanda Agazio for scientific discussion and Judith Spiegel for proofreading the manuscript. This work was funded by a grant from the National Institutes of Health: R01AI093822. The authors declare no conflict of interest. The authors now wish to add the following and apologise for any inconvenience caused: “The authors thank Dr. Tasuku Honjo for the AID-deficient B6 mice that made this study possible.”
Journal of Autoimmunity, 2015
Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark ... more Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLEassociated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda −/− mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM.
Proceedings of the National Academy of Sciences, 2014
Significance This study changes our understanding of the relationship between T cells and B cells... more Significance This study changes our understanding of the relationship between T cells and B cells. Although it is known that T cells provide help for specific B-cell responses, it is unclear if and to what extent T cells also influence preimmune B-cell functions. We show here that γδ T cells modulate systemic antibody levels in nonimmunized mice, including all major subclasses and especially IgE antibodies. One mouse strain deficient in certain γδ T cells developed various autoantibodies, whereas mice deficient in all γδ T cells had relatively normal antibodies. Based on these and other findings, we conclude that γδ T cells, influenced by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.
Lupus, 2009
We describe a unique spontaneous mouse model of autoimmunity, which occurs on a non-autoimmune–pr... more We describe a unique spontaneous mouse model of autoimmunity, which occurs on a non-autoimmune–prone SWR genetic background. In this model, SWR mice carry an IghV partial transgene ( pTg) encoding only the heavy chain variable domain of an antibody directed against chromatin. Autoimmune disease in pTg mice was manifested by some of the features of systemic lupus erythematosus (SLE), including the presence of serum anti-nuclear antibodies, splenomegaly, skin lesions and a moderate degree of kidney pathology, in various combinations among individuals. Autoimmunity was observed in three independent transgenic lines, but not in three control lines carrying a nearly identical pTg, in which a VHCDR3 codon for Arg was replaced by one for Ser to ablate chromatin reactivity. Various features of disease were often but not always accompanied by anti-chromatin antibodies. Unexpectedly, the anti-chromatin antibodies detected in seropositive animals were not encoded by the pTg. These observations...
The Journal of Immunology, 2011
Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquir... more Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquired by CD4 T cells to germlineencoded sequences within the B cell Ag receptor (BCR). We sought to determine how such tolerance is attained when a peptide from the BCR variable (V) region is expressed by small numbers of B cells as it is in the physiological state. Mixed bone marrow (BM) chimeras were generated using donor BM from mice with B cells that expressed a transgene (Tg)-encoded k L chain and BM from TCR Tg mice in which the CD4 T cells (CA30) were specific for a Vk peptide encoded by the kTg. In chimeras where few B cells express the kTg, many CA30 cells were deleted in the thymus. However, a substantial fraction survived to the CD4 singlepositive stage. Among single-positive CA30 thymocytes, few reached maturity and migrated to the periphery. Maturation was strongly associated with, and likely promoted by, expression of an endogenous TCR a-chain. CD4 + CA30 cells that reached peripheral lymphoid tissues were Ag-experienced and anergic, and some developed into regulatory cells. These findings reveal several checkpoints and mechanisms that enforce a state of self-tolerance in developing T cells specific for BCR V region sequences, thus ensuring that T cell help to B cells occurs through linked recognition of foreign Ag.
The Journal of Immunology, 2012
Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for ... more Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive ane...
The Journal of Immunology, 2003
A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/d... more A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vκ4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vκ4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vκ4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of VHCDR3 Arg residues in dsDNA binding and the near identity of the SN5–18 sequence to other dsDNA-specific Ab, we tested the contributions of two VHCDR3 Arg residues in SN5–18 to chromatin specificity. We found that both these Arg r...
Journal of Experimental Medicine, 2010
Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (A... more Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell–dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a VH gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings rev...
European Journal of Immunology, 2014
Somatic gene rearrangement generates a diverse repertoire of B cells, including B cell receptors ... more Somatic gene rearrangement generates a diverse repertoire of B cells, including B cell receptors (BCR) possessing a range of affinities for self-Ag. Newly generated B cells express high and relatively uniform amounts of surface IgM (sIgM), while follicular (FO) B cells express sIgM at widely varying levels. It is plausible, therefore, that down-modulation of sIgM serves as a mechanism to maintain weakly self-reactive B cells in a responsive state by decreasing their avidity for self-Ag. We tested this hypothesis by performing comparative functional tests with FO IgM hi and IgM lo B cells from the unrestricted repertoire of wildtype (WT) mice. We found that FO IgM lo B cells mobilized Ca 2+ equivalently to IgM hi B cells when the same number of sIgM molecules was engaged. In agreement, FO IgM lo B cells were functionally competent to produce an antibody response following adoptive transfer. The FO IgM lo cell population had elevated levels of Nur77 transcript, and was enriched with nuclear-reactive specificities. Hybridoma sampling revealed that these BCR were of low affinity. Collectively, these results suggest that sIgM down-modulation by low-affinity, self-reactive B cells preserves their immunocompetence and circumvents classical peripheral tolerance mechanisms that would otherwise reduce diversity within the B cell compartment.
Clinical and Experimental Immunology, 2004
SUMMARY Cytokines are key modulators of the immune responses that take place in the inflamed syno... more SUMMARY Cytokines are key modulators of the immune responses that take place in the inflamed synovium of arthritis patients. Consequently, substances that can reverse the inflammatory profile of the inflamed joint are potential tools for clinical management of the disease. Mycobacterial heat shock protein 70 (MTBHSP70) has been found to protect rats from experimentally induced arthritis through the induction of interleukin (IL)-10-producing T cells. In this study, we have demonstrated that MTBHSP70 induces IL-10 production in synoviocytes from arthritis patients and peripheral blood monoculear cells (PBMCs) from both patients and healthy controls. IL-10 production was accompanied by a decrease in tumour necrosis factor (TNF)-α production by synovial cells. Separation studies showed that the target cells were mainly monocytes. Accordingly, we observed that MTBHSP70 delayed maturation of murine bone marrow-derived dendritic cells. Our results suggest that MTBHSP may act on antigen-pre...
Autoimmunity, 2013
Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemi... more Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemic lupus erythematosus (SLE). SLE is associated with high titers of serum autoantibodies of the IgG class that are predominantly directed against nuclear antigens, with pathological manifestations that are considered by many to be characteristic of an immune-complex mediated disease. In this review, we focus on the known and potential roles of somatic mutagenesis in SLE. We will argue that antinuclear antibodies (ANA) arise predominantly from nonautoreactive B cells that are transformed into autoreactive cells by the process of somatic hypermutation (SHM), which is normally associated with affinity maturation during the germinal center reaction. We will also discuss the role of SHM in creating antigenic peptides in the V region of the B cell receptor (BCR) and its potential to open an avenue of unregulated T cell help to autoreactive B cells. Finally, we will end this review with new experimental evidence suggesting that spontaneous somatic mutagenesis of genes that regulate B cell survival and activation is a rate-limiting causative factor in the development of ANA.
<p>(A) Adoptive transfer and immunization scheme. Primary and secondary injections were as ... more <p>(A) Adoptive transfer and immunization scheme. Primary and secondary injections were as specified in the figure. Naïve mice were B6AF1 mice that received CA30 cells, but no primary or secondary injection. (B) Mean titration curves (above) and concentrations (below) of serum IgG anti-Id at day 21. Each point represents an experimental mouse (n = 5/group). Results representative of 2 independent experiments with n = 5 mice per group.</p
<p>(A) Adoptive transfer scheme designed to test early CA30 T cell proliferation after B ce... more <p>(A) Adoptive transfer scheme designed to test early CA30 T cell proliferation after B cell depletion. (B) B220+ cell numbers at day 5. (C) CA30 T cell numbers at day 5. Data are representative of two independent experiments. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test with n = 5 mice per group. Data are representative of 2 independent experiments using n ≥ 5 mice per group.</p
<p>(A) Adoptive transfer scheme. (B) Representative FACS plots of CFSE and CD45.1 staining ... more <p>(A) Adoptive transfer scheme. (B) Representative FACS plots of CFSE and CD45.1 staining in the CD4+, MHC II-, CD19-, CD8α-, F4/80- gate and representative CFSE histograms for CD4+ CD45.1+ (CA30) cells. (C) Graph of percentage or total numbers (D) of CD4+ CD45.1+ (CA30) cells with SEM in each cell cycle division gate as defined by the FlowJo CFSE proliferation algorithm. Data presented are from a single experiment with mice treated with deaggregated (n = 7), immune complex (n = 7), or heat aggregated (n = 4) mAb 36–71. Statistical differences were determined by a two-tailed Mann-Whitney <i>U</i> test (* = p<0.05, ** = p<0.005). Data are representative of 3 independent experiments using n ≥ 4 mice per group.</p
<p>(A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies d... more <p>(A) Adoptive transfer and immunization scheme. (B) Serum IgG anti-idiotypic antibodies directed against the Vκ of mAb 36–71 were quantified using DELFIA as described in Materials and Methods. Connected lines denote a single mouse. Data are representative of two independent experiments with n = 5 mice per group. (C) Table summarizing results from 2 experiments with n = 5 mice per group with positive titers at each time point by treatment. Both experiments had 3 mice with positive titers at d42 and 5 mice with positive titers at d63 in both the heat-aggregated and immune complex groups.</p
As visões atuais do processo inflamatório articular, demonstram que a articulação é um local de r... more As visões atuais do processo inflamatório articular, demonstram que a articulação é um local de resposta imune, apresentando células apresentadoras de antígeno (APCs), células T e B, e intensa produção de citocinas. Em diversas artropatias, o perfil de citocinas exerce um papel fundamental. Na artrite reumatóide, a neutralização local de TNF-alfa induz uma melhora sintomática nestes indivíduos, enquanto em artrites infecciosas, produção INF-gama no sinovio é correlacionado com a melhora clínica. Proteínas de choque de calor (HSPs) são historicamente relacionadas com autoimunidade, porém poucos trabalhos descrevem como as HSPs correlacionam-se com a autoimunidade. Estudos recentes afirmam que a HSP70 autóloga induz a produção de citocinas pró-inflamatórias. O objetivo deste trabalho foi de montar um sistema para o estudo da imunomodulação em pacientes com artrite utilizando a HSP70 de micobactéria. O material foi coletado por punção articular, e após realizada separação de células por Ficoll-Histopaque. As células foram postas em cultura com fitohemaglutinina, HSP70 de Mycobacterium tuberculosis e após 48horas as células foram analisadas por citometria de fluxo para HLA de classe I e II. O sobrenadante foi analisado para as seguintes citocinas: INF-gama, IL-10, IL-12 e TGF-beta. HSP70 micobacteriana induziu a produção de IL-10 com a sem produção de INF-gama, foi visto também que a proteína bacteriana aumenta a expressão de HLA I e II nos linfócitos. Os resultados preliminares sugerem que a HSP70 micobacteriana pode modular o perfil de citocinas, pela produção de IL-10 pelas APCs que inibe a produção de INF gama pelos linfócitos. Estamos atualmente testando esta hipótese.
PloS one, 2017
A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic pr... more A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36-71. We found that heat-aggregated or immune complexes (IC) of mAb 36-71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36-71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between ...
Frontiers in Immunology, 2016
In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by s... more In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase, we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in antiviral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses and found that mutations producing Arg codons in antiviral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with Ag. In many cases, mutations producing codons for these alternative amino acids in antiviral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which two-thirds of random mutations generate codons for these key residues. Finally, by directly analyzing X-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via SHM occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences.
Rev Bras Reumatol, Oct 1, 2002
Journal of immunology (Baltimore, Md. : 1950), Jan 18, 2015
We previously reported that selective ablation of certain γδ T cell subsets, rather than removal ... more We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations we...
Journal of Autoimmunity, 2015
Unfortunately some text was missing from the Acknowledgments section We thank Desiree Straign for... more Unfortunately some text was missing from the Acknowledgments section We thank Desiree Straign for help with the Southern blots; Katja Aviszus, James B. St. Clair, Greg Kirchenbaum and Amanda Agazio for scientific discussion and Judith Spiegel for proofreading the manuscript. This work was funded by a grant from the National Institutes of Health: R01AI093822. The authors declare no conflict of interest. The authors now wish to add the following and apologise for any inconvenience caused: “The authors thank Dr. Tasuku Honjo for the AID-deficient B6 mice that made this study possible.”
Journal of Autoimmunity, 2015
Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark ... more Serum IgG anti-nuclear antibodies (ANA) directed to complexes of DNA and histones are a hallmark of systemic lupus erythematosus (SLE) and reflect a failure in lymphocyte self-tolerance. A prior study utilizing spontaneously autoimmune B6.Nba2 mice deficient in terminal deoxynucleotidyl transferase (TdT) and with heterozygous deficiencies in Jh and Igk loci underscored the importance of somatic hypermutation (SHM) as a major generator of SLEassociated ANA. This interpretation had to be qualified because of severely limited opportunities for receptor editing and restricted VHCDR3 diversity. Therefore, we performed the converse study using mice that carried functional Tdt genes and wild type Jh and Igk loci but that could not undergo SHM. Analyses of ANA and ANA-producing hybridomas from B6.Nba2 Aicda −/− mice revealed that few animals produced high titers of the prototypical ANA directed to complexes of histones and DNA, that this response was delayed and that those cells that did produce such antibody exhibited limited clonal expansion, unusual Jk use and only infrequent dual receptor expression. This, together with the additional finding of an intrinsic propensity for SHM to generate Arg codons selectively in CDRs, reinforce the view that most IgG autoimmune clones producing prototypical anti-nucleosome antibodies in wild type mice are created by SHM.
Proceedings of the National Academy of Sciences, 2014
Significance This study changes our understanding of the relationship between T cells and B cells... more Significance This study changes our understanding of the relationship between T cells and B cells. Although it is known that T cells provide help for specific B-cell responses, it is unclear if and to what extent T cells also influence preimmune B-cell functions. We show here that γδ T cells modulate systemic antibody levels in nonimmunized mice, including all major subclasses and especially IgE antibodies. One mouse strain deficient in certain γδ T cells developed various autoantibodies, whereas mice deficient in all γδ T cells had relatively normal antibodies. Based on these and other findings, we conclude that γδ T cells, influenced by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.
Lupus, 2009
We describe a unique spontaneous mouse model of autoimmunity, which occurs on a non-autoimmune–pr... more We describe a unique spontaneous mouse model of autoimmunity, which occurs on a non-autoimmune–prone SWR genetic background. In this model, SWR mice carry an IghV partial transgene ( pTg) encoding only the heavy chain variable domain of an antibody directed against chromatin. Autoimmune disease in pTg mice was manifested by some of the features of systemic lupus erythematosus (SLE), including the presence of serum anti-nuclear antibodies, splenomegaly, skin lesions and a moderate degree of kidney pathology, in various combinations among individuals. Autoimmunity was observed in three independent transgenic lines, but not in three control lines carrying a nearly identical pTg, in which a VHCDR3 codon for Arg was replaced by one for Ser to ablate chromatin reactivity. Various features of disease were often but not always accompanied by anti-chromatin antibodies. Unexpectedly, the anti-chromatin antibodies detected in seropositive animals were not encoded by the pTg. These observations...
The Journal of Immunology, 2011
Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquir... more Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquired by CD4 T cells to germlineencoded sequences within the B cell Ag receptor (BCR). We sought to determine how such tolerance is attained when a peptide from the BCR variable (V) region is expressed by small numbers of B cells as it is in the physiological state. Mixed bone marrow (BM) chimeras were generated using donor BM from mice with B cells that expressed a transgene (Tg)-encoded k L chain and BM from TCR Tg mice in which the CD4 T cells (CA30) were specific for a Vk peptide encoded by the kTg. In chimeras where few B cells express the kTg, many CA30 cells were deleted in the thymus. However, a substantial fraction survived to the CD4 singlepositive stage. Among single-positive CA30 thymocytes, few reached maturity and migrated to the periphery. Maturation was strongly associated with, and likely promoted by, expression of an endogenous TCR a-chain. CD4 + CA30 cells that reached peripheral lymphoid tissues were Ag-experienced and anergic, and some developed into regulatory cells. These findings reveal several checkpoints and mechanisms that enforce a state of self-tolerance in developing T cells specific for BCR V region sequences, thus ensuring that T cell help to B cells occurs through linked recognition of foreign Ag.
The Journal of Immunology, 2012
Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for ... more Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive ane...
The Journal of Immunology, 2003
A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/d... more A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vκ4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vκ4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vκ4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of VHCDR3 Arg residues in dsDNA binding and the near identity of the SN5–18 sequence to other dsDNA-specific Ab, we tested the contributions of two VHCDR3 Arg residues in SN5–18 to chromatin specificity. We found that both these Arg r...
Journal of Experimental Medicine, 2010
Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (A... more Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell–dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a VH gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings rev...
European Journal of Immunology, 2014
Somatic gene rearrangement generates a diverse repertoire of B cells, including B cell receptors ... more Somatic gene rearrangement generates a diverse repertoire of B cells, including B cell receptors (BCR) possessing a range of affinities for self-Ag. Newly generated B cells express high and relatively uniform amounts of surface IgM (sIgM), while follicular (FO) B cells express sIgM at widely varying levels. It is plausible, therefore, that down-modulation of sIgM serves as a mechanism to maintain weakly self-reactive B cells in a responsive state by decreasing their avidity for self-Ag. We tested this hypothesis by performing comparative functional tests with FO IgM hi and IgM lo B cells from the unrestricted repertoire of wildtype (WT) mice. We found that FO IgM lo B cells mobilized Ca 2+ equivalently to IgM hi B cells when the same number of sIgM molecules was engaged. In agreement, FO IgM lo B cells were functionally competent to produce an antibody response following adoptive transfer. The FO IgM lo cell population had elevated levels of Nur77 transcript, and was enriched with nuclear-reactive specificities. Hybridoma sampling revealed that these BCR were of low affinity. Collectively, these results suggest that sIgM down-modulation by low-affinity, self-reactive B cells preserves their immunocompetence and circumvents classical peripheral tolerance mechanisms that would otherwise reduce diversity within the B cell compartment.
Clinical and Experimental Immunology, 2004
SUMMARY Cytokines are key modulators of the immune responses that take place in the inflamed syno... more SUMMARY Cytokines are key modulators of the immune responses that take place in the inflamed synovium of arthritis patients. Consequently, substances that can reverse the inflammatory profile of the inflamed joint are potential tools for clinical management of the disease. Mycobacterial heat shock protein 70 (MTBHSP70) has been found to protect rats from experimentally induced arthritis through the induction of interleukin (IL)-10-producing T cells. In this study, we have demonstrated that MTBHSP70 induces IL-10 production in synoviocytes from arthritis patients and peripheral blood monoculear cells (PBMCs) from both patients and healthy controls. IL-10 production was accompanied by a decrease in tumour necrosis factor (TNF)-α production by synovial cells. Separation studies showed that the target cells were mainly monocytes. Accordingly, we observed that MTBHSP70 delayed maturation of murine bone marrow-derived dendritic cells. Our results suggest that MTBHSP may act on antigen-pre...
Autoimmunity, 2013
Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemi... more Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemic lupus erythematosus (SLE). SLE is associated with high titers of serum autoantibodies of the IgG class that are predominantly directed against nuclear antigens, with pathological manifestations that are considered by many to be characteristic of an immune-complex mediated disease. In this review, we focus on the known and potential roles of somatic mutagenesis in SLE. We will argue that antinuclear antibodies (ANA) arise predominantly from nonautoreactive B cells that are transformed into autoreactive cells by the process of somatic hypermutation (SHM), which is normally associated with affinity maturation during the germinal center reaction. We will also discuss the role of SHM in creating antigenic peptides in the V region of the B cell receptor (BCR) and its potential to open an avenue of unregulated T cell help to autoreactive B cells. Finally, we will end this review with new experimental evidence suggesting that spontaneous somatic mutagenesis of genes that regulate B cell survival and activation is a rate-limiting causative factor in the development of ANA.