RESUMO
Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Humanos , Borrelia burgdorferi/metabolismo , Receptor Toll-Like 9/metabolismo , Macrófagos , Doença de Lyme/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/metabolismoRESUMO
RATIONALE: Accumulating evidence implicates inflammation in pulmonary arterial hypertension (PAH) and therapies targeting immunity are under investigation, although it remains unknown if distinct immune phenotypes exist. OBJECTIVE: Identify PAH immune phenotypes based on unsupervised analysis of blood proteomic profiles. METHODS AND RESULTS: In a prospective observational study of group 1 PAH patients evaluated at Stanford University (discovery cohort; n=281) and University of Sheffield (validation cohort; n=104) between 2008 and 2014, we measured a circulating proteomic panel of 48 cytokines, chemokines, and factors using multiplex immunoassay. Unsupervised machine learning (consensus clustering) was applied in both cohorts independently to classify patients into proteomic immune clusters, without guidance from clinical features. To identify central proteins in each cluster, we performed partial correlation network analysis. Clinical characteristics and outcomes were subsequently compared across clusters. Four PAH clusters with distinct proteomic immune profiles were identified in the discovery cohort. Cluster 2 (n=109) had low cytokine levels similar to controls. Other clusters had unique sets of upregulated proteins central to immune networks-cluster 1 (n=58; TRAIL [tumor necrosis factor-related apoptosis-inducing ligand], CCL5 [C-C motif chemokine ligand 5], CCL7, CCL4, MIF [macrophage migration inhibitory factor]), cluster 3 (n=77; IL [interleukin]-12, IL-17, IL-10, IL-7, VEGF [vascular endothelial growth factor]), and cluster 4 (n=37; IL-8, IL-4, PDGF-ß [platelet-derived growth factor beta], IL-6, CCL11). Demographics, PAH clinical subtypes, comorbidities, and medications were similar across clusters. Noninvasive and hemodynamic surrogates of clinical risk identified cluster 1 as high-risk and cluster 3 as low-risk groups. Five-year transplant-free survival rates were unfavorable for cluster 1 (47.6%; 95% CI, 35.4%-64.1%) and favorable for cluster 3 (82.4%; 95% CI, 72.0%-94.3%; across-cluster P<0.001). Findings were replicated in the validation cohort, where machine learning classified 4 immune clusters with comparable proteomic, clinical, and prognostic features. CONCLUSIONS: Blood cytokine profiles distinguish PAH immune phenotypes with differing clinical risk that are independent of World Health Organization group 1 subtypes. These phenotypes could inform mechanistic studies of disease pathobiology and provide a framework to examine patient responses to emerging therapies targeting immunity.
Assuntos
Aprendizado de Máquina , Hipertensão Arterial Pulmonar/imunologia , Adulto , Idoso , Estudos de Coortes , Citocinas/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteômica , Hipertensão Arterial Pulmonar/mortalidadeRESUMO
Rheumatoid arthritis (RA) is characterized by the production of anti-citrullinated protein antibodies (ACPAs). To gain insights into the relationship between ACPA-expressing B cells in peripheral blood (PB) and synovial tissue (ST), we sequenced the B cell repertoire in paired PB and ST samples from five individuals with established, ACPA+ RA. Bioinformatics analysis of paired heavy- and light-chain sequences revealed clonally-related family members shared between PB and ST. ST-derived antibody repertoires exhibited reduced diversity and increased normalized clonal family size compared to PB-derived repertoires. Functional characterization showed that seven recombinant antibodies (rAbs) expressed from subject-derived sequences from both compartments bound citrullinated antigens and immune complexes (ICs) formed using one ST-derived rAb stimulated macrophage TNF-α production. Our findings demonstrate B cell trafficking between PB and ST in subjects with RA and ST repertoires include B cells that encode ACPA capable of forming ICs that stimulate cellular responses implicated in RA pathogenesis.
Assuntos
Anticorpos Antiproteína Citrulinada/imunologia , Complexo Antígeno-Anticorpo/imunologia , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Macrófagos/imunologia , Membrana Sinovial/imunologia , Fator de Necrose Tumoral alfa/imunologia , Diversidade de Anticorpos/imunologia , Biologia Computacional , Humanos , Ativação de Macrófagos/imunologia , Membrana Sinovial/citologiaRESUMO
Idiopathic pulmonary arterial hypertension (IPAH) is a devastating pulmonary vascular disease in which autoimmune and inflammatory phenomena are implicated. B cells and autoantibodies have been associated with IPAH and identified as potential therapeutic targets. However, the specific populations of B cells involved and their roles in disease pathogenesis are not clearly defined. We aimed to assess the levels of activated B cells (plasmablasts) in IPAH, and to characterize recombinant antibodies derived from these plasmablasts. Blood plasmablasts are elevated in IPAH, remain elevated over time, and produce IgA autoantibodies. Single-cell sequencing of plasmablasts in IPAH revealed repertoires of affinity-matured antibodies with small clonal expansions, consistent with an ongoing autoimmune response. Recombinant antibodies representative of these clonal lineages bound known autoantigen targets and displayed an unexpectedly high degree of polyreactivity. Representative IPAH plasmablast recombinant antibodies stimulated human umbilical vein endothelial cells to produce cytokines and overexpress the adhesion molecule ICAM-1. Together, our results demonstrate an ongoing adaptive autoimmune response involving IgA plasmablasts that produce anti-endothelial cell autoantibodies in IPAH. These antibodies stimulate endothelial cell production of cytokines and adhesion molecules, which may contribute to disease pathogenesis. These findings suggest a role for mucosally-driven autoimmunity and autoimmune injury in the pathogenesis of IPAH.
Assuntos
Autoanticorpos/imunologia , Autoimunidade/imunologia , Células Endoteliais/imunologia , Hipertensão Pulmonar Primária Familiar/imunologia , Plasmócitos/imunologia , Formação de Anticorpos/imunologia , Citocinas/biossíntese , Hipertensão Pulmonar Primária Familiar/sangue , Hipertensão Pulmonar Primária Familiar/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Plasmócitos/citologiaRESUMO
Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70-89) and young (aged 20-29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.
Assuntos
Envelhecimento/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Plasmócitos/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Diversidade de Anticorpos , Humanos , Ligação Proteica , Receptores de Antígenos de Linfócitos B/genética , Vacinação , Adulto JovemRESUMO
We developed a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of the antibodies expressed by individual B cells. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy-chain VJ and light-chain VJ sequence usage, do so most effectively. Vaccine-induced heavy-chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years' seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years' seasonal influenza, suggesting that 'original antigenic sin' shapes the antibody response to influenza vaccination.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Linfócitos B/imunologia , Hemaglutininas Virais/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/biossíntese , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Memória Imunológica , Vírus da Influenza A Subtipo H1N2/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Dados de Sequência Molecular , Tipagem Molecular , Vacinação , Vacinas de Subunidades AntigênicasRESUMO
Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.
Assuntos
Antígenos CD , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana , Células Mieloides , Receptores Imunológicos , Microambiente Tumoral , Receptores Imunológicos/metabolismo , Animais , Humanos , Camundongos , Microambiente Tumoral/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Glicoproteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismoRESUMO
Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15+HLADRhi and CD48highS100A2pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.
Assuntos
Artrite Reumatoide , Doenças Periodontais , Humanos , Autoanticorpos , Mucosa Bucal , Formação de Anticorpos , Epitopos , BactériasRESUMO
Our aim was to elucidate the contribution of mucosal mast cells to the effector phase of a secondary immune response to Trichinella spiralis. During secondary infection, rats expel 90-99% of T. spiralis first-stage larvae from the intestine in a matter of hours. This phenomenon appears to be unique to rats and has been called rapid expulsion. Primary intestinal infection by T. spiralis induces mastocytosis, and mast cell degranulation occurs when challenged rats exhibit rapid expulsion. These observations have engendered the view that mast cells mediate rapid expulsion. In this study, we report that immunization of adult Albino Oxford rats by an infection limited to the muscle phase did not induce intestinal mastocytosis, yet such rats exhibited rapid expulsion when challenged orally. Although mastocytosis was absent, the protease unique to mucosal mast cells, rat mast cell protease II (RMCPII), was detected in sera at the time of expulsion. We further evaluated mast cell activity in neonatal rats that display rapid expulsion. Pups born to infected dams displayed rapid expulsion, and RMCPII was detected in their sera. By feeding pups parasite-specific mAbs or polyclonal Abs before challenge infection, it was possible to dissociate mast cell degranulation from parasite expulsion. These results indicate that rapid expulsion can occur in the absence of either intestinal mastocytosis or RMCPII release. Furthermore, release of RMCPII is not sufficient to cause expulsion. The data argue against a role for mast cells in the mechanism underlying the effector phase of protective immunity against T. spiralis in rats.
Assuntos
Quimases/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mastócitos/enzimologia , Mastócitos/imunologia , Trichinella spiralis/imunologia , Triquinelose/enzimologia , Triquinelose/imunologia , Animais , Degranulação Celular/imunologia , Quimases/sangue , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Larva/crescimento & desenvolvimento , Larva/imunologia , Masculino , Mastócitos/metabolismo , Mastocitose/enzimologia , Mastocitose/imunologia , Mastocitose/parasitologia , Ratos , Ratos Endogâmicos , Ratos Nus , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/parasitologiaRESUMO
This study evaluated long-term immunophenotypic changes in circulating levels of 24 immune cell subsets through 4 years of continuous treatment with first-line ibrutinib (420 mg once daily) in 31 patients with chronic lymphocytic leukemia (CLL) from the RESONATE-2 study, and compared them with untreated age-matched healthy donors (n = 20). Ibrutinib progressively decreased total B-cell counts and preferentially targeted malignant CLL B cells over normal B cells. Elevated counts of chronically activated, exhausted, and effector memory T cells were normalized within 6-16 months, while naive T cells remained mostly within healthy donor range (HDR). Immunosuppressive regulatory T cells and myeloid-derived suppressor cells were normalized within the first 1-2 years and then plateaued. Additionally, ibrutinib restored low counts of innate cell populations associated with antitumor immunity: plasmacytoid dendritic cells were restored to HDR after 2 years, and classical monocyte counts progressively and continuously increased toward HDR. Ibrutinib also consistently preserved circulating mature natural killer cell counts. The data indicate that ibrutinib continuously exerted positive effects on immune cell populations throughout 4 years of treatment, consistent with improved clinical outcomes observed in patients. The normalization of adaptive and innate immune cell populations suggests that long-term ibrutinib treatment mediates restoration of immunity.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Piperidinas/uso terapêutico , Adenina/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunofenotipagem , MasculinoRESUMO
Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.
Assuntos
Imunoterapia , Neoplasias , Imunidade Adaptativa , Animais , Antígenos de Neoplasias , Imunoterapia/métodos , Camundongos , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.
Assuntos
Vacinas contra Influenza/imunologia , Tonsila Palatina/imunologia , Adjuvantes Imunológicos , Linfócitos B/citologia , Linfócitos B/imunologia , Vacinas contra COVID-19/imunologia , Centro Germinativo/citologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Técnicas In Vitro , Tecido Linfoide/imunologia , Vacina contra Sarampo-Caxumba-Rubéola/imunologia , Organoides/citologia , Organoides/imunologia , Vacina Antirrábica/imunologia , Linfócitos T/imunologiaRESUMO
Ibrutinib positively modulates many T-cell subsets in chronic lymphocytic leukemia (CLL). To understand ibrutinib's effects on the broader landscape of immune cell populations, we comprehensively characterized changes in circulating counts of 21 immune blood cell subsets throughout the first year of treatment in patients with relapsed/refractory (R/R) CLL (n = 55, RESONATE) and previously untreated CLL (n = 50, RESONATE-2) compared with untreated age-matched healthy donors (n = 20). Ibrutinib normalized abnormal immune cell counts to levels similar to those of age-matched healthy donors. Ibrutinib significantly decreased pathologically high circulating B cells, regulatory T cells, effector/memory CD4+ and CD8+ T cells (including exhausted and chronically activated T cells), natural killer (NK) T cells, and myeloid-derived suppressor cells; preserved naive T cells and NK cells; and increased circulating classical monocytes. T-cell function was assessed in response to T-cell receptor stimulation in patients with R/R CLL (n = 21) compared with age-matched healthy donors (n = 18). Ibrutinib significantly restored T-cell proliferative ability, degranulation, and cytokine secretion. Over the same period, ofatumumab or chlorambucil did not confer the same spectrum of normalization as ibrutinib in multiple immune subsets. These results establish that ibrutinib has a significant and likely positive impact on circulating malignant and nonmalignant immune cells and restores healthy T-cell function.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/imunologia , Terapia de Salvação , Linfócitos T Reguladores/imunologia , Adenina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/administração & dosagem , Estudos de Casos e Controles , Clorambucila/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Feminino , Seguimentos , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Recidiva Local de Neoplasia/patologia , Piperidinas , Prognóstico , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
OBJECTIVE: Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). While epitope spreading of the serum ACPA response is believed to contribute to RA pathogenesis, little is understood regarding how this phenomenon occurs. This study was undertaken to analyze the antibody repertoires of individuals with RA to gain insight into the mechanisms leading to epitope spreading of the serum ACPA response in RA. METHODS: Plasmablasts from the blood of 6 RA patients were stained with citrullinated peptide tetramers to identify ACPA-producing B cells by flow cytometry. Plasmablasts were single-cell sorted and sequenced to obtain antibody repertoires. Sixty-nine antibodies were recombinantly expressed, and their anticitrulline reactivities were characterized using a cyclic citrullinated peptide enzyme-linked immuosorbent assay and synovial antigen arrays. Thirty-six mutated antibodies designed either to represent ancestral antibodies or to test paratope residues critical for binding, as determined from molecular modeling studies, were also tested for anticitrulline reactivities. RESULTS: Clonally related monoclonal ACPAs and their shared ancestral antibodies each exhibited differential reactivity against citrullinated antigens. Molecular modeling identified residues within the complementarity-determining region loops and framework regions predicted to be important for citrullinated antigen binding. Affinity maturation resulted in mutations of these key residues, which conferred binding to different citrullinated epitopes and/or increased polyreactivity to citrullinated epitopes. CONCLUSION: These results demonstrate that the different somatic hypermutations accumulated by clonally related B cells during affinity maturation alter the antibody paratope to mediate epitope spreading and polyreactivity of the ACPA response in RA, suggesting that these may be key properties that likely contribute to the pathogenicity of ACPAs.
Assuntos
Anticorpos Antiproteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Sítios de Ligação de Anticorpos/imunologia , Epitopos/imunologia , Peptídeos Cíclicos/imunologia , Adulto , Artrite Reumatoide/sangue , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Feminino , Humanos , Masculino , Plasmócitos/imunologiaRESUMO
OBJECTIVE: Rheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA. METHODS: Blood samples were obtained at up to 4 serial time points from 8 individuals with established RA who were positive for ACPAs by the anti-cyclic citrullinated peptide test. CD19+CD3-IgD-CD14-CD20-CD27+CD38++ plasmablasts were isolated by single-cell sorting and costained with citrullinated peptide tetramers to identify ACPA-expressing plasmablasts. Cell-specific oligonucleotide barcodes were utilized, followed by large-scale sequencing and bioinformatics analysis, to obtain error-corrected, paired heavy- and light-chain antibody gene sequences for each B cell. RESULTS: Bioinformatics analysis revealed 170 persistent plasmablast lineages in the RA blood, of which 19% included multiple isotypes. Among IgG- and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages were observed (P < 0.01). Shared complementarity-determining region 3 sequence motifs were identified across subjects. A subset of the plasmablast lineages included members derived from later time points with divergent somatic hypermutations that encoded antibodies that bind an expanded set of citrullinated antigens. Furthermore, these recombinant, differentially mutated plasmablast antibodies formed immune complexes that stimulated higher macrophage production of tumor necrosis factor (TNF) compared to antibodies representing earlier time point-derived lineage members that were less mutated. CONCLUSION: These findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.
Assuntos
Anticorpos Antiproteína Citrulinada/imunologia , Afinidade de Anticorpos/fisiologia , Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Epitopos/imunologia , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/sangue , Linfócitos B/imunologia , Biologia Computacional , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasmócitos/imunologiaRESUMO
Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity worldwide. Here, we show that blood plasmablasts and CD27- memory B cells are elevated in untreated Lyme disease, with higher plasmablast levels associated with more rapid resolution of clinical symptoms. Stronger serum reactivity to surface proteins and peptides from B. burgdorferi was also associated with faster resolution of clinical symptoms. Through molecular identifier-enabled antibody heavy-chain sequencing of bulk B cells and single-cell paired-chain antibody sequencing of blood plasmablasts, we characterized immunoglobulin gene usage patterns specific to B. burgdorferi infection. Recombinantly expressed antibodies from expanded lineages bound B. burgdorferi antigens, confirming that these clones are driven by the infection. Furthermore, recombinant sequence-derived antibodies were functional, inhibiting growth of B. burgdorferi in vitro. Elevations and clonal expansion of blood plasmablasts were associated with rapid return to health, while poor plasmablast responses were associated with a longer duration of symptoms following treatment. Plasmablasts induced by B. burgdorferi infection showed preferential antibody gene segment usage, while bulk sequencing of total B cells revealed convergent CDR3 motifs specific to B. burgdorferi-infected patients. Our results show that robust plasmablast responses encoding Bb-static antibodies are associated with more rapid resolution of Lyme disease, and these antibodies could provide the basis for next-generation therapeutics for Lyme disease.
RESUMO
Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell-sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Rotavirus/imunologia , Adulto , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Antígenos Virais/imunologia , Linfócitos B/imunologia , Proteínas do Capsídeo/imunologia , Células Cultivadas , Citometria de Fluxo , Humanos , Rotavirus/patogenicidade , Infecções por Rotavirus/tratamento farmacológico , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/prevenção & controleRESUMO
OBJECTIVE: The disease process in rheumatoid arthritis (RA) starts years before the clinical diagnosis is made, and elevated levels of disease-specific autoantibodies can be detected during this period. Early responses to known or novel autoantigens likely drive the eventual production of pathogenic autoimmunity. Importantly, the presence of disease-specific autoantibodies can identify individuals who are at high risk of developing RA but who do not currently have arthritis. The goal of the current study was to characterize plasmablasts from individuals at risk of developing RA. METHODS: We investigated antibody-secreting plasmablasts derived from a well-characterized cohort of individuals who were at risk of developing RA, based on RA-related serum autoantibody positivity, as compared to patients with early (<1 year) seropositive RA as well as healthy control subjects. The plasmablast antibody repertoires of at-risk subjects were analyzed using DNA barcode-based methods with paired heavy- and light-chain gene sequencing. Cells were single-cell sorted, the cell- and plate-specific DNA barcodes were sequentially added, and next-generation sequencing was performed. RESULTS: Total plasmablast levels were similar in the antibody-positive (1%) and control (0.4-1.6%) groups. However, increased frequencies of IgA+ versus IgG+ plasmablasts were observed in the antibody-positive group (39% IgA+ and 37% IgG+) as compared to other groups (1-9% IgA+ and 71-87% IgG+). Paired antibody sequences from antibody-positive subjects revealed cross-isotype clonal families and similar sequence characteristics in the IgA and IgG plasmablast repertoires. Antibody-positive individuals also demonstrated elevated serum levels of IgA isotype anti-cyclic citrullinated peptide 3 antibodies. CONCLUSION: The IgA plasmablast dominance in these antibody-positive individuals suggests that a subset of RA-related autoantibodies may arise from mucosal immune responses and may be involved in early disease pathogenesis in individuals who are at risk of developing RA.
Assuntos
Artrite Reumatoide/imunologia , Imunoglobulina A/imunologia , Peptídeos Cíclicos/imunologia , Plasmócitos/imunologia , Fator Reumatoide/imunologia , Adulto , Autoanticorpos/genética , Autoanticorpos/imunologia , Feminino , Genes de Cadeia Pesada de Imunoglobulina/genética , Genes de Cadeia Leve de Imunoglobulina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina A/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Risco , Análise de Sequência de DNARESUMO
OBJECTIVE: A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti-citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into plasmablasts, which are released into the blood. We undertook this study to sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA. METHODS: We developed a novel DNA barcoding method to sequence the cognate heavy- and light-chain pairs of antibodies expressed by individual blood plasmablasts in RA. The method uses a universal 5' adapter that enables full-length sequencing of the antibodies' variable regions and recombinant expression of the paired antibody chains. The sequence data sets were bioinformatically analyzed to generate phylogenetic trees that identify clonal families of antibodies sharing heavy- and light-chain VJ sequences. Representative antibodies were expressed, and their binding properties were characterized using anti-cyclic citrullinated peptide 2 (anti-CCP-2) enzyme-linked immunosorbent assay (ELISA) and antigen microarrays. RESULTS: We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts from 4 individuals with anti-CCP+ RA, and recombinantly expressed 14 antibodies that were either "singleton" antibodies or representative of clonal antibody families. Anti-CCP-2 ELISA identified 4 ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α-enolase, citrullinated fibrinogen, and citrullinated histone H2B. CONCLUSION: Our data provide evidence that autoantibodies targeting α-enolase, citrullinated fibrinogen, and citrullinated histone H2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/sangue , Linfócitos B/imunologia , Código de Barras de DNA Taxonômico , Plasmócitos/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/genética , Epitopos/genética , HumanosRESUMO
Toll-like receptor 9 (TLR9) promiscuously binds self- and microbial DNA, but only microbial DNA elicits an inflammatory response. How TLR9 discriminates between self- and foreign DNA is unclear, but inappropriate localization of TLR9 permits response to self-DNA, suggesting that TLR9 localization and trafficking are critical components. The molecular mechanisms controlling the movement of TLR9 may provide new insight into the recognition of DNA in normal and in pathological conditions such as autoimmune systemic lupus erythematosus. We have shown earlier that TLR9 is retained in the endoplasmic reticulum (ER) and it moves to endolysosomes to recognize CpG DNA. Other studies have suggested that TLR9 bypasses the Golgi complex to access endolysosomes. Here, we show that TLR9 translocates from ER to endolysosomes through the Golgi complex and that Golgi export is required for optimal TLR9 signaling. In all, 6-13% of TLR9 constitutively exits the ER, moves through the Golgi complex and resides in lysosomal-associated membrane protein-1-positive vesicles. TLR9 bound to CpG DNA had glycan modifications indicative of Golgi processing confirming that TLR9 travels through the Golgi complex to access CpG DNA in endolysosomes. Together, these data support a model where TLR9 uses traditional secretory pathways and does not bypass the Golgi complex.