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1.
Nat Commun ; 11(1): 3989, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778653

RESUMO

Upon stimulation, B cells assume heterogeneous cell fates, with only a fraction differentiating into antibody-secreting cells (ASC). Here we investigate B cell fate programming and heterogeneity during ASC differentiation using T cell-independent models. We find that maximal ASC induction requires at least eight cell divisions in vivo, with BLIMP-1 being required for differentiation at division eight. Single cell RNA-sequencing of activated B cells and construction of differentiation trajectories reveal an early cell fate bifurcation. The ASC-destined branch requires induction of IRF4, MYC-target genes, and oxidative phosphorylation, with the loss of CD62L expression serving as a potential early marker of ASC fate commitment. Meanwhile, the non-ASC branch expresses an inflammatory signature, and maintains B cell fate programming. Finally, ASC can be further subseted based on their differential responses to ER-stress, indicating multiple development branch points. Our data thus define the cell division kinetics of B cell differentiation in vivo, and identify the molecular trajectories of B cell fate and ASC formation.


Assuntos
Células Produtoras de Anticorpos/metabolismo , Linfócitos B/imunologia , Ativação Linfocitária/fisiologia , Animais , Antígenos CD19/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Imunidade , Fatores Reguladores de Interferon/metabolismo , Selectina L , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Transcriptoma
4.
Ann Rheum Dis ; 79(1): 150-158, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31611218

RESUMO

OBJECTIVES: Recent evidences have revealed that anti-SSA/SSB antibodies, the major autoantibodies in Sjögren's syndrome (SS), are produced in salivary glands. This study aims to clarify overall of autoantibody production at lesion site, including anti-centromere antibody (ACA)-positive SS. METHODS: Antibodies of antibody-secreting cells in human salivary glands were produced as recombinant antibodies. The reactivity of these antibodies and their revertants were investigated by ELISA and newly developed antigen-binding beads assay, which can detect conformational epitopes. The target of uncharacterised antibodies was identified by immunoprecipitation and mass spectrometry. Autoantibody-secreting cells in salivary gland tissue were identified by immunohistochemistry using green fluorescent protein-autoantigen fusion proteins. RESULTS: A total of 256 lesion antibodies were generated, and 69 autoantibodies including 24 ACAs were identified among them. Beads assay could detect more autoantibodies than ELISA, suggesting autoantibodies target to antigens with native conformation. After somatic hypermutations were reverted, autoantibodies drastically decreased antigen reactivity. We showed that MIS12 complex, a novel target of ACA, and CENP-C are major targets of ACA produced in salivary glands by examining cloned antibodies and immunohistochemistry, whereas few anti-CENP-B antibodies were detected. The target profiling of serum ACA from 269 patients with SS, systemic sclerosis (SSc), primary biliary cirrhosis (PBC) and healthy controls revealed that ACA-positive patients have antibodies against various sites of centromere complex regardless of disease. CONCLUSION: We showed direct evidences of antigen-driven maturation of anti-SSA/SSB antibody and ACA in SS lesion. ACA recognises centromere 'complex' rather than individual protein, and this feature is common among patients with SS, SSc and PBC.


Assuntos
Anticorpos Antinucleares/imunologia , Formação de Anticorpos/imunologia , Centrômero/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Adulto , Idoso , Células Produtoras de Anticorpos , Autoanticorpos/imunologia , Proteína Centromérica A/imunologia , Proteína B de Centrômero/imunologia , Proteínas Cromossômicas não Histona/imunologia , Feminino , Humanos , Cirrose Hepática Biliar/imunologia , Masculino , Pessoa de Meia-Idade , Glândulas Salivares/citologia , Escleroderma Sistêmico/imunologia
5.
PLoS Negl Trop Dis ; 13(8): e0007634, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31369553

RESUMO

BACKGROUND: Oral cholera vaccine (OCV) containing killed Vibrio cholerae O1 and O139 organisms (Bivalent-OCV; Biv-OCV) are playing a central role in global cholera control strategies. OCV is currently administered in a 2-dose regimen (day 0 and 14). There is a growing body of evidence that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae mediate protection against cholera. There are limited data on anti-OSP responses in recipients of Biv-OCV. We assessed serum antibody responses against O1 OSP, as well as antibody secreting cell (ASC) responses (a surrogate marker for mucosal immunity) and memory B cell responses in blood of adult recipients of Biv-OCV in Dhaka, Bangladesh. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 30 healthy adults in this study and administered two doses of OCV (Shanchol) at days 0 and 14. Blood samples were collected before vaccination (day 0) and 7 days after each vaccination (day 7 and day 21), as well as on day 44. Serum responses were largely IgA with minimal IgG and IgM responses in this population. There was no appreciable boosting following day 14 vaccination. There were significant anti-OSP IgA ASC responses on day 7 following the first vaccination, but none after the second immunization. Anti-OSP IgA memory B cell responses were detectable 30 days after completion of the vaccination series, with no evident induction of IgG memory responses. In this population, anti-Ogawa OSP responses were more prominent than anti-Inaba responses, perhaps reflecting impact of previous exposure. Serum anti-OSP responses returned to baseline within 30 days of completing the vaccine series. CONCLUSION: Our results call into question the utility of the 2-dose regimen separated by 14 days in adults in cholera endemic areas, and also suggest that Biv-OCV-induced immune responses targeting OSP are largely IgA in this highly endemic cholera area. Studies in children in cholera-endemic areas need to be performed. Protective efficacy that extends for more than a month after vaccination presumably is mediated by direct mucosal immune response which is not assessed in this study. Our results suggest a single dose of OCV in adults in a cholera endemic zone may be sufficient to mediate at least short-term protection.


Assuntos
Formação de Anticorpos , Vacinas contra Cólera/imunologia , Cólera/prevenção & controle , Memória Imunológica/imunologia , Membrana Mucosa/imunologia , Antígenos O/imunologia , Vacinação , Vibrio cholerae O1/imunologia , Administração Oral , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Bangladesh , Vacinas contra Cólera/administração & dosagem , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Vibrio cholerae O139/imunologia , Adulto Jovem
7.
BMC Immunol ; 20(1): 16, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159728

RESUMO

BACKGROUND: Both antibody secreting cells (ASCs) and memory B cells are essential for the maintenance of humoral immunity. To date, limit studies have focused on the two populations in Kawasaki disease (KD). To address the status of humoral immunity during KD, our current concentration is on the variations of ASCs and memory B cells, as well as their subsets in both acute and remission stages of KD. METHODS: ASCs were defined as the population with high expressions of CD27 and CD38 among CD3-CD20- lymphocytes. Based on the expression of surface marker CD138 and intracellular marker IgG, ASCs were further divided into two subsets. Memory B cells were characterized by the expressions of IgD, CD27 and IgM, upon which memory B cells were further categorized into CD27 + IgD- (switched memory, Sm), CD27-IgD- (Double negative, DN) and CD27 + IgD + IgM+ (marginal zone, MZ) B cells. Collectively, six populations were analyzed using flow cytometry. The blood samples were collected from KD patients in different stages and healthy controls. RESULTS: In the acute stage, the percentages of ASCs, CD138+ ASCs, and IgG+ ASCs were significantly increased. In contrast, the percentages of memory B cells including Sm and MZ B cells were significantly decreased. Correlation analysis found ASCs positively correlated with the level of serum IgM, whereas MZ B cells not only positively correlated with the level of serum IgG, IgA, and IgM, but also positively correlated with the level of serum complement C3 and C4 and negatively correlated with the value of C-reactive protein (CRP). In the remission stage, the percentages of IgG+ ASCs and MZ B cells were significantly reduced, whereas other subsets presented heterogeneous variations. CONCLUSIONS: Our study provided direct evidence that ASCs contributed to the pathogenesis of KD, and it was the first time to describe the variation of memory B cells in this disease. Among the subsets, only IgG+ ASCs presented a significant increase in the acute stage and decreased after IVIG administration, indicating the involvement of IgG+ ASCs in the inflammation of KD and also suggesting that IVIG played an inhibitory role in the expression of cytoplasmic IgG.


Assuntos
Células Produtoras de Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Síndrome de Linfonodos Mucocutâneos/imunologia , Doença Aguda , Antígenos CD/metabolismo , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Imunidade Humoral , Memória Imunológica , Lactente , Masculino
8.
J Immunol Res ; 2019: 3658215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183387

RESUMO

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease associated with immune dysregulation and increased risk of infections. The presence of autoantibodies and immunoglobulin abnormalities indicates B-cell and antibody-secreting cell (ASC) dysfunction. We hypothesize that soluble factors associated with B-cell and ASC activity are decreased in RA patients and that this is linked to higher susceptibility to infections. Methods: Using the Johns Hopkins Arthritis Cohort and Biorepository, we contrasted serum protein levels of soluble factors involved in B-cell activation (CD40, CD40L) and B-cell/ASC homing (CXCL10, CXCL11, and CXCL13) or survival (BAFF, APRIL, TACI, and BCMA) in 10 healthy subjects and 23 adult RA patients (aged 24-65 years). We subdivided RA patients into those with (n = 17) and those without infections (n = 6) within a 2-year period. In order to reduce the effect of RA treatment, we only included patients receiving methotrexate monotherapy or no RA treatments at baseline. Soluble serum protein levels of B-cell/ASC factors were quantified by multiplex immunoassays. Results: We identified that (1) serum levels of soluble BCMA, APRIL, CD40, and CD40L were significantly decreased in RA patients relative to healthy individuals; (2) serum soluble BCMA, predominantly released by ASC, correlated with serum concentrations of class-switched immunoglobulins, IgG and IgA; and (3) RA patients with a history of infections had significantly lower soluble BCMA levels compared with healthy donors and with RA patients without infections. Conclusions: Our study using soluble factors linked to B-cell/ASC activation and survival suggests that there is a paucity of ASC in a subset of RA patients and that this may be linked to altered antibody production and increased risk of infections. Further delineating the link between ASC and infection susceptibility in RA may optimize disease management and provide novel insights into disease pathogenesis that are susceptible to intervention.


Assuntos
Células Produtoras de Anticorpos/imunologia , Artrite Reumatoide/diagnóstico , Linfócitos B/imunologia , Infecções/diagnóstico , Adolescente , Adulto , Idoso , Artrite Reumatoide/epidemiologia , Autoanticorpos/sangue , Antígeno de Maturação de Linfócitos B/sangue , Biomarcadores/sangue , Antígenos CD40/sangue , Sobrevivência Celular , Feminino , Humanos , Infecções/epidemiologia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Risco , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue , Estados Unidos/epidemiologia , Adulto Jovem
9.
Immunity ; 50(5): 1172-1187.e7, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31076359

RESUMO

Although viral infections elicit robust interferon-γ (IFN-γ) and long-lived antibody-secreting cell (ASC) responses, the roles for IFN-γ and IFN-γ-induced transcription factors (TFs) in ASC development are unclear. We showed that B cell intrinsic expression of IFN-γR and the IFN-γ-induced TF T-bet were required for T-helper 1 cell-induced differentiation of B cells into ASCs. IFN-γR signaling induced Blimp1 expression in B cells but also initiated an inflammatory gene program that, if not restrained, prevented ASC formation. T-bet did not affect Blimp1 upregulation in IFN-γ-activated B cells but instead regulated chromatin accessibility within the Ifng and Ifngr2 loci and repressed the IFN-γ-induced inflammatory gene program. Consistent with this, B cell intrinsic T-bet was required for formation of long-lived ASCs and secondary ASCs following viral, but not nematode, infection. Therefore, T-bet facilitates differentiation of IFN-γ-activated inflammatory effector B cells into ASCs in the setting of IFN-γ-, but not IL-4-, induced inflammatory responses.


Assuntos
Linfócitos B/imunologia , Interferon gama/imunologia , Receptores de Interferon/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Linfócitos B/citologia , Diferenciação Celular/imunologia , Células Cultivadas , Cromatina/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nematospiroides dubius/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/biossíntese , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Proteínas com Domínio T/genética
10.
Mol Biol Rep ; 46(4): 3865-3876, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31016614

RESUMO

Classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family causes contagious fatal disease in swine. Antibodies against E2, Erns and NS3 proteins of virus can be detected in infected animals. Development of an ELISA coating antigen to improve the sensitivity of detecting Erns-specific antibodies in pig sera is always desirable for diagnosis as well as for differentiation of infected from vaccinated animals. In present study, a lentivirus-based gene delivery system was used to develop a stable PK-15 cell line expressing Erns (PK-Erns) for production of diagnostic antigen. The Lenti-Erns virus was purified from the supernatant of co-transfected 293LTV cells and used to transduce PK-15 cells. The homogenous PK-Erns cell line was produced by single cell cloning by monitoring eGFP expression. The Erns gene in the genomic DNA and RNA transcripts in total RNA isolated from PK-Erns cells were detected by PCR and RT-PCR, respectively. Expression of 45 kDa Erns glycoprotein was detected in western blot using CSFV-specific hyperimmune sera. The use of PK-Erns cell lysate as antigen in serial dilution and single dilution ELISAs with known positive and negative pig sera was investigated. The PK-Erns ELISA revealed sensitivity equivalent to commercial HerdChek ELISA kit. The sensitivity, specificity and accuracy of the PK-Erns ELISA was 95%, 100% and 96.66%, respectively compared to ELISA using purified CSFV as coating antigen. When field pig sera (n = 69) were tested in PK-Erns ELISA, a significant correlation between the titers from serial dilution and single dilution ELISA was observed. This indicated that PK-Erns cell line can serve as continuous source of ELISA diagnostic antigen for detection of CSFV-specific antibodies in pig sera.


Assuntos
Vírus da Febre Suína Clássica/genética , Técnicas de Diagnóstico Molecular/métodos , Proteínas Estruturais Virais/imunologia , Animais , Especificidade de Anticorpos/genética , Células Produtoras de Anticorpos/metabolismo , Linhagem Celular , Vírus da Febre Suína Clássica/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Transferência de Genes , Lentivirus/metabolismo , Proteínas , Proteínas Recombinantes , Sensibilidade e Especificidade , Suínos/genética , Suínos/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas Estruturais Virais/genética
11.
Immunity ; 50(3): 616-628.e6, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30850343

RESUMO

Humoral immunity depends on efficient activation of B cells and their subsequent differentiation into antibody-secreting cells (ASCs). The transcription factor NFκB cRel is critical for B cell proliferation, but incorporating its known regulatory interactions into a mathematical model of the ASC differentiation circuit prevented ASC generation in simulations. Indeed, experimental ectopic cRel expression blocked ASC differentiation by inhibiting the transcription factor Blimp1, and in wild-type (WT) cells cRel was dynamically repressed during ASC differentiation by Blimp1 binding the Rel locus. Including this bi-stable circuit of mutual cRel-Blimp1 antagonism into a multi-scale model revealed that dynamic repression of cRel controls the switch from B cell proliferation to ASC generation phases and hence the respective cell population dynamics. Our studies provide a mechanistic explanation of how dysregulation of this bi-stable circuit might result in pathologic B cell population phenotypes and thus offer new avenues for diagnostic stratification and treatment.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Proliferação de Células/fisiologia , NF-kappa B/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Linhagem Celular , Feminino , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Imunidade Humoral/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL
12.
Pediatr Infect Dis J ; 38(4): 431-438, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30882741

RESUMO

BACKGROUND: Kawasaki disease (KD) is an acute, systemic vasculitis syndrome that occurs in children. The clinical symptoms and epidemiologic features of KD strongly suggest that KD is triggered by unidentified infectious agents in genetically predisposed patients. In addition, a number of studies have described the role of B cells in the development of KD. To obtain a mechanistic insight into the humoral immune response of B-lineage cells in KD patients, we examined peripheral blood antibody secreting cells (ASCs) and inhibitory immunoreceptors, immunoglobulin-like transcript (ILT)/leukocyte immunoglobulin-like receptor (LILR), on each B cell subpopulation. METHODS: Eighteen Japanese KD patients and thirteen healthy control subjects were recruited for this study. Their peripheral blood mononuclear cells were examined by flow cytometry for the number of CD19 B cells, the size of each B cell subset and the expression of the inhibitory isoforms of ILT/LILR on the B cell subset. RESULTS: The frequency of CD19CD27 ASCs was significantly increased in the acute phase of KD and reduced after high-dose intravenous immunoglobulin (IVIG) treatment. Interestingly, while ILT2/LILRB1 expression was ubiquitously observed on every B cell/ASCs subset and the level was not significantly different after IVIG, ILT3/LILRB4 (B4) was uniquely expressed on only ASCs, and its expression was significantly decreased after IVIG. CONCLUSIONS: In the acute phase of KD, the frequency of ASCs is high with augmented B4 expression, whereas it is lower with decreased B4 expression after IVIG. Further studies of B4 expression on ASCs in autoimmune and infectious diseases will be needed to confirm the significance of our findings.


Assuntos
Células Produtoras de Anticorpos/química , Glicoproteínas de Membrana/análise , Síndrome de Linfonodos Mucocutâneos/patologia , Receptores Imunológicos/análise , Antígenos CD19/análise , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Lactente , Japão , Leucócitos Mononucleares/química , Masculino
13.
Int Immunol ; 31(6): 397-406, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30768140

RESUMO

AbstractImmune homeostasis is critically regulated by the balance between activating and inhibitory receptors expressed on various immune cells such as T and B lymphocytes, and myeloid cells. The inhibitory receptors play a fundamental role in the immune checkpoint pathway, thus maintaining peripheral tolerance. We recently found that expression of leukocyte immunoglobulin-like receptor (LILR)B4, an inhibitory member of the human LILR family, is augmented in auto-antibody-producing plasmablasts/plasma cells of systemic lupus erythematosus (SLE) patients. However, the mechanism behind the 'paradoxical' up-regulation of this inhibitory receptor upon pathogenic antibody-secreting cells is yet to be known. To this end, in this study, we examined if glycoprotein 49B (gp49B), the murine counterpart of human LILRB4, is also elevated in auto-antibody-producing cells in several SLE mouse models, and tried to clarify the underlying mechanism. We found that gp49B is expressed on plasma cells of lupus-prone models but not of healthy C57BL/6 mice, and the level was positively correlated to the anti-double-stranded DNA IgG titer in serum. Gp49B genetic deletion, however, did not abolish the serum auto-antibodies or fully ameliorate the lethal glomerulonephritis, indicating that gp49B is not the sole regulator of lupus but a pathogenic element in the disease. We conclude that the elevated expression of this inhibitory receptor on pathogenic plasma cells was also relevant upon the murine SLE model. The mechanism of gp49B underlying the disease progression in lupus-prone mice has been discussed.


Assuntos
Células Produtoras de Anticorpos/imunologia , Biomarcadores/metabolismo , Glomerulonefrite/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/imunologia , Glicoproteínas de Membrana/metabolismo , Plasmócitos/imunologia , Receptores Imunológicos/metabolismo , Animais , Anticorpos Antinucleares/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Imunológicos/genética
14.
J Immunol ; 202(4): 1287-1300, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30642980

RESUMO

Recurrent mutational activation of the MAP kinase pathway in plasma cell myeloma implicates growth factor-like signaling responses in the biology of Ab-secreting cells (ASCs). Physiological ASCs survive in niche microenvironments, but how niche signals are propagated and integrated is poorly understood. In this study, we dissect such a response in human ASCs using an in vitro model. Applying time course expression data and parsimonious gene correlation network analysis (PGCNA), a new approach established by our group, we map expression changes that occur during the maturation of proliferating plasmablast to quiescent plasma cell under survival conditions including the potential niche signal TGF-ß3. This analysis demonstrates a convergent pattern of differentiation, linking unfolded protein response/endoplasmic reticulum stress to secretory optimization, coordinated with cell cycle exit. TGF-ß3 supports ASC survival while having a limited effect on gene expression including upregulation of CXCR4. This is associated with a significant shift in response to SDF1 in ASCs with amplified ERK1/2 activation, growth factor-like immediate early gene regulation and EGR1 protein expression. Similarly, ASCs responding to survival conditions initially induce partially overlapping sets of immediate early genes without sustaining the response. Thus, in human ASCs growth factor-like gene regulation is transiently imposed by niche signals but is not sustained during subsequent survival and maturation.


Assuntos
Células Produtoras de Anticorpos/imunologia , Quimiocina CXCL12/imunologia , Fator de Crescimento Transformador beta3/imunologia , Sobrevivência Celular , Células Cultivadas , Quimiocina CXCL12/genética , Voluntários Saudáveis , Humanos , Fator de Crescimento Transformador beta3/genética
15.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30463981

RESUMO

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.


Assuntos
Herpesvirus Humano 2/fisiologia , Eliminação de Partículas Virais/imunologia , Eliminação de Partículas Virais/fisiologia , Animais , Anticorpos Antivirais/imunologia , Células Produtoras de Anticorpos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Feminino , Cobaias/virologia , Herpes Simples/imunologia , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/patogenicidade , Imunidade Celular/imunologia , Proteínas do Envelope Viral/imunologia
16.
Transpl Immunol ; 53: 34-37, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30552996

RESUMO

Transplant recipients become immunocompromised through the use of immunosuppressive therapy to prevent allograft rejection. These recipients readily experience human cytomegalovirus (CMV) infection or reactivation. Therefore, CMV represents a life-threatening pathogen in transplant recipients. To demonstrate the serostatus and course of IgG maturation against CMV in transplant patients, we measured the transition of anti-CMV IgG and its affinity (avidity index; AI) as criteria for antibody maturation. Among 31 lung transplant recipients, 26 were infected with CMV before transplantation and maintained anti-CMV IgG and high AI values throughout the study period. Four of the 31 experienced primary infection with CMV through the allograft, with two of the 4 recipients presented high AI values even after 6 month post-transplantation. A significant portion of donor-derived plasma cells were detectable in one recipient. These results suggested that the plasma cells from donors are carried in through the transplanted lung and lymph nodes and produce matured high-avidity IgG from the early stage of transplantation.


Assuntos
Células Produtoras de Anticorpos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Transplante de Pulmão , Plasmócitos/imunologia , Adulto , Anticorpos Antivirais/metabolismo , Afinidade de Anticorpos , Feminino , Humanos , Imunoglobulina G/metabolismo , Imunossupressão , Isoantígenos/imunologia , Masculino , Pessoa de Meia-Idade , Ativação Viral
17.
J Immunol ; 202(2): 401-405, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30552165

RESUMO

The T follicular helper (Tfh) cell subset of CD4+ Th cells promotes affinity maturation by B cells in germinal centers. The contribution of other Th cell subsets to B cell responses has not been fully explored in vivo. We addressed this issue by analyzing the T cell-dependent B cell response to the protein Ag PE in mice lacking specific Th cell subsets. As expected, PE-specific germinal center B cell production required Tfh cells. However, Tfh, Th1, or Th17 cell-deficient mice produced as many PE-specific, isotype-switched plasmablasts as wild-type mice. This response depended on Th cell expression of CD154 and Ag presentation by B cells. These results indicate that many Th cell subsets can promote plasmablast formation by providing CD40 signals to naive B cells.


Assuntos
Células Produtoras de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Centro Germinativo/imunologia , Plasmócitos/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Afinidade de Anticorpos , Apresentação do Antígeno , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Feminino , Switching de Imunoglobulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais
18.
Eur J Immunol ; 49(1): 30-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30273443

RESUMO

Antibodies are an essential component of our immune system, underpinning the effectiveness of both the primary immune response to microbial pathogens and the protective and long-lived immunity against re-challenge. All antibodies are produced by relatively rare populations of plasmablasts and plasma cells, collectively termed antibody-secreting cells (ASCs). It is now apparent that ASCs are unique in the body in terms of their gene expression program and metabolic pathways that enable these cells to have an extraordinary rate of immunoglobulin gene transcription, translation, assembly and secretion. In this review we will discuss the cellular, metabolic and molecular specialization that allows ASCs to maintain such high rates of antibody production, in some cases for the life of the individual. Throughout the review we will link these exquisite cellular and molecular adaptations to the major regulators of ASC gene expression, in an attempt to define how the ASC phenotype and function is genetically programmed.


Assuntos
Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Plasmócitos/imunologia , Animais , Formação de Anticorpos , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Imunoglobulinas/metabolismo
19.
J Infect Dis ; 219(2): 323-334, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30289460

RESUMO

Protection against encapsulated bacteria can be elicited using polysaccharide vaccines. These antigens often behave as T-cell-independent type 2 antigens (TI-2 Ags). However, TI-2 Ags, including pneumococcal polysaccharides, often elicit weak immunoglobulin G (IgG) responses and are refractive to boosting. Conjugate vaccines have not completely overcome this challenge and hence, alternative strategies are required to enhance polysaccharide vaccine responses. Herein, we describe an adjuvant consisting of a Toll-like receptor and C-type lectin receptor agonist pairing that significantly increases primary immunoglobulin M and IgG responses to TI-2 Ags as well as enables significant boosting when coadministered with polysaccharide vaccines. Consistent with this, the adjuvant significantly increased the generation of both TI-2 memory B cells and long-lived antibody secreting cells. Adjuvant effects were highly dependent on B-cell-intrinsic MyD88, but not Trif expression. Importantly, coadministration of the adjuvant with the Pneumovax vaccine significantly increased the protective efficacy of vaccination in a lethal challenge mouse model of pneumococcal respiratory infection. Collectively, these data provide evidence that B-cell-directed adjuvants have promise in significantly improving the quality and quantity of serologic and B-cell memory responses to clinically relevant polysaccharide vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Polissacarídeos/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Anticorpos Antibacterianos , Células Produtoras de Anticorpos/imunologia , Linfócitos B/imunologia , Vacinas Bacterianas/imunologia , Modelos Animais de Doenças , Feminino , Imunoglobulina G/imunologia , Imunoglobulina M , Lectinas Tipo C/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Receptores Toll-Like/imunologia , Vacinação , Vacinas Conjugadas/imunologia
20.
Methods Mol Biol ; 1904: 147-162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539469

RESUMO

Antigen-specific monoclonal antibodies are useful tools to detect very small amounts of antigenic materials and are applicable for antibody therapeutics. To produce mouse monoclonal antibodies, a hybridoma between B lymphocytes and myeloma cells is used to produce antigen-specific monoclonal antibodies. However, a good hybridoma system is not available to obtain human monoclonal antibodies. To produce antigen-specific human monoclonal antibodies, transformation of B lymphocytes with Epstein-Barr viruses or a phage-display system is used. Here, we describe the screening of antigen-specific, antibody-secreting cells using microwell array chips to obtain antigen-specific human monoclonal antibodies. The system can be applied to screen antigen-specific, antibody-secreting cells from any animal species.


Assuntos
Formação de Anticorpos/imunologia , Células Produtoras de Anticorpos/imunologia , Antígenos/imunologia , Epitopos/imunologia , Imunoensaio , Análise em Microsséries , Formação de Anticorpos/genética , Células Produtoras de Anticorpos/metabolismo , Biomarcadores , Expressão Gênica , Vetores Genéticos/genética , Hibridomas/imunologia , Imunoensaio/métodos , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Análise em Microsséries/métodos
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