RESUMO
Germinal center B cells (GCBCs) are critical for generating long-lived humoral immunity. How GCBCs meet the energetic challenge of rapid proliferation is poorly understood. Dividing lymphocytes typically rely on aerobic glycolysis over oxidative phosphorylation for energy. Here we report that GCBCs are exceptional among proliferating B and T cells, as they actively oxidize fatty acids (FAs) and conduct minimal glycolysis. In vitro, GCBCs had a very low glycolytic extracellular acidification rate but consumed oxygen in response to FAs. [13C6]-glucose feeding revealed that GCBCs generate significantly less phosphorylated glucose and little lactate. Further, GCBCs did not metabolize glucose into tricarboxylic acid (TCA) cycle intermediates. Conversely, [13C16]-palmitic acid labeling demonstrated that GCBCs generate most of their acetyl-CoA and acetylcarnitine from FAs. FA oxidation was functionally important, as drug-mediated and genetic dampening of FA oxidation resulted in a selective reduction of GCBCs. Hence, GCBCs appear to uncouple rapid proliferation from aerobic glycolysis.
Assuntos
Linfócitos B/metabolismo , Ácidos Graxos/metabolismo , Centro Germinativo/metabolismo , Animais , Linfócitos B/imunologia , Proliferação de Células , Metabolismo Energético , Ácidos Graxos não Esterificados/metabolismo , Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/imunologia , Glucose/metabolismo , Glicólise/genética , Técnicas In Vitro , Metaboloma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxirredução , Fosforilação Oxidativa , Consumo de OxigênioRESUMO
Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.
Assuntos
Betacoronavirus/fisiologia , Vacinas contra COVID-19/imunologia , Infecções por Coronavirus/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Sequência Conservada/genética , Evolução Molecular , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Desenvolvimento de VacinasRESUMO
The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of ß-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23-TH17 cell-dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/metabolismo , Linfócitos B/imunologia , Tolerância Imunológica , Imunoglobulina G/metabolismo , Interleucina-23/metabolismo , Células Th17/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Glicosilação , Humanos , Interleucinas/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Sialiltransferases/genética , Sialiltransferases/metabolismo , Transdução de Sinais , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , Interleucina 22RESUMO
The intestinal epithelium harbours a unique lymphocyte population, the intraepithelial lymphocytes (IELs). A large fraction of IELs is represented by γδ T cells. Their role in epithelial homeostasis and immune response is well documented, but a conclusive view of their developmental pathway is still missing. In this review, we discuss the existing literature as well as recent advances regarding the tissue adaptation of γδ IELs, both for the characteristic cytotoxic subset and the newly described noncytotoxic subset. We particularly highlight the environmental cues and the transcriptional regulation that equip γδ T cells with their IEL phenotype.
RESUMO
Human cytomegalovirus is a medically important pathogen. Previously, using murine CMV (MCMV), we provided evidence that both neutralizing and nonneutralizing antibodies can confer protection from viral infection in vivo. In this study, we report that serum derived from infected animals had a greater protective capacity in MCMV-infected RAG-/- mice than serum from animals immunized with purified virus. The protective activity of immune serum was strictly dependent on functional Fcγ receptors (FcγR). Deletion of individual FcγRs or combined deletion of FcγRI and FcγRIV had little impact on the protection afforded by serum. Adoptive transfer of CD115-positive cells from noninfected donors demonstrated that monocytes represent important cellular mediators of the protective activity provided by immune serum. Our studies suggest that Fc-FcγR interactions and monocytic cells are critical for antibody-mediated protection against MCMV infection in vivo. These findings may provide new avenues for the development of novel strategies for more effective CMV vaccines or antiviral immunotherapies.
RESUMO
The potency of antibody neutralization in cell culture has been used as the key criterion for selection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for clinical development. As other aspects may also influence the degree of protection in vivo, we compared the efficacy of two neutralizing monoclonal antibodies (TRES6 and 4C12) targeting different epitopes of the receptor binding domain (RBD) of SARS-CoV-2 in a prophylactic setting in rhesus monkeys. All four animals treated with TRES6 had reduced viral loads in the upper respiratory tract 2 days after naso-oropharyngeal challenge with the Alpha SARS-CoV-2 variant. Starting 2 days after challenge, mutations conferring resistance to TRES6 were dominant in two of the rhesus monkeys, with both animals failing to maintain reduced viral loads. Consistent with its lower serum neutralization titer at the day of challenge, prophylaxis with 4C12 tended to suppress viral load at day 2 less efficiently than TRES6. However, a week after challenge, mean viral loads in the lower respiratory tract in 4C12-treated animals were lower than in the TRES6 group and no mutations conferring resistance to 4C12 could be detected in viral isolates from nasal or throat swabs. Thus, genetic barrier to resistance seems to be a critical parameter for the efficacy of prophylaxis with monoclonal antibodies against SARS-CoV-2. Furthermore, comparison of antibody concentrations in respiratory secretions to those in serum shows reduced distribution of the 4C12 antibody into respiratory secretions and a delay in the appearance of antibodies in bronchoalveolar lavage fluid compared to their appearance in secretions of the upper respiratory tract.IMPORTANCEMonoclonal antibodies are a powerful tool for the prophylaxis and treatment of acute viral infections. Hence, they were one of the first therapeutic agents licensed for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oftentimes, the main criterion for the selection of antibodies for clinical development is their potency of neutralization in cell culture. By comparing two antibodies targeting the Spike protein of SARS-CoV-2, we now observed that the antibody that neutralized SARS-CoV-2 more efficiently in cell culture suppressed viral load in challenged rhesus monkeys to a lesser extent. Extraordinary rapid emergence of mutants of the challenge virus, which had lost their sensitivity to the antibody, was identified as the major reason for the reduced efficacy of the antibody in rhesus monkeys. Therefore, the viral genetic barrier to resistance to antibodies also affects their efficacy.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Modelos Animais de Doenças , Macaca mulatta , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Carga Viral , Animais , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Monoclonais/imunologia , COVID-19/imunologia , COVID-19/virologia , COVID-19/prevenção & controle , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Humanos , Mutação , Epitopos/imunologia , Testes de NeutralizaçãoRESUMO
Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults in the Western world. B cell receptor (BCR) signaling is known to be crucial for the pathogenesis and maintenance of CLL cells which develop from mature CD5+ B cells. BCR signaling is regulated by the inhibitory co-receptor Siglec-G and Siglec-G-deficient mice have an enlarged CD5+ B1a cell population. Here, we determine how Siglec-G expression influences the severity of CLL. Our results show that Siglec-G deficiency leads to earlier onset and more severe course of the CLL-like disease in the murine Eµ-TCL1 model. In contrast, mice overexpressing Siglec-G on the B cell surface are almost completely protected from developing CLL-like disease. Furthermore, we observe a downmodulation of the human ortholog Siglec-10 from the surface of human CLL cells. These results demonstrate a critical role for Siglec-G in disease progression in mice, and suggest that a similar mechanism for Siglec-10 in human CLL may exist.
Assuntos
Leucemia Linfocítica Crônica de Células B , Camundongos , Animais , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Camundongos Transgênicos , Proteínas Proto-Oncogênicas , Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Camundongos , SARS-CoV-2RESUMO
Germinal center reactions are established during a thymus-dependent immune response. Germinal center (GC) B cells are rapidly proliferating and undergo somatic hypermutation in Ab genes. This results in the production of high-affinity Abs and establishment of long-lived memory cells. GC B cells show lower BCR-induced signaling when compared with naive B cells, but the functional relevance is not clear. CD22 is a member of the Siglec family and functions as an inhibitory coreceptor on B cells. Interestingly, GC B cells downregulate sialic acid forms that serve as high-affinity ligands for CD22, indicating a role for CD22 ligand binding during GC responses. We studied the role of CD22 in the GC with mixed bone marrow chimeric mice and found a disadvantage of CD22-/- GC B cells during the GC reaction. Mechanistic investigations ruled out defects in dark zone/light zone distribution and affinity maturation. Rather, an increased rate of apoptosis in CD22-/- GC B cells was responsible for the disadvantage, also leading to a lower GC output in plasma cells and memory B cells. CD22-/- GC B cells showed a clearly increased calcium response upon BCR stimulation, which was almost absent in wild-type GC B cells. We conclude that the differential expression of the low-affinity cis CD22 ligands in the GC normally results in a strong attenuation of BCR signaling in GC B cells, probably due to higher CD22-BCR interactions. Therefore, attenuation of BCR signaling by CD22 is involved in GC output and B cell fate.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Memória Imunológica/imunologia , Plasmócitos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Transdução de Sinais/imunologia , Animais , Apoptose/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologiaRESUMO
COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected are to date not well understood. Comparing the immunological phenotype in healthy donors and patients with mild versus severe COVID-19 shows that in COVID-19 patients, NK-/B-cell activation and proliferation are enhanced independent of severity. As an important precondition for effective antibody responses, T-follicular helper cells and antibody secreting cells are increased both in patients with mild and severe SARS-CoV-2 infection. Beyond this, T cells in COVID-19 patients exhibit a stronger activation profile with differentiation toward effector cell phenotypes. Importantly, when looking at the rates of pulmonary complications in COVID-19 patients, the chemokine receptor CCR4 is higher expressed by both CD4 and CD8 T cells of patients with severe COVID-19. This raises the hypothesis that CCR4 upregulation on T cells in the pathogenesis of COVID-19 promotes stronger T-cell attraction to the lungs leading to increased immune activation with presumably higher pulmonary toxicity. Our study contributes significantly to the understanding of the immunological changes during COVID-19, as new therapeutic agents, preferentially targeting the immune system, are highly warranted.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Pulmão/imunologia , Ativação Linfocitária , Receptores CCR4/imunologia , SARS-CoV-2/imunologia , Regulação para Cima/imunologia , Adulto , Linfócitos T CD8-Positivos/patologia , COVID-19/patologia , Feminino , Humanos , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe clinical disease in immunosuppressed patients and congenitally infected newborn infants. Viral envelope glycoproteins represent attractive targets for vaccination or passive immunotherapy. To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (MAbs) were generated following immunization of mice with HCMV virions. Hybridoma supernatants were screened for in vitro neutralization activity, yielding three potent MAbs, 6E3, 3C11, and 2B10. MAbs 6E3 and 3C11 blocked infection of all viral strains that were tested, while MAb 2B10 neutralized only 50% of the HCMV strains analyzed. Characterization of the MAbs using indirect immunofluorescence analyses demonstrated their reactivity with recombinantly derived gH. While MAbs 6E3 and 3C11 reacted with gH when expressed alone, 2B10 detected gH only when it was coexpressed with gB and gL. Recognition of gH by 3C11 was dependent on the expression of the entire ectodomain of gH, whereas 6E3 required residues 1 to 629 of gH. The strain-specific determinant for neutralization by Mab 2B10 was identified as a single MetâIle amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is evenly distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. IMPORTANCE HCMV infections are life threatening to people with compromised or immature immune systems. Understanding the antiviral antibody repertoire induced during HCMV infection is a necessary prerequisite to define protective antibody responses. Here, we report three novel anti-gH MAbs that potently neutralized HCMV infectivity. One of these MAbs (2B10) targets a novel strain-specific conformational epitope on gH that only becomes accessible upon coexpression of the minimal fusion machinery gB/gH/gL. Strain specificity is dependent on a single amino acid polymorphism within gH. Our data highlight the importance of strain-specific neutralizing antibody responses against HCMV. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. In addition, the dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Citomegalovirus/classificação , Infecções por Citomegalovirus/virologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.
Assuntos
Artrite Reumatoide/imunologia , Reabsorção Óssea/imunologia , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Animais , Artrite Experimental/sangue , Artrite Experimental/complicações , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Reumatoide/sangue , Artrite Reumatoide/complicações , Artrite Reumatoide/genética , Reabsorção Óssea/patologia , Osso e Ossos/imunologia , Osso e Ossos/patologia , Células Cultivadas , Feminino , Humanos , Imunoglobulinas/genética , Leucócitos Mononucleares , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Osteoclastos/imunologia , Cultura Primária de CélulasRESUMO
Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.
Assuntos
Anticorpos Neutralizantes/farmacologia , Citomegalovirus/genética , Proteínas Mutantes Quiméricas/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Fusão Celular , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Células Gigantes/ultraestrutura , Células Gigantes/virologia , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/virologia , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.
Assuntos
Anticorpos Antivirais/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2 , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Two-photon microscopy enables monitoring cellular dynamics and communication in complex systems, within a genuine environment, such as living tissues and, even, living organisms. Particularly, its application to understand cellular interactions in the immune system has brought unique insights into pathophysiologic processes in vivo. Simultaneous multiplexed imaging is required to understand the dynamic orchestration of the multiple cellular and non-cellular tissue compartments defining immune responses. Here, we present an improvement of our previously developed method, which allowed us to achieve multiplexed dynamic intravital two-photon imaging, by using a synergistic strategy. This strategy combines a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an unmixing algorithm based on the calculation of spectral similarities with previously measured fluorophore fingerprints. The improvement of the similarity spectral unmixing algorithm here described is based on dimensionality reduction of the mixing matrix. We demonstrate its superior performance in the correct pixel-based assignment of probes to tissue compartments labeled by single fluorophores with similar spectral fingerprints, as compared to the full-dimensional similarity spectral unmixing approach.
Assuntos
Comunicação Celular/genética , Microambiente Celular/genética , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Algoritmos , Linhagem Celular , Corantes Fluorescentes/química , FótonsRESUMO
After allogeneic hematopoietic stem cell transplantation (HSCT), patients are repetitively vaccinated to reduce the risk of infection caused by the immune deficiency following allogeneic HSCT. By the vaccination of transplanted patients, the humoral memory function can be restored in the majority of cases. It is unknown, however, to what extent memory B cells derived from the donor contribute to the mobilization of antibody-secreting cells and long-term humoral memory in patients after allogeneic HSCT. We therefore analyzed patients after allogeneic HSCT for memory B cell responses 7 days after single vaccination against tetanus toxoid (TT), diphtheria toxoid (DT), pertussis toxoid (PT), Haemophilus influenzae type b (Hib), and poliovirus. Patients showed an insufficient mobilization of plasmablasts (PB) after vaccination, whereas healthy subjects (HD, n = 13) exhibited a significant increase of PB in the peripheral blood. Regarding vaccine-specific antibody-secreting PB, all HD responded against all vaccine antigens, as expected. However, only 65% of the patients responded with a measurable increase in IgG-secreting PB against TT, 65% against DT, 33% against PT, and 53% against poliovirus. Correspondingly, the antibody titers on day 7 after vaccination did not increase in patients. A significant increase of serum titers for the vaccine antigens was detectable in the majority of patients only after repetitive vaccinations. In contrast to the low mobilization of vaccine-specific PB after vaccination, a high number of PB before vaccination was detectable in patients following allogeneic HSCT. High frequencies of circulating PB correlated with the incidence of moderate/severe chronic GVHD. In summary, patients showed a weak mobilization of antigen-specific PB and an inadequate increase in antibody titers 7 days after the first vaccination. Patients with moderate or severe chronic GVHD in their history had a significantly higher percentage of IgG-secreting PB prior to vaccination. The antigen specificity of these IgG-secreting PB is currently unknown.
Assuntos
Linfócitos B/imunologia , Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Vacinas Anti-Haemophilus/administração & dosagem , Transplante de Células-Tronco Hematopoéticas , Memória Imunológica , Vacinas Pneumocócicas/administração & dosagem , Vacina Antipólio de Vírus Inativado/administração & dosagem , Vacinação , Adolescente , Adulto , Idoso , Aloenxertos , Anticorpos Antibacterianos/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Vacinas Combinadas/administração & dosagemRESUMO
Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra Citomegalovirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Modelos Animais de Doenças , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The function of CD83 during GC responses is unclear. CD83(-/-) mice have a strong reduction of CD4(+) T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell-specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo.
Assuntos
Antígenos CD/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulinas/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Animais , Antígenos CD/genética , Linfócitos B/fisiologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Borrelia burgdorferi/imunologia , Células Dendríticas/imunologia , Genes MHC da Classe II/genética , Genes MHC da Classe II/imunologia , Centro Germinativo/citologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulinas/deficiência , Imunoglobulinas/genética , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Células Th2/imunologia , Antígeno CD83RESUMO
BACKGROUND: We have recently shown that memory B cells from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of immunodeficiency after allogeneic hematopoietic stem cell transplantation. As adoptive transfer of B cells has not been used before in a clinical setting it was necessary to establish a technology for the generation of good manufacturing practice (GMP)-grade B cell products. METHODS: Starting from the leukapheresis product of healthy blood donors, B cells were purified by two different separation strategies using GMP-grade microbeads and the CliniMACS system. A one-step protocol was used for positive enrichment of B lymphocytes with anti-CD19 microbeads. In a two-step enrichment protocol, first T lymphocytes were depleted by anti-CD3 microbeads and the remaining fraction was positively selected by anti-CD19 microbeads. RESULTS: The purity and recovery after enrichment of B lymphocytes from the leukapheresis material in both separations strategies was not statistically different. However, contamination of the B-cell product with T cells was significantly lower after the two-step protocol (0.16%, range 0.01-0.43% after two-step separation and 0.55%, range 0.28-0.85% after one-step separation, p < 0.05). Therefore, a combined CD3 depletion and CD19 enrichment was used for the production of GMP-conform B-cell products from the leukapheresis material of 17 healthy stem cell donors. The absolute B-cell numbers obtained in the final product was 4.70 ± 3.64 × 108 with a purity of 95.98 ± 3.31% B lymphocytes and a recovery of 18.9 ± 10.6%. Importantly, the contamination with CD3+ T cells was extremely low in the final B- cell products (0.10 ± 0.20%). Purified B cells exhibited normal antibody production after in vitro stimulation and showed excellent viability after cryopreservation. CONCLUSIONS: A GMP-grade B-cell product can be obtained with high purity and very low T-cell contamination using the two-step enrichment protocol based on CliniMACS® technology.
Assuntos
Transferência Adotiva , Linfócitos B/metabolismo , Separação Celular/métodos , Separação Celular/normas , Transplante de Células-Tronco Hematopoéticas , Controle de Qualidade , Antígenos CD/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imunofenotipagem , Transplante HomólogoRESUMO
Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αß-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1(-/-) mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1(-/-) mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.