RESUMEN
Although absence of interleukin-7 (IL-7) signaling completely abrogates T and B lymphopoiesis in mice, patients with severe combined immunodeficiency caused by mutations in the IL-7 receptor α chain (IL-7Rα) still generate peripheral blood B cells. Consequently, human B lymphopoiesis has been thought to be independent of IL-7 signaling. Using flow cytometric analysis and single-cell RNA sequencing of bone marrow samples from healthy controls and patients who are IL-7Rα deficient, in combination with in vitro modeling of human B-cell differentiation, we demonstrate that IL-7R signaling plays a crucial role in human B lymphopoiesis. IL-7 drives proliferation and expansion of early B-cell progenitors but not of pre-BII large cells and has a limited role in the prevention of cell death. Furthermore, IL-7 guides cell fate decisions by enhancing the expression of BACH2, EBF1, and PAX5, which jointly orchestrate the specification and commitment of early B-cell progenitors. In line with this observation, early B-cell progenitors of patients with IL-7Rα deficiency still expressed myeloid-specific genes. Collectively, our results unveil a previously unknown role for IL-7 signaling in promoting the B-lymphoid fate and expanding early human B-cell progenitors while defining important differences between mice and humans. Our results have implications for hematopoietic stem cell transplantation strategies in patients with T- B+ severe combined immunodeficiency and provide insights into the role of IL-7R signaling in leukemogenesis.
Asunto(s)
Interleucina-7 , Inmunodeficiencia Combinada Grave , Humanos , Animales , Ratones , Interleucina-7/metabolismo , Receptores de Interleucina-7/genética , Diferenciación Celular , HematopoyesisRESUMEN
In past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and disease. For such studies, it is paramount that isolation strategies yield a population of bona fide naive B cells, i.e., B cells that are phenotypically and functionally naive, clonally non-expanded, and have non-mutated BCR variable regions. In this study different combinations of common as well as recently identified B cell markers were compared to isolate naive B cells from human peripheral blood. High-throughput BCR sequencing was performed to analyze levels of somatic hypermutation and clonal expansion. Additionally, contamination from mature mutated B cells intrinsic to each cell-sorting strategy was evaluated and how this impacts the purity of obtained populations. Our results show that current naive B cell isolation strategies harbor contamination from non-naive B cells, and use of CD27-IgD+ is adequate but can be improved by including markers for CD45RB glycosylation and IgM. The finetuning of naive B cell classification provided herein will harmonize research lines using naive B cells, and will improve B cell profiling during health and disease, e.g. during diagnosis, treatment, and vaccination strategies.
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Subgrupos de Linfocitos B , Subgrupos de Linfocitos B/metabolismo , Separación Celular , Glicosilación , Humanos , Inmunoglobulina D/metabolismo , Isotipos de Inmunoglobulinas/metabolismo , Inmunoglobulina M/metabolismo , Memoria Inmunológica/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Glycosylation of CD45RB (RB+) has recently been identified to mark antigen-experienced B cells, independent of their CD27 expression. By using a novel combination of markers including CD45RB glycosylation, CD27 and IgM/IgD isotype expression we segregated human peripheral blood B cell subsets and investigated their IGHV repertoire and in vitro functionality. We observed distinct maturation stages for CD27-RB+ cells, defined by differential expression of non-switched Ig isotypes. CD27-RB+ cells, which only express IgM, were more matured in terms of Ig gene mutation levels and function as compared to CD27-RB+ cells that express both IgM and IgD or cells that were CD27-RB-. Moreover, CD27-RB+IgM+ cells already showed remarkable rigidity in IgM isotype commitment, different from CD27-RB+IgMD+ and CD27-RB- cells that still demonstrated great plasticity in B cell fate decision. Thus, glycosylation of CD45RB is indicative for antigen-primed B cells, which are, dependent on the Ig isotype, functionally distinct.
Asunto(s)
Subgrupos de Linfocitos B , Antígenos Comunes de Leucocito/inmunología , Subgrupos de Linfocitos B/metabolismo , Glicosilación , Humanos , Inmunoglobulina D/metabolismo , Isotipos de Inmunoglobulinas/metabolismo , Inmunoglobulina M/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Inborn errors of immunity (IEI) are genetic defects that can affect both the innate and the adaptive immune system. Patients with IEI usually present with recurrent infections, but many also suffer from immune dysregulation, autoimmunity, and malignancies.Inborn errors of the immune system can cause defects in the development and selection of the B-cell receptor (BCR ) repertoire. Patients with IEI can have a defect in one of the key processes of immune repertoire formation like V(D)J recombination, somatic hypermutation (SHM), class switch recombination (CSR), or (pre-)BCR signalling and proliferation. However, also other genetic defects can lead to quantitative and qualitative differences in the immune repertoire.In this chapter, we will give an overview of protocols that can be used to study the immune repertoire in patients with IEI, provide considerations to take into account before setting up experiments, and discuss analysis of the immune repertoire data using Antigen Receptor Galaxy (ARGalaxy).
Asunto(s)
Sistema Inmunológico , Cambio de Clase de Inmunoglobulina , Autoinmunidad , Humanos , Fenotipo , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
BACKGROUND: Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). PURPOSE: To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. METHODS: Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. RESULTS: At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. CONCLUSIONS: This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. CLINICAL IMPLICATIONS: This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices.
Asunto(s)
Síndrome de Deleción 22q11 , Linfopenia , Inmunodeficiencia Combinada Grave , Adolescente , ADN , Estudios de Seguimiento , Humanos , Recién Nacido , Linfopenia/diagnóstico , Tamizaje Neonatal , Receptores de Antígenos de Linfocitos T/genética , Inmunodeficiencia Combinada Grave/diagnósticoRESUMEN
Reduced total and memory B-cell numbers in peripheral blood long term after hematopoietic stem cell transplantation (HSCT) are associated with an increased incidence of infections and immune complications. Using novel modelling strategies, baseline factors influencing B-cell reconstitution can be comprehensively studied. This study aims to investigate the numerical total and memory B-cell reconstitution in children and the association with baseline determinants 0.5-2 years after allogeneic HSCT. Eligible for inclusion were children transplanted in our center between 2004-2017 who received a first HSCT for malignant or non-malignant disorders. The continuous absolute counts of total and memory B-cells were evaluated as outcome measure. Exploratory analysis at one year was done to identify possible determinants. Linear mixed effect modelling was used to analyze the association of these determinants with total and memory B-cell reconstitution 0.5-2 years after HSCT. In a cohort of 223 evaluable patients analyzed at 1-year after HSCT donor age, stem cell source, donor type, recipient age and conditioning were identified as significant determinants for total and memory B-cell numbers. Multivariable analysis revealed that both donor and recipient age were inversely correlated with the size of total and memory B-cell reconstitution. In contrast, no correlation was found with stem cell source, donor type and conditioning. Making use of linear mixed modelling both stem cell donor and recipient age were identified as independent determinants of total and memory B-cell reconstitution 0.5-2 years after HSCT.
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Linfocitos B/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Reconstitución Inmune , Adolescente , Factores de Edad , Linfocitos B/metabolismo , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Lactante , Infecciones/etiología , Modelos Lineales , Masculino , Análisis Multivariante , Acondicionamiento Pretrasplante/efectos adversos , Acondicionamiento Pretrasplante/métodosRESUMEN
ATM is often dubbed the master regulator of the DNA double stranded break (DSB) response. Since proper induction and repair of DNA DSBs forms the core of immunological diversity, it is surprising that patients with ataxia telangiectasia generally have a mild immunodeficiency in contrast to other DSB repair syndromes. In this review, we address this discrepancy by delving into the functions of ATM in DSB repair and cell cycle control and translate these to adaptive immunity. We conclude that ATM, despite its myriad functions, is not an absolute requirement for acquiring sufficient levels of immunological diversity to prevent severe viral and opportunistic infections. There is, however, a more clinically pronounced antibody deficiency in ataxia telangiectasia due to disturbed class switch recombination.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Inmunidad Adaptativa , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Cambio de Clase de InmunoglobulinaRESUMEN
Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors. Mutation patterns were congruent with SHM-derived single nucleotide mutations, delineating initiation of the tandem substitution by AID. Tandem substitutions comprised 5,7% of AID-induced mutations. The majority of tandem substitutions represents single nucleotide juxtalocations of directly adjacent sequences. These observations were confirmed in an independent cohort of healthy donors. We propose a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Tandem substitutions may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.
Asunto(s)
Mutación , Secuencias Repetidas en Tándem , Alelos , Codón , Reparación de la Incompatibilidad de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Receptores de Antígenos de Linfocitos B/genética , Hipermutación Somática de Inmunoglobulina , Recombinación V(D)JRESUMEN
The generation of high-affinity antibodies depends on somatic hypermutation (SHM). SHM is initiated by the activation-induced cytidine deaminase (AID), which generates uracil (U) lesions in the B-cell receptor (BCR) encoding genes. Error-prone processing of U lesions creates a typical spectrum of point mutations during SHM. The aim of this study was to determine the molecular mechanism of SHM in humans; currently available knowledge is limited by the number of mutations analyzed per patient. We collected a unique cohort of 10 well-defined patients with bi-allelic mutations in genes involved in base excision repair (BER) (UNG) or mismatch repair (MMR) (MSH2, MSH6, or PMS2) and are the first to present next-generation sequencing (NGS) data of the BCR, allowing us to study SHM extensively in humans. Analysis using ARGalaxy revealed selective skewing of SHM mutation patterns specific for each genetic defect, which are in line with the five-pathway model of SHM that was recently proposed based on mice data. However, trans-species comparison revealed differences in the role of PMS2 and MSH2 in strand targeting between mice and man. In conclusion, our results indicate a role for UNG, MSH2, MSH6, and PMS2 in the generation of SHM in humans comparable to their function in mice. However, we observed differences in strand targeting between humans and mice, emphasizing the importance of studying molecular mechanisms in a human setting. The here developed method combining NGS and ARGalaxy analysis of BCR mutation data forms the basis for efficient SHM analyses of other immune deficiencies.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Reparación de la Incompatibilidad de ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Hipermutación Somática de Inmunoglobulina , Estudios de Casos y Controles , Humanos , MutaciónRESUMEN
Common variable immune deficiency disorder (CVID) is the most clinically relevant cause of antibody failure. It is a highly heterogeneous disease with different underlying etiologies. CVID has been associated with a quantitative B cell defect, however, little is known about the quality of B cells present. Here, we studied the naïve and antigen selected B-cell receptor (BCR) repertoire in 33 CVID patients using next generation sequencing, to investigate B cells quality. Analysis for each individual patient revealed whether they have a defect in immune repertoire formation [V(D)J recombination] or specification (somatic hypermutation, subclass distribution, or selection). The naïve BCR repertoire was normal in most of the patients, although alterations in repertoire diversity and the junctions were found in a limited number of patients indicating possible defects in early B-cell development or V(D)J recombination in these patients. In contrast, major differences were found in the antigen selected BCR repertoire. Here, most patients (15/17) showed a reduced frequency of somatic hypermutation (SHM), changes in subclass distribution and/or minor alterations in antigen selection. Together these data show that in our CVID cohort only a small number of patients have a defect in formation of the naïve BCR repertoire, whereas the clear majority of patients have disturbances in their antigen selected repertoire, suggesting a defect in repertoire specification in the germinal centers of these patients. This highlights that CVID patients not only have a quantitative B cell defect, but that also the quality of, especially post germinal center B cells, is impaired.
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Linfocitos B/inmunología , Inmunodeficiencia Variable Común/inmunología , Centro Germinal/inmunología , Receptores de Antígenos de Linfocitos B/genética , Adolescente , Adulto , Anciano , Diferenciación Celular , Niño , Selección Clonal Mediada por Antígenos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Hipermutación Somática de Inmunoglobulina/genética , Adulto JovenAsunto(s)
Linfocitos B/patología , Factor de Transcripción Ikaros/genética , Lupus Eritematoso Sistémico/diagnóstico , Mutación/genética , Animales , Anticuerpos Antinucleares/metabolismo , Autoinmunidad/genética , Diferenciación Celular/genética , Niño , Femenino , Frecuencia de los Genes , Humanos , Inhibidor de Coagulación del Lupus/metabolismo , Lupus Eritematoso Sistémico/genética , Ratones , Células 3T3 NIH , Linaje , Polimorfismo Genético , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
Antigen Receptor Galaxy (ARGalaxy) is a Web-based tool for analyses and visualization of TCR and BCR sequencing data of 13 species. ARGalaxy consists of four parts: the demultiplex tool, the international ImMunoGeneTics information system (IMGT) concatenate tool, the immune repertoire pipeline, and the somatic hypermutation (SHM) and class switch recombination (CSR) pipeline. Together they allow the analysis of all different aspects of the immune repertoire. All pipelines can be run independently or combined, depending on the available data and the question of interest. The demultiplex tool allows data trimming and demultiplexing, whereas with the concatenate tool multiple IMGT/HighV-QUEST output files can be merged into a single file. The immune repertoire pipeline is an extended version of our previously published ImmunoGlobulin Galaxy (IGGalaxy) virtual machine that was developed to visualize V(D)J gene usage. It allows analysis of both BCR and TCR rearrangements, visualizes CDR3 characteristics (length and amino acid usage) and junction characteristics, and calculates the diversity of the immune repertoire. Finally, ARGalaxy includes the newly developed SHM and CSR pipeline to analyze SHM and/or CSR in BCR rearrangements. It analyzes the frequency and patterns of SHM, Ag selection (including BASELINe), clonality (Change-O), and CSR. The functionality of the ARGalaxy tool is illustrated in several clinical examples of patients with primary immunodeficiencies. In conclusion, ARGalaxy is a novel tool for the analysis of the complete immune repertoire, which is applicable to many patient groups with disturbances in the immune repertoire such as autoimmune diseases, allergy, and leukemia, but it can also be used to address basic research questions in repertoire formation and selection.
Asunto(s)
Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos T/genética , Programas Informáticos , Biología Computacional/métodos , Humanos , Internet , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Hipermutación Somática de InmunoglobulinaRESUMEN
BACKGROUND: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. METHODS: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. RESULTS: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. CONCLUSIONS: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.
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Agammaglobulinemia/genética , Agammaglobulinemia/inmunología , Linfocitos B/inmunología , Diferenciación Celular/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasas/genética , Adolescente , Adulto , Diferenciación Celular/inmunología , Niño , Preescolar , Fosfatidilinositol 3-Quinasa Clase Ia , Femenino , Humanos , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Infecciones/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Mutación/genética , Mutación/inmunología , Fosforilación/genética , Células Plasmáticas/inmunología , Células Precursoras de Linfocitos B/inmunología , Proteínas Proto-Oncogénicas c-akt/genética , Recurrencia , Transducción de Señal/genética , Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunología , Adulto JovenRESUMEN
Upon antigen recognition via their B cell receptor (BR), B cells migrate to the germinal center where they undergo somatic hypermutation (SHM) to increase their affinity for the antigen, and class switch recombination (CSR) to change the effector function of the secreted antibodies. These steps are essential to create an antigen-experienced BR repertoire that efficiently protects the body against pathogens. At the same time, the BR repertoire should be selected to protect against responses to self-antigen or harmless antigens. Insights into the processes of SHM, selection, and CSR can be obtained by studying the antigen-experienced BR repertoire. Currently, a large reference data set of healthy children and adults, which ranges from neonates to the elderly, is not available. In this study, we analyzed the antigen-experienced repertoire of 38 healthy donors (HD), ranging from cord blood to 74 years old, by sequencing IGA and IGG transcripts using next generation sequencing. This resulted in a large, freely available reference data set containing 412,890 IGA and IGG transcripts. We used this data set to study mutation levels, SHM patterns, antigenic selection, and CSR from birth to elderly HD. Only small differences were observed in SHM patterns, while the mutation levels increase in early childhood and stabilize at 6 years of age at around 7%. Furthermore, comparison of the antigen-experienced repertoire with sequences from the naive immune repertoire showed that features associated with autoimmunity such as long CDR3 length and IGHV4-34 usage are reduced in the antigen-experienced repertoire. Moreover, IGA2 and IGG2 usage was increased in HD in higher age categories, while IGG1 usage was decreased. In addition, we studied clonal relationship in the different samples. Clonally related sequences were found with different subclasses. Interestingly, we found transcripts with the same CDR1-CDR3 sequence, but different subclasses. Together, these data suggest that a single antigen can provoke a B-cell response with BR of different subclasses and that, during the course of an immune response, some B cells change their isotype without acquiring additional SHM or can directly switch to different isotypes.
RESUMEN
Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.
Asunto(s)
Adalimumab/sangre , Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales/sangre , Inmunoglobulina G/sangre , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Unión Proteica/fisiología , Estándares de ReferenciaRESUMEN
Loss-of-function mutations in DOCK8 are linked to hyper-IgE syndrome. Patients typically present with recurrent sinopulmonary infections, severe cutaneous viral infections, food allergies and elevated serum IgE. Although patients may present with a spectrum of disease-related symptoms, molecular mechanisms explaining phenotypic variability in patients are poorly defined. Here we characterized a novel compound heterozygous mutation in DOCK8 in a patient diagnosed with primary combined immunodeficiency which was not typical of classical DOCK8 deficiency. In contrast to previously identified mutations in DOCK8 which result in complete loss of function, the newly identified single nucleotide insertion results in expression of a truncated DOCK8 protein. Functional evaluation of the truncated DOCK8 protein revealed its hypomorphic function. In addition we found somatic reversion of DOCK8 predominantly in T cells. The combination of somatic reversion and hypomorphic DOCK8 function explains the milder and atypical phenotype of the patient and further broadens the spectrum of DOCK8-associated disease.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/genética , Inmunoglobulina E/inmunología , Inmunoglobulina M/inmunología , Síndromes de Inmunodeficiencia/inmunología , Bronquiectasia/etiología , Bronquiectasia/inmunología , Niño , Femenino , Heterocigoto , Humanos , Inmunoglobulina G/inmunología , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/genética , Mutación , Recurrencia , Infecciones del Sistema Respiratorio/etiología , Infecciones del Sistema Respiratorio/inmunologíaRESUMEN
Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways.
Asunto(s)
Linfocitos B/metabolismo , Inmunodeficiencia Variable Común/genética , Genoma/genética , ARN Mensajero/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Adulto , Receptor del Factor Activador de Células B/genética , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Persona de Mediana Edad , Herencia Multifactorial , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Adulto JovenRESUMEN
The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales/inmunología , Adalimumab , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Mutación PuntualRESUMEN
Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.