RESUMEN
Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Centro Germinal/inmunología , Interleucina-17/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Quimiotaxis de Leucocito/inmunología , Interleucina-17/antagonistas & inhibidores , Ratones , Ratones Mutantes , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismoRESUMEN
VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 µHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Células Precursoras de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Antígenos CD19/metabolismo , Secuencia de Bases , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Activación Enzimática , Reordenamiento Génico , Humanos , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Tonsila Palatina/citología , Células Precursoras de Linfocitos B/citología , Proteínas Tirosina Quinasas/metabolismo , Quinasa Syk , Familia-src Quinasas/metabolismoRESUMEN
CD19-deficient mice were used as a model to study follicular dendritic cell (FDC) activation because these mice have normal numbers of FDC-containing primary follicles, but lack the ability to activate FDCs or form GCs. It was hypothesized that CD19 expression is necessary for B-cell activation and upregulation of membrane lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM-1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19(+) wild-type B cells into CD19-deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19(+) donor B cells lacking mLT were unable to induce VCAM-1 expression on FDCs, furthermore FcγRII/III upregulation was impaired in FDCs stimulated with mLT-deficient B cells. VCAM-1 expression on FDCs, but not FcγRII/III, was rescued when CD19-deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19(+) , mLT-deficient B cells, suggesting that FDC activation requires the CD19-dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Dendríticas Foliculares/inmunología , Células Dendríticas Foliculares/metabolismo , Centro Germinal/inmunología , Centro Germinal/metabolismo , Heterotrímero de Linfotoxina alfa1 y beta2/biosíntesis , Animales , Antígenos CD19/biosíntesis , Antígenos CD19/genética , Antígenos CD19/inmunología , Activación de Linfocitos , Heterotrímero de Linfotoxina alfa1 y beta2/genética , Heterotrímero de Linfotoxina alfa1 y beta2/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de IgG/biosíntesis , Receptores de IgG/genética , Receptores de IgG/inmunología , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
The marginal zone (MZ) of the mouse spleen contains macrophages that express receptors that trap pathogens, including the scavenger receptor macrophage receptor with a collagenous structure and the C-type lectin specific intracellular adhesion molecule-grabbing nonintegrin receptor 1 (SIGN-R1). We previously reported that expression of SIGN-R1 was decreased in CD19-deficient mice. In this study, we demonstrate that SIGN-R1 is expressed on a subset of macrophage receptor with a collagenous structure (MARCO)(+) macrophages. This subset is diminished when MZ B cells are absent due to either genetic developmental defects or following transient migration of B cells out of the MZ. When B cells return to the MZ, there is a delay in recovery of SIGN-R1-expressing macrophages. During this period, capture of Ficoll, which for the macrophages requires SIGN-R1, remains defective not only by the macrophages, but also by the B cells. Thus, MZ B cells regulate expression of molecules on macrophages that are important for trapping Ag, which, in turn, is required for Ag capture by the B cells.
Asunto(s)
Antígenos/metabolismo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Macrófagos/citología , Macrófagos/inmunología , Bazo/citología , Bazo/inmunología , Animales , Antígenos/genética , Antígenos CD19/genética , Subgrupos de Linfocitos B/metabolismo , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Movimiento Celular/genética , Movimiento Celular/inmunología , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Bazo/metabolismoRESUMEN
BACKGROUND: Functional limitations and physical disabilities associated with aging and chronic disease are major concerns for human societies and expeditious development of function-promoting therapies is a public health priority. METHODS: Expert panel discussion. RESULTS: The remarkable success of Operation Warp Speed for the rapid development of COVID-19 vaccines, COVID-19 therapeutics, and of oncology drug development programs over the past decade have taught us that complex public health problems such as the development of function-promoting therapies will require collaboration among many stakeholders, including academic investigators, the National Institutes of Health, professional societies, patients and patient advocacy organizations, the pharmaceutical and biotechnology industry, and the U.S. Food and Drug Administration. CONCLUSIONS: There was agreement that the success of well designed, adequately powered clinical trials will require careful definitions of indication/s, study population, and patient-important endpoints that can be reliably measured using validated instruments, commensurate resource allocation, and versatile organizational structures such as those used in Operation Warp Speed.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Estados Unidos , Humanos , National Institutes of Health (U.S.) , Desarrollo de MedicamentosRESUMEN
Death Receptor 5 (DR5) induces apoptosis in various types of cells and is a potential therapeutic target. We have investigated whether targeting DR5 could be used to eliminate pathogenic B lymphocytes from systemic lupus erythematosus (SLE) patients. We examined DR5 expression and function on B lymphocytes from healthy controls subjects, SLE patients, and human tonsil. DR5 was expressed similarly on all B cell subpopulations, including resting and activated B cells. Expression of DR5 was equivalent on B cells from SLE patients and healthy subjects. Additionally, DR5 expression was unchanged after B lymphocyte stimulation. However, B cells were resistant to DR5-induced apoptosis, including after in vitro activation. No changes in subsets of B cells were observed in subjects of a trial of CS-1008, an agonist anti-DR5. While DR5 shows promise as a way to selectively eliminate tumor cells and activated synoviocytes, these data suggest DR5 alone cannot be used as a target to remove pathogenic SLE B cells.
Asunto(s)
Apoptosis/fisiología , Linfocitos B/fisiología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Adolescente , Adulto , Anticuerpos Monoclonales , Regulación de la Expresión Génica/fisiología , Humanos , Lupus Eritematoso Sistémico , Masculino , Persona de Mediana Edad , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Adulto JovenRESUMEN
Marginal zone (MZ) B cells are absent in CD19(-/-) mice. Possible causes include an intrinsic defect in B cells and/or a secondary defect in the extrinsic MZ microenvironment as a result of changes in B cell differentiation in mice lacking CD19. Cells in the MZ also include MZ macrophages (MZM) and MZ dendritic cells (DC). Although CD19 is only expressed on B cells, SIGN-R1(+) MZM are absent and CD11c(+) MZ DC distribution is abnormal in CD19(-/-) mice. Adoptively transferred B cells from normal mice are able to reconstitute MZ B cells in CD19(-/-) mice. In contrast, CD19(-/-) B cells could not enter the MZ of the normal mice. Furthermore, MZM distribution and MZ DC distribution are restored following MZ B cell reconstitution in CD19(-/-) mice. Thus, MZ B cells are required for MZM differentiation and MZ DC localization, but the deficiency of MZ B cells in CD19(-/-) mice is caused by a defect of intrinsic B cell signaling.
Asunto(s)
Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Bazo/citología , Bazo/inmunología , Animales , Antígenos CD19/genética , Linfocitos B/citología , Linfocitos B/inmunología , Diferenciación Celular/inmunología , Ratones , Ratones Noqueados , Transducción de Señal/inmunología , Bazo/metabolismoAsunto(s)
Antígenos CD19/historia , Antígenos de Diferenciación de Linfocitos B/historia , Linfocitos B/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD19/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Linfocitos B/citología , Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Replicación del ADN , Historia del Siglo XXRESUMEN
B cells play a key role in regulating the immune system by producing antibodies, acting as antigen-presenting cells, providing support to other mononuclear cells, and contributing directly to Inflammatory pathways. Accumulating evidence points to disruption of these tightly regulated processes in the pathogenesis of autoimmune disorders. Although the exact mechanisms involved remain to be elucidated, a fundamental feature of many autoimmune disorders is a loss of B-cell tolerance and the inappropriate production of autoantibodies. Dysfunctional immune responses resulting from genetic mutations that cause intrinsic B-cell abnormalities and induction of autoimmunity in the T-cell compartment by B cells that have broken tolerance may also contribute to these disorders. These findings provide the rationale for B-cell depletion as a potential therapeutic strategy in autoimmune disorders and other disease states characterized by inappropriate immune responses. Preliminary results with the CD20-targeted monoclonal antibody rituximab indicate that rituximab can improve symptoms in a number of autoimmune and neurologic disorders (including rheumatoid arthritis, systemic lupus erythematosus, and paraneoplastic neurologic syndromes). Additional studies are warranted to further characterize the role of B cells in autoimmune diseases and the therapeutic utility of B-cell depletion.
Asunto(s)
Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/terapia , Linfocitos B/fisiología , Estado de Salud , Inmunidad/fisiología , Enfermedades Autoinmunes/fisiopatología , Humanos , Factores Inmunológicos/uso terapéuticoRESUMEN
Early studies, largely based on in vitro models, revealed potential functional roles for the components of the CD19/21/81 complex in B cell proliferation and antibody production. These studies also identified signal transduction pathways linked to these receptors. Over the last decade, studies on knockout mice defined the biologic functions of CD19, CD21 and CD81. This review focuses on current attempts to use these receptors as tools to understand how the immune system regulates responsiveness and tolerance, while correlating specific biochemical pathways with biologic function.
Asunto(s)
Antígenos CD19/fisiología , Linfocitos B/fisiología , Receptores de Complemento 3d/fisiología , Transducción de Señal/fisiología , Animales , Formación de Anticuerpos , Presentación de Antígeno , Calcio/metabolismo , Centro Germinal/fisiología , Humanos , Activación de Linfocitos , Microdominios de Membrana/fisiología , FosforilaciónRESUMEN
Knockout studies have established an important role for the B lymphocyte surface protein CD19 in physiology. Previous studies by us and others have examined how CD19 might function at the biochemical level, with focus on the cytoplasmic tyrosines. We have mapped multiple different molecular associations with these tyrosines. However, the major question in CD19 signaling is, how do these tyrosines relate to what happens in vivo? To address this issue, we created mice expressing only mutant forms of CD19. Our initial studies have found that most CD19 function in vivo is dependent on two of these cytoplasmic tyrosines, Y482 and Y513. However, the signaling defects in cells expressing this mutation are subtle. They demonstrate that how CD19 signals depends on how it is ligated, and that CD19 signals by more than one mechanism. These observations in primary cells give insights into how CD19 functions at the molecular level and likely explain some of the differences in CD19 function that have been reported in vivo.
Asunto(s)
Antígenos CD19/metabolismo , Linfocitos B/inmunología , Animales , Formación de Anticuerpos , Antígenos CD19/química , Antígenos CD19/genética , Linfocitos B/citología , Diferenciación Celular , Humanos , Ratones , Ratones Noqueados , Modelos Inmunológicos , Mutación , Transducción de Señal , Tirosina/químicaRESUMEN
The germinal center is a critical locus in the production of protective immunity, but its function is poorly understood. Studies of mutant forms of CD19 revealed differences in signaling in different compartments inside the germinal center, and structural findings indicate a selective role in the interaction with follicular dendritic cells in the GC. Loss of these signals leads to surprising changes in germinal center B cells that challenge previous models of GC function.
Asunto(s)
Antígenos CD19/inmunología , Subgrupos de Linfocitos B/inmunología , Células Dendríticas Foliculares/citología , Células Dendríticas Foliculares/inmunología , Centro Germinal/inmunología , Comunicación Paracrina , Animales , Afinidad de Anticuerpos , Antígenos CD19/genética , Subgrupos de Linfocitos B/citología , Diferenciación Celular , Regulación de la Expresión Génica , Centro Germinal/citología , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina D , Inmunoglobulina M , Ratones , Ratones Noqueados , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Transducción de Señal/inmunología , TransgenesRESUMEN
Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.
Asunto(s)
Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/etiología , Linfocitos B/enzimología , Linfocitos B/inmunología , Citidina Desaminasa/fisiología , Abatacept , Adenoviridae/genética , Animales , Antígeno B7-2/análisis , Linfocitos T CD4-Positivos/fisiología , Citidina Desaminasa/antagonistas & inhibidores , Femenino , Inmunoconjugados/genética , Inmunoconjugados/uso terapéutico , Cambio de Clase de Inmunoglobulina/genética , Ratones , Ratones Endogámicos C57BL , Receptores de Complemento 3d/análisis , Receptores de IgE/análisis , Hipermutación Somática de InmunoglobulinaRESUMEN
Mice with mutations in CD19 Y482/Y513 form germinal centers (GC) but fail to produce high-affinity antibodies. In these mice, GC B cell differentiation, proliferation, and class switching occur but are defective. Altered CD19 signaling results in retention of early GC B cells and reduced proliferation in the follicular dendritic cell (FDC) zone of GC, and causes failure to select for high-affinity mutations. In normal mice, the earliest detectable aggregates of GC B cells are in contact with FDC and IgM+ cells are only found in the FDC zone, further evidence that the FDC zone is the site of initial GC B cell proliferation, differentiation, and class switching. Proliferation in the non-FDC zone and somatic mutation are not dependent on CD19, indicating separate signaling requirements for the two GC compartments, but these CD19-independent GC functions are not sufficient to generate high-affinity antibodies and B cell memory.
Asunto(s)
Antígenos CD19/inmunología , Linfocitos B/citología , Diferenciación Celular/inmunología , Proliferación Celular , Centro Germinal/inmunología , Animales , Antígenos CD19/metabolismo , Linfocitos B/inmunología , Células Dendríticas/inmunología , Citometría de Flujo , Centro Germinal/citología , Inmunohistoquímica , Activación de Linfocitos/inmunología , Ratones , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Hipermutación Somática de Inmunoglobulina/inmunología , Bazo/citología , Bazo/inmunologíaRESUMEN
OBJECTIVE: To determine whether receptors for B lymphocyte stimulator (BLyS) are altered on B cells of patients with systemic lupus erythematosus (SLE). METHODS: Total available receptors for BLyS were measured by analysis of binding of recombinant soluble BLyS to peripheral blood B cells in 36 SLE patients, 29 healthy controls, and 10 disease controls. Antibodies to the receptors BAFF-R, BCMA, and TACI were used to define expression of the individual BLyS receptors on subsets of B cells in blood, spleen, and tonsils. Two different antibodies to BAFF-R, which were differentially sensitive to BAFF-R occupancy, were used to compare BAFF-R on B cells in an additional 20 healthy subjects and 25 SLE patients. Assays of B cell survival after stimulation in vitro were used to determine the sensitivity of B cells to exogenous BLyS. RESULTS: Total available receptors for BLyS were decreased in patients with SLE, independent of changes of subsets in the blood in these patients. The decrease correlated with changes in disease activity. Although total surface BAFF-R was not significantly different between healthy controls and SLE patients, BAFF-R was occupied in SLE patients. B cells from these patients were less responsive to exogenous BLyS. CONCLUSION: BAFF-R is consistently occupied on blood B cells in SLE. Occupancy of BAFF-R on blood B cells is likely to contribute to disease mechanisms in SLE and could serve as a biomarker of disease activity. Targeting BLyS as a therapeutic strategy will require overcoming the persistent binding of BLyS to BAFF-R.
Asunto(s)
Linfocitos B/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Adulto , Anticuerpos Monoclonales , Receptor del Factor Activador de Células B , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The apparent efficacy of B-cell depletion in autoimmune diseases has increased interest in targeting B cells. One goal of next generation therapies is to develop treatments that block B-cell activation and preserve resting nonautoimmune cells that maintain B cell memory. To do so, one needs to understand how B cells are activated and what receptors and intracellular signaling pathways regulate this process. This paper will summarize B-cell activation pathways and illustrate how these are being targeted in the development of new treatments for rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Activación de Linfocitos/efectos de los fármacos , Femenino , Humanos , Inmunidad Celular , Memoria Inmunológica , Inmunosupresores/uso terapéutico , Cooperación Linfocítica , Masculino , Medición de Riesgo , Sensibilidad y Especificidad , Transducción de Señal/fisiologíaRESUMEN
The development of biologic agents has provided rheumatologists with a variety of new and effective treatment options. The success of early biologics, especially etanercept and infliximab for the treatment of rheumatoid arthritis, has spurred research into novel targets for the management of systemic inflammatory and autoimmune diseases. In addition, existing biologics approved for use in other diseases, such as rituximab, are now under study for the treatment of new indications. This article reviews ongoing research on the treatment of rheumatic diseases with new and existing biologic agents.
Asunto(s)
Productos Biológicos/administración & dosificación , Terapia Biológica/tendencias , Enfermedades Reumáticas/tratamiento farmacológico , Adalimumab , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Productos Biológicos/efectos adversos , Terapia Biológica/normas , Ensayos Clínicos como Asunto , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Quimioterapia Combinada , Etanercept , Femenino , Predicción , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/efectos adversos , Infliximab , Masculino , Pronóstico , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Enfermedades Reumáticas/diagnóstico , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
CD19 is required for the development of B1 and marginal zone B cells, for Ab responses, and for B cell memory. CD19 immunoprecipitates contain a complex of cytoplasmic proteins, including Lyn, Vav, phospholipase Cgamma2 (PLCgamma2), Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Which of these bind directly to CD19 and the strengths of the interactions are unknown. These issues are important in understanding the signaling functions of CD19, which are crucial for normal B cell physiology. Using purified, recombinant proteins, we now show that each of these signaling proteins contains at least one Src homology 2 (SH2) domain that interacts directly with the phosphorylated CD19 cytoplasmic domain. The affinities of binding of the SH2 domains of Vav, p85, and Grb2 to CD19 are each in the nanomolar range by surface plasmon resonance (Biacore) analysis. Binding of Lyn and PLCgamma2 do not fit 1:1 modeling. However, analyses of binding data (Lyn) and competition experiments (PLCgamma2) suggest that these bind with comparable affinity. Competition experiments demonstrate that SH2 domains whose binding is dependent on the same CD19 tyrosine(s) compete for binding, but these SH2 domains do not impede binding of different SH2 domains to other CD19 tyrosines. We conclude that binding to the CD19 cytoplasmic domain is multimeric, high affinity, and competitive. The high affinity of the interactions also suggests that tyrosines that were nonessential in vivo are nevertheless functional. A preliminary structural model suggests that CD19 forms a signaling complex containing multiple cytoplasmic proteins in close proximity to each other and to the plasma membrane.
Asunto(s)
Antígenos CD19/metabolismo , Citoplasma/química , Proteínas/metabolismo , Transducción de Señal , Antígenos CD19/química , Unión Competitiva , Línea Celular Tumoral , Dimerización , Humanos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión , Resonancia por Plasmón de Superficie , Tirosina , Dominios Homologos srcRESUMEN
OBJECTIVE: The low-affinity receptor Fcgamma receptor IIb (FcgammaRIIb), with an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic domain, down-regulates humoral immune responses and modulates the risk of autoimmunity in animal models. The transmembrane domain of FcgammaRIIb may also contribute to receptor signaling. Therefore, we investigated the biologic significance of single-nucleotide polymorphisms (SNPs) throughout the coding region. METHODS: Discovery of SNPs in FCGR2B was performed by direct cycle sequencing of complementary DNA samples derived by reverse transcriptase-polymerase chain reaction. To assess the biologic significance of the nonsynonymous transmembrane SNP, we studied 3 functions influenced by the FcgammaRIIb transmembrane domain: tyrosine dephosphorylation of CD19, inhibition of B cell receptor (BCR)-induced calcium response, and modulation of BCR- or anti-Fas-induced apoptosis. RESULTS: The nonsynonymous C-to-T transition in the first cytoplasmic exon, originally reported in the Raji cell line, was not found in either the African-American or the Caucasian population, but a nonsynonymous T-to-C transition at nucleotide 775 in exon 4 of FCGR2B, which changes isoleucine to threonine at residue 187 in the transmembrane domain, was significantly more common in African Americans. Using the FcgammaRIIb-negative mouse B cell line IIA1.6, we expressed both allelic forms as both full-length and truncated cytoplasmic domain constructs. The FCGR2B-187T allele mediated a higher level of CD19 dephosphorylation (P = 0.029) and a greater degree of inhibition of the calcium response (P = 0.003) when co-engaged with BCR than did FCGR2B-187I, independent of the presence of the ITIM. In contrast, FcgammaRIIb modulation of BCR-induced and anti-Fas antibody-induced cell death rates were similar in IIA1.6 cells expressing either the 187I or the 187T allelic form. CONCLUSION: The differential activity of FCGR2B alleles suggests a novel mechanism of FcgammaRIIb regulation that may influence the risk of autoimmune disease.