RESUMEN
Targeting Ags to the CD180 receptor activates both B cells and dendritic cells (DCs) to become potent APCs. After inoculating mice with Ag conjugated to an anti-CD180 Ab, B cell receptors were rapidly internalized. Remarkably, all B cell subsets, including even transitional 1 B cells, were programed to process, present Ag, and stimulate Ag-specific CD4+ T cells. Within 24-48 hours, Ag-specific B cells were detectable at T-B borders in the spleen; there, they proliferated in a T cell-dependent manner and induced the maturation of T follicular helper (TFH) cells. Remarkably, immature B cells were sufficient for the maturation of TFH cells after CD180 targeting: TFH cells were induced in BAFFR-/- mice (with only transitional 1 B cells) and not in µMT mice (lacking all B cells) following CD180 targeting. Unlike CD180 targeting, CD40 targeting only induced DCs but not B cells to become APCs and thus failed to efficiently induce TFH cell maturation, resulting in slower and lower-affinity IgG Ab responses. CD180 targeting induces a unique program in Ag-specific B cells and to our knowledge, is a novel strategy to induce Ag presentation in both DCs and B cells, especially immature B cells and thus has the potential to produce a broad range of Ab specificities. This study highlights the ability of immature B cells to present Ag to and induce the maturation of cognate TFH cells, providing insights toward vaccination of mature B cell-deficient individuals and implications in treating autoimmune disorders.
Asunto(s)
Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Antígenos CD/inmunología , Linfocitos B/inmunología , Antígenos CD40/inmunología , Animales , Células Presentadoras de Antígenos/citología , Antígenos CD/genética , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/inmunología , Linfocitos B/citología , Antígenos CD40/genética , Ratones , Ratones Noqueados , Ratas , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
The caspase (Casp) family of proteases regulate both lymphocyte apoptosis and activation. Here, we show that Casp6 regulates early B-cell development. One-week-old Casp6 knockout (Casp6 KO) mice have significantly more splenic B-cell subsets than wild-type (WT) mice. Adult Casp6 KO mice have normal levels of total splenic B cells but have increased numbers of B1a B cells and CD43+ "transitional" or splenic red pulp (RP) B cells. These results suggested that Casp6 may function to control B-cell numbers under nonhomeostatic conditions and during B-cell development. Consistent with this model, reconstitution of B cells was dysregulated in Casp6 KO mice after sublethal irradiation. Furthermore, bone marrow pro-B, pre-B and immature B-cell numbers were significantly higher in 1-week-old Casp6 KO mice than in 1-week-old WT mice. Casp6 KO pro-B cells proliferated more in response to IL-7 than WT pro-B cells, suggesting that Casp6 regulates early B-cell responses to IL-7. Indeed, adult and aged Casp6 KO mice had elevated numbers of IL-7αR+ Sca1+ precursors of common lymphoid progenitors, suggesting Casp6 may help regulate progenitors of B cells and early B-lineage cells. Casp6 regulates B-cell programs both during early development and after antigen stimulation in the periphery.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Caspasa 6/metabolismo , Inmunomodulación , Animales , Animales Recién Nacidos , Caspasa 6/genética , Interleucina-7/metabolismo , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Linfopoyesis , Masculino , Ratones , Ratones Noqueados , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
Caspase (Casp) family proteases regulate not only lymphocyte apoptosis but also lymphocyte activation and development. In this study, we show that Casp6 regulates B cell activation and differentiation into plasma cells by modifying cell cycle entry. B cells from Casp6 knockout (Casp6 KO) mice examined ex vivo have more cells in G(1) than wild-type B cells, and mitogen-induced G(1) entry of Casp6 KO B cells is much faster than that of wild-type B cells. Even so, S phase entry and proliferation are not increased in Casp6 KO B cells. Rather than proliferating, activated Casp6 KO B cells preferentially differentiate into syndecan-1(+) plasma cells and produce Abs. In Casp6 KO mice compared with WT mice, serum levels of IgG1, IgG2a, and IgG2b are increased and Ag-specific Ab responses are also enhanced along with increased percentages of syndecan-1(+) plasma cells. Casp6 may regulate both B cell activation and differentiation by modifying requirements for G(0) B cells to enter G(1).
Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Caspasa 6/metabolismo , Activación de Linfocitos/inmunología , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Animales , Linfocitos B/metabolismo , Western Blotting , Caspasa 6/inmunología , Ciclo Celular/inmunología , Diferenciación Celular/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Ratones , Ratones Noqueados , Células Plasmáticas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Abnormal B-cell activation is implicated in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). The B-cell surface molecule CD22, which regulates activation through the B-cell receptor (BCR), is a potential target for inhibiting pathogenic B cells; however, the regulatory functions of CD22 remain poorly understood. In this study, we determined how targeting of CD22 with epratuzumab (Emab), a humanized anti-CD22 IgG1 monoclonal antibody, affects the activation of human B-cell subsets in response to Toll-like receptor 7 (TLR7) and BCR engagement. METHODS: B-cell subsets were isolated from human tonsils and stimulated with F(ab')2 anti-human IgM and/or the TLR7 agonist R848 in the presence of Emab or a human IgG1 isotype control. Changes in mRNA levels of genes associated with B-cell activation and differentiation were analyzed by quantitative PCR. Cytokine production was measured by ELISA. Cell proliferation, survival, and differentiation were assessed by flow cytometry. RESULTS: Pretreatment of phenotypically naïve CD19+CD10-CD27- cells with Emab led to a significant increase in IL-10 expression, and in some but not all patient samples to a reduction of IL-6 production in response to TLR7 stimulation alone or in combination with anti-IgM. Emab selectively inhibited the expression of PRDM1, the gene encoding B-lymphocyte-induced maturation protein 1 (Blimp-1) in activated CD10-CD27- B cells. CD10-CD27-IgD- cells were highly responsive to stimulation through TLR7 as evidenced by the appearance of blasting CD27hiCD38hi cells. Emab significantly inhibited the activation and differentiation of CD10-CD27-IgD- B cells into plasma cells. CONCLUSIONS: Emab can both regulate cytokine expression and block Blimp1-dependent B-cell differentiation, although the effects of Emab may depend on the stage of B-cell development or activation. In addition, Emab inhibits the activation of CD27-IgD- tonsillar cells, which correspond to so-called double-negative memory B cells, known to be increased in SLE patients with more active disease. These data may be relevant to the therapeutic effect of Emab in vivo via modulation of the production of pro-inflammatory and anti-inflammatory cytokines by B cells. Because Blimp-1 is required by B cells to mature into antibody-producing cells, inhibition of Blimp1 may reduce autoantibody production.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Linfocitos B/metabolismo , Citocinas/biosíntesis , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Receptor Toll-Like 7/metabolismo , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/metabolismo , Linfocitos B/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Sistemas de Liberación de Medicamentos/métodos , Humanos , Factor 1 de Unión al Dominio 1 de Regulación Positiva/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Receptor Toll-Like 7/antagonistas & inhibidoresRESUMEN
Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G(0)) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad specificity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G(1). In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G(0) B cells. These findings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle.