RESUMEN
Passively administered anti-tumor monoclonal antibodies (mAbs) rapidly kill tumor targets via FcγR-mediated cytotoxicity (ADCC), a short-term process. However, anti-tumor mAb treatment can also induce a vaccinal effect, in which mAb-mediated tumor death induces a long-term anti-tumor cellular immune response. To determine how such responses are generated, we utilized a murine model of an anti-tumor vaccinal effect against a model neoantigen. We demonstrate that FcγR expression by CD11c(+) antigen-presenting cells is required to generate anti-tumor T cell responses upon ADCC-mediated tumor clearance. Using FcγR-humanized mice, we demonstrate that anti-tumor human (h)IgG1 must engage hFcγRIIIA on macrophages to mediate ADCC, but also engage hFcγRIIA, the sole hFcγR expressed by human dendritic cells (DCs), to generate a potent vaccinal effect. Thus, while next-generation anti-tumor antibodies with enhanced binding to only hFcγRIIIA are now in clinical use, ideal anti-tumor antibodies must be optimized for both cytotoxic effects as well as hFcγRIIA engagement on DCs to stimulate long-term anti-tumor cellular immunity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Neoplasias/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos , Presentación de Antígeno , Antígeno CD11c/inmunología , Vacunas contra el Cáncer/inmunología , Modelos Animales de Enfermedad , Humanos , Macrófagos/inmunología , RatonesRESUMEN
Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibody's Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.
Asunto(s)
Inmunidad Adaptativa , Inmunidad Innata , Fragmentos Fc de Inmunoglobulinas/inmunología , Receptores de IgG/inmunología , Secuencia de Aminoácidos , Anticuerpos/inmunología , Presentación de Antígeno/inmunología , Enfermedades Autoinmunes/inmunología , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/clasificación , Inmunoglobulina G/inmunología , Neoplasias/inmunología , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de IgG/química , Receptores de IgG/clasificación , VacunaciónRESUMEN
Ebola virus (EBOV) continues to pose significant threats to global public health, requiring ongoing development of multiple strategies for disease control. To date, numerous monoclonal antibodies (mAbs) that target the EBOV glycoprotein (GP) have demonstrated potent protective activity in animal disease models and are thus promising candidates for the control of EBOV. However, recent work in a variety of virus diseases has highlighted the importance of coupling Fab neutralization with Fc effector activity for effective antibody-mediated protection. To determine the contribution of Fc effector activity to the protective function of mAbs to EBOV GP, we selected anti-GP mAbs targeting representative, protective epitopes and characterized their Fc receptor (FcγR) dependence in vivo in FcγR humanized mouse challenge models of EBOV disease. In contrast to previous studies, we find that anti-GP mAbs exhibited differential requirements for FcγR engagement in mediating their protective activity independent of their distance from the viral membrane. Anti-GP mAbs targeting membrane proximal epitopes or the GP mucin domain do not rely on Fc-FcγR interactions to confer activity, whereas antibodies against the GP chalice bowl and the fusion loop require FcγR engagement for optimal in vivo antiviral activity. This complexity of antibody-mediated protection from EBOV disease highlights the structural constraints of FcγR binding for specific viral epitopes and has important implications for the development of mAb-based immunotherapeutics with optimal potency and efficacy.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/metabolismo , Interacciones Huésped-Patógeno/inmunología , Receptores de IgG/metabolismo , Animales , Afinidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Fiebre Hemorrágica Ebola/virología , Humanos , Inmunoglobulina G/inmunología , Ratones , Mucinas/antagonistas & inhibidores , Mucinas/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de IgG/químicaRESUMEN
The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, posttranslational site-selective modifications of glycans in antibodies and other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial α1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the FcγIIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/química , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Citotoxicidad Celular Dependiente de Anticuerpos/fisiología , Cetuximab/metabolismo , Glicósido Hidrolasas/metabolismo , Glicosilación , Humanos , Polisacáridos/metabolismo , Especificidad por SustratoRESUMEN
The goal of cancer immunotherapy is to stimulate the host immune system to attack malignant cells. Antibody-dependent cellular cytotoxicity (ADCC) is a pivotal mechanism of antitumor action of clinically employed antitumor antibodies. IL-15 administered to patients with metastatic malignancy by continuous i.v. infusion at 2 µg/kg/d for 10 days was associated with a 38-fold increase in the number and activation status of circulating natural killer (NK) cells and activation of macrophages which together are ADCC effectors. We investigated combination therapy of IL-15 with rituximab in a syngeneic mouse model of lymphoma transfected with human CD20 and with alemtuzumab (Campath-1H) in a xenograft model of human adult T cell leukemia (ATL). IL-15 greatly enhanced the therapeutic efficacy of both rituximab and alemtuzumab in tumor models. The additivity/synergy was shown to be associated with augmented ADCC. Both NK cells and macrophages were critical elements in the chain of interacting effectors involved in optimal therapeutic responses mediated by rituximab with IL-15. We provide evidence supporting the hypothesis that NK cells interact with macrophages to augment the NK-cell activation and expression of FcγRIV and the capacity of these cells to become effectors of ADCC. The present study supports clinical trials of IL-15 combined with tumor-directed monoclonal antibodies.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Interleucina-15/administración & dosificación , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Alemtuzumab/administración & dosificación , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Interleucina-15/inmunología , Células Asesinas Naturales/efectos de los fármacos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Rituximab/administración & dosificaciónRESUMEN
IgG antibodies contain a conserved N-glycosylation site on the Fc domain to which a complex, biantennary glycan is attached. The fine structures of this glycan modulate antibody effector functions by affecting the binding affinity of the Fc to diverse Fc receptor family members. For example, core fucosylation significantly decreases antibody-dependent cellular cytotoxicity (ADCC), whereas terminal α2,6-sialylation plays a critical role in the anti-inflammatory activity of human i.v. immunoglobulin therapy. The effect of specific combinations of sugars in the glycan on ADCC remains to be further addressed, however. Therefore, we synthesized structurally well-defined homogeneous glycoforms of antibodies with different combinations of fucosylation and sialylation and performed side-by-side in vitro FcγR-binding analyses, cell-based ADCC assays, and in vivo IgG-mediated cellular depletion studies. We found that core fucosylation exerted a significant adverse effect on FcγRIIIA binding, in vitro ADCC, and in vivo IgG-mediated cellular depletion, regardless of sialylation status. In contrast, the effect of sialylation on ADCC was dependent on the status of core fucosylation. Sialylation in the context of core fucosylation significantly decreased ADCC in a cell-based assay and suppressed antibody-mediated cell killing in vivo. In contrast, in the absence of fucosylation, sialylation did not adversely impact ADCC.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Polisacáridos/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Glicosilación , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Ingeniería de Proteínas , Receptores Fc/inmunologíaRESUMEN
B cells regulate immune responses by producing antigen-specific antibodies. However, specific B-cell subsets can also negatively regulate T-cell immune responses, and have been termed regulatory B cells. Human and mouse regulatory B cells (B10 cells) with the ability to express the inhibitory cytokine interleukin-10 (IL-10) have been identified. Although rare, B10 cells are potent negative regulators of antigen-specific inflammation and T-cell-dependent autoimmune diseases in mice. How B10-cell IL-10 production and regulation of antigen-specific immune responses are controlled in vivo without inducing systemic immunosuppression is unknown. Using a mouse model for multiple sclerosis, here we show that B10-cell maturation into functional IL-10-secreting effector cells that inhibit in vivo autoimmune disease requires IL-21 and CD40-dependent cognate interactions with T cells. Moreover, the ex vivo provision of CD40 and IL-21 receptor signals can drive B10-cell development and expansion by four-million-fold, and generate B10 effector cells producing IL-10 that markedly inhibit disease symptoms when transferred into mice with established autoimmune disease. The ex vivo expansion and reinfusion of autologous B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos B Reguladores/inmunología , Interleucinas/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linfocitos B Reguladores/citología , Linfocitos B Reguladores/metabolismo , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Antígenos CD5/metabolismo , División Celular , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Receptores de Interleucina-21/inmunología , Receptores de Interleucina-21/metabolismoRESUMEN
B cells provide humoral protection against pathogens and promote cellular immunity through diverse nonclassical effector functions. To assess B cell function in promoting T cell homeostasis, mature B cells were either acutely or chronically depleted in mice using CD20 mAb. Acute B cell depletion in either 2- or 4-mo-old mice significantly reduced spleen and lymph node CD4(+) and CD8(+) T cell numbers, including naive, activated, and Foxp3(+)CD25(+)CD4(+) regulatory T cell subsets. The numbers of IFN-γ- and TNF-α-producing T cells were also significantly reduced. Chronic B cell depletion for 6 mo in aged naive mice resulted in a 40-70% reduction in activated CD4(+) and CD8(+) T cell numbers and 20-50% reductions in IFN-γ-producing T cells. Therefore, B cells were necessary for maintaining naive CD4(+) and CD8(+) T cell homeostasis for subsequent optimal T cell expansion in young and old mice. To determine the significance of this finding, a week of B cell depletion in 4-mo-old mice was followed by acute viral infection with lymphocytic choriomeningitis virus Armstrong. Despite their expansion, activated and cytokine-producing CD4(+) and CD8(+) T cell numbers were still significantly reduced 1 wk later. Moreover, viral peptide-specific CD4(+) and CD8(+) T cell numbers and effector cell development were significantly reduced in mice lacking B cells, whereas lymphocytic choriomeningitis virus titers were dramatically increased. Thus, T cell function is maintained in B cell-depleted mice, but B cells are required for optimal CD4(+) and CD8(+) T cell homeostasis, activation, and effector development in vivo, particularly during responses to acute viral infection.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Homeostasis/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Enfermedad Aguda , Animales , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Interacciones Huésped-Patógeno/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Depleción Linfocítica , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Passive immunotherapy represents a promising therapeutic intervention for a number of neoplastic, chronic inflammatory, and infectious diseases, with several monoclonal antibodies currently under development or already in use in the clinic. While Fab-antigen interactions play a crucial role in the activity of an antibody, it has become clear that Fc-mediated effector functions are involved during antibody-mediated activities in vivo. A complete understanding of the contributions of effector activities mediated by an antibody during its in vivo function is required for the development of antibodies with improved therapeutic efficacies. Animal models that are commonly used for the preclinical evaluation of antibodies include murine and non-human primate species, whose FcγRs present substantial structural, functional, and genetic variation compared with their human counterparts. Therefore, the use of such animal models provides limited information on the role of human IgG Fc-FcγR interactions during the in vivo activities of antibodies intended for human therapeutics. In this chapter, we describe the development and evaluation of an FcγR-humanized mouse model for the study of human FcγR function in vivo. In this model, endogenous mouse FcγR genes have been deleted and human FcγRs are expressed as transgenes that faithfully recapitulate the unique pattern of human FcγR expression. Evaluation of the in vivo activities of a number of cytotoxic or therapeutic antibodies using FcγR-humanized mice provided useful insights into human IgG Fc effector function. This mouse model has become a vital preclinical model for testing therapeutic human antibodies to treat malignancies, autoimmunity, inflammation, and infectious disease.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Receptores de IgG/fisiología , Animales , Humanos , Ratones , Modelos AnimalesRESUMEN
Pathogens use numerous methods to subvert host immune responses, including the modulation of host IL-10 production by diverse cell types. However, the B cell sources of IL-10 and their overall influence on innate and cellular immune responses have not been well characterized during infections. Using Listeria as a model pathogen, infection drove the acute expansion of a small subset of regulatory B cells (B10 cells) that potently suppress inflammation and autoimmunity through the production of IL-10. Unexpectedly, spleen bacteria loads were 92-97% lower in B10 cell-deficient CD19(-/-) mice, in mice depleted of mature B cells, and in mice treated with CD22 mAb to preferentially deplete B10 cells before infection. By contrast, the adoptive transfer of wild-type B10 cells reduced bacterial clearance by 38-fold in CD19(-/-) mice through IL-10-dependent pathways. B10 cell depletion using CD22 mAb significantly enhanced macrophage phagocytosis of Listeria and their production of IFN-γ, TNF-α, and NO ex vivo. Accelerated bacteria clearance following B10 cell depletion significantly reduced Ag-specific CD4(+) T cell proliferation and cytokine production, but did not alter CD8(+) T cell responses. B10 cell regulatory function during innate immune responses was nonetheless dependent on cognate interactions with CD4(+) T cells because B10 cells deficient in IL-10, MHC-II, or IL-21R expression did not influence Listeria clearance. Thus, Listeria manipulates immune responses through a strategy of immune evasion that involves the preferential expansion of endogenous B10 cells that regulate the magnitude and duration of both innate and cellular immune responses.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Evasión Inmune/inmunología , Interleucina-10/biosíntesis , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Traslado Adoptivo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Antígenos CD19/genética , Antígenos CD20/inmunología , Subgrupos de Linfocitos B/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Inmunidad Celular , Inmunidad Innata , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-10/genética , Interleucina-10/inmunología , Listeria monocytogenes/aislamiento & purificación , Listeriosis/microbiología , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico/biosíntesis , Óxido Nítrico/genética , Ovalbúmina/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Organismos Libres de Patógenos Específicos , Subgrupos de Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The in vivo biological activities of IgG antibodies result from their bifunctional nature, in which antigen recognition by the Fab is coupled to the effector and immunomodulatory diversity found in the Fc domain. This diversity, resulting from both amino acid and glycan heterogeneity, is translated into cellular responses through Fcγ receptors (FcγRs), a structurally and functionally diverse family of cell surface receptors found throughout the immune system. Although many of the overall features of this system are maintained throughout mammalian evolution, species diversity has precluded direct analysis of human antibodies in animal species, and, thus, detailed investigations into the unique features of the human IgG antibodies and their FcγRs have been limited. We now report the development of a mouse model in which all murine FcγRs have been deleted and human FcγRs, encoded as transgenes, have been inserted into the mouse genome resulting in recapitulation of the unique profile of human FcγR expression. These human FcγRs are shown to function to mediate the immunomodulatory, inflammatory, and cytotoxic activities of human IgG antibodies and Fc engineered variants and provide a platform for the detailed mechanistic analysis of therapeutic and pathogenic IgG antibodies.
Asunto(s)
Variación Genética/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Masculino , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismoRESUMEN
Dendritic cells (DCs) play an essential role in regulation of immune responses. In the periphery, Ag presentation by DCs is critical for adaptive responses; for this reason, DCs are often targets of adjuvants that enhance vaccine responses. Activated mature DCs enhance B cell activation and differentiation by providing cytokines like BAFF and a proliferation-inducing ligand. However, the role of immature DCs in B cell tolerance is not well studied. Recently, mouse immature bone marrow-derived DCs (iBMDCs) have been shown to suppress anti-IgM-induced B cell activation. In this study, we tested the ability of mouse DCs to modulate B cell functions during TLR activation. We found that iBMDCs potently suppressed proliferation and differentiation of various B cell subsets on TLR stimulation. However, iBMDCs did not affect CD40-mediated B cell activation. Optimal suppression of B cell activation by iBMDCs required cell contact via the CD22 receptor on B cells. The B cell suppression was a property of iBMDCs or DCs resident in the bone marrow (BM), but not mature BM-derived DCs or DCs resident in the spleen. Presence of iBMDCs also enhanced the Ag-induced apoptotic response of BM B cells, suggesting that the suppressive effects of iBMDCs may have a role in B cell tolerance.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Animales , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/inmunología , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Bazo/citología , Bazo/inmunología , Bazo/metabolismoRESUMEN
Regulatory B cells that are functionally defined by their capacity to express IL-10 (B10 cells) downregulate inflammation and autoimmunity. In studies using well-defined IL-10 reporter mice, this rare B10 cell subset was also found to maintain a capacity for plasma cell differentiation. During a transient period of il10 transcription, the blimp1 and irf4 transcription factors were induced in B10 cells, whereas pax5 and bcl6 were downregulated as a significant fraction of B10 cells completed the genetic and phenotypic program leading to Ab-secreting cell differentiation in vitro and in vivo. B10 cell-derived IgM reacted with both self- and foreign Ags, whereas B10 cells generated Ag-specific IgG in response to immunizations. Moreover, B10 cells represented a significant source of serum IgM and IgG during adoptive-transfer experiments and produced Ag-specific, polyreactive and autoreactive Ab specificities that were consistent with their expression of a diverse AgR repertoire. Thereby, B10 cells limit inflammation and immune responses by the transient production of IL-10, and may facilitate clearance of their eliciting Ags through an inherent capacity to quickly generate polyreactive and/or Ag-specific Abs during humoral immune responses.
Asunto(s)
Células Productoras de Anticuerpos/citología , Subgrupos de Linfocitos B/citología , Linfocitos B Reguladores/inmunología , Diferenciación Celular/inmunología , Interleucina-10/biosíntesis , Animales , Antígenos , Autoinmunidad , Subgrupos de Linfocitos B/inmunología , Linfocitos B Reguladores/citología , Regulación de la Expresión Génica , Inmunidad Humoral , Inmunoglobulina G , Inmunoglobulina M , Inflamación , RatonesRESUMEN
The presence of high endothelial venules (HEV) and tertiary lymphoid structures (TLS) in solid tumors is correlated with favorable prognosis and better responses to immune-checkpoint blockade (ICB) in many cancer types. Elucidation of the molecular mechanisms underlying intratumoral HEV and TLS formation and their contribution to anti-tumor responses may facilitate development of improved treatment strategies. Lymphotoxin beta receptor (LTßR) signaling is a critical regulator of lymph node organogenesis and can cooperate with antiangiogenic and ICB treatment to augment tumor-associated HEV formation. Here, we demonstrated that LTßR signaling modulates the tumor microenvironment via multiple mechanisms to promote anti-tumor T cell responses. Systemic activation of the LTßR pathway via agonistic antibody treatment induced tumor-specific HEV formation, upregulated the expression of TLS-related chemokines, and enhanced dendritic cell (DC) and T cell infiltration and activation in syngeneic tumor models. In vitro studies confirmed direct effects of LTßR agonism on DC activation and maturation and associated DC-mediated T cell activation. Single agent LTßR agonist treatment inhibited syngeneic tumor growth in a CD8+ T cell- and HEV-dependent manner, and the LTßR agonist enhanced anti-tumor effects of anti-PD-1 and CAR T cell therapies. An in vivo tumor screen for TLS-inducing cytokines revealed that the combination of LTßR agonism and lymphotoxin alpha (LTâº) expression promoted robust intratumoral TLS induction and enhanced tumor responses to anti-CTLA-4 treatment. Collectively, this study highlights crucial functions of LTßR signaling in modulating the tumor microenvironment and could inform future HEV/TLS-based strategies for cancer treatments.
RESUMEN
Regulatory B cells control inflammation and autoimmunity in mice, including the recently identified IL-10-competent B10 cell subset that represents 1% to 3% of spleen B cells. In this study, a comparable IL-10-competent B10 cell subset was characterized in human blood. B10 cells were functionally identified by their ability to express cytoplasmic IL-10 after 5 hours of ex vivo stimulation, whereas progenitor B10 (B10pro) cells required 48 hours of in vitro stimulation before they acquired the ability to express IL-10. B10 and B10pro cells represented 0.6% and approximately 5% of blood B cells, respectively. Ex vivo B10 and B10pro cells were predominantly found within the CD24(hi)CD27(+) B-cell subpopulation that was able to negatively regulate monocyte cytokine production through IL-10-dependent pathways during in vitro functional assays. Blood B10 cells were present in 91 patients with rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren syndrome, autoimmune vesiculobullous skin disease, or multiple sclerosis, and were expanded in some cases as occurs in mice with autoimmune disease. Mean B10 + B10pro-cell frequencies were also significantly higher in patients with autoimmune disease compared with healthy controls. The characterization of human B10 cells will facilitate their identification and the study of their regulatory activities during human disease.
Asunto(s)
Enfermedades Autoinmunes/sangre , Subgrupos de Linfocitos B/inmunología , Interleucina-10/inmunología , Células Precursoras de Linfocitos B/inmunología , Adolescente , Adulto , Anciano , Animales , Enfermedades Autoinmunes/inmunología , Subgrupos de Linfocitos B/citología , Separación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Interleucina-10/metabolismo , Ratones , Persona de Mediana Edad , Células Precursoras de Linfocitos B/citología , Adulto JovenRESUMEN
The relative contributions of B lymphocytes and plasma cells during allograft rejection remain unclear. Therefore, the effects of B cell depletion on acute cardiac rejection, chronic renal rejection, and skin graft rejection were compared using CD20 or CD19 mAbs. Both CD20 and CD19 mAbs effectively depleted mature B cells, and CD19 mAb treatment depleted plasmablasts and some plasma cells. B cell depletion did not affect acute cardiac allograft rejection, although CD19 mAb treatment prevented allograft-specific IgG production. Strikingly, CD19 mAb treatment significantly reduced renal allograft rejection and abrogated allograft-specific IgG development, whereas CD20 mAb treatment did not. By contrast, B cell depletion exacerbated skin allograft rejection and augmented the proliferation of adoptively transferred alloantigen-specific CD4(+) T cells, demonstrating that B cells can also negatively regulate allograft rejection. Thereby, B cells can either positively or negatively regulate allograft rejection depending on the nature of the allograft and the intensity of the rejection response. Moreover, CD19 mAb may represent a new approach for depleting both B cells and plasma cells to concomitantly impair T cell activation, inhibit the generation of new allograft-specific Abs, or reduce preexisting allograft-specific Ab levels in transplant patients.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Rechazo de Injerto/inmunología , Trasplante de Corazón/inmunología , Trasplante de Riñón/inmunología , Trasplante de Piel/inmunología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD19/inmunología , Antígenos CD20/inmunología , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/patología , Línea Celular Tumoral , Enfermedad Crónica , Relación Dosis-Respuesta Inmunológica , Rechazo de Injerto/patología , Trasplante de Corazón/patología , Humanos , Trasplante de Riñón/patología , Depleción Linfocítica/métodos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Trasplante de Piel/patologíaRESUMEN
B lymphocytes can both positively and negatively regulate cellular immune responses. Previous studies have demonstrated augmented T cell-mediated tumor immunity in genetically B cell-deficient mice, suggesting that therapeutic B cell depletion would enhance tumor immunity. To test this hypothesis and quantify B cell contributions to T cell-mediated anti-tumor immune responses, mature B cells were depleted from wild-type adult mice using CD20 mAb prior to syngeneic B16 melanoma tumor transfers. Remarkably, s.c. tumor volume and lung metastasis were increased 2-fold in B cell-depleted mice. Effector-memory and IFN-gamma-or TNF-alpha-secreting CD4(+) and CD8(+) T cell induction was significantly impaired in B cell-depleted mice with tumors. Tumor Ag-specific CD8(+) T cell proliferation was also impaired in tumor-bearing mice that lacked B cells. Thus, B cells were required for optimal T cell activation and cellular immunity in this in vivo nonlymphoid tumor model. Although B cells may not have direct effector roles in tumor immunity, impaired T cell activation, and enhanced tumor growth in the absence of B cells argue against previous proposals to augment tumor immunity through B cell depletion. Rather, targeting tumor Ags to B cells in addition to dendritic cells is likely to optimize tumor-directed vaccines and immunotherapies.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/inmunología , Traslado Adoptivo , Animales , Separación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.
Asunto(s)
Anticuerpos Biespecíficos , Mieloma Múltiple , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antígeno de Maduración de Linfocitos B , Humanos , Inmunoterapia Adoptiva , Ratones , Mieloma Múltiple/tratamiento farmacológico , Linfocitos TRESUMEN
T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.
Asunto(s)
Anticuerpos Biespecíficos , Complejo CD3 , Citocinas , Citocinas/metabolismo , Activación de Linfocitos , Distribución TisularRESUMEN
Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I and MHC-IIrestricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.