RESUMO
B cells and the antibodies they produce are critical in host defense against pathogens and contribute to various immune-mediated diseases. B cells responding to activating signals in vitro release extracellular vesicles (EV) that carry surface antibodies, yet B cell production of EVs that express antibodies and their function in vivo is incompletely understood. Using transgenic mice expressing the Cre recombinase in B cells switching to IgG1 to induce expression of fusion proteins between emerald green fluorescent protein (emGFP) and the EV tetraspanin CD63 as a model, we identify emGFP expression in B cells responding to foreign antigen in vivo and characterize the emGFP+ EVs they release. Our data suggests that emGFP+ germinal center B cells undergoing immunoglobulin class switching to express IgG and their progeny memory B cells and plasma cells, also emGFP+, are sources of circulating antigen-specific IgG+ EVs. Furthermore, using a mouse model of influenza virus infection, we find that IgG+ EVs specific for the influenza hemagglutinin antigen protect against virus infection. In addition, crossing the B cell Cre driver EV reporter mice onto the Nba2 lupus-prone strain revealed increased circulating emGFP+ EVs that expressed surface IgG against nuclear antigens linked to autoimmunity. These data identify EVs loaded with antibodies as a novel route for antibody secretion in B cells that contribute to adaptive immune responses, with important implications for different functions of IgG+ EVs in infection and autoimmunity.
Assuntos
Linfócitos B , Vesículas Extracelulares , Imunoglobulina G , Camundongos Transgênicos , Animais , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Camundongos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Orthomyxoviridae/imunologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Antígenos/imunologia , Switching de Imunoglobulina , Camundongos Endogâmicos C57BL , Centro Germinativo/imunologia , Centro Germinativo/metabolismoRESUMO
Introduction: Bone morphogenetic proteins (BMPs) are used as key therapeutic agents for the treatment of difficult fractures. While their effects on osteoprogenitors are known, little is known about their effects on the immune system. Methods: We used permutations of BMP-6 (B), vascular endothelial growth factor (V), and Hedgehog signaling pathway activator smoothened agonist (S), to treat a rat mandibular defect and investigated healing outcomes at week 8, in correlation with the cellular landscape of the immune cells in the fracture callus at week 2. Results: Maximum recruitment of immune cells to the fracture callus is known to occur at week 2. While the control, S, V, and VS groups remained as nonunions at week 8; all BMP-6 containing groups - B, BV, BS and BVS, showed near-complete to complete healing. This healing pattern was strongly associated with significantly higher ratios of CD4 T (CD45+CD3+CD4+) to putative CD8 T cells (CD45+CD3+CD4-), in groups treated with any permutation of BMP-6. Although, the numbers of putative M1 macrophages (CD45+CD3-CD11b/c+CD38high) were significantly lower in BMP-6 containing groups in comparison with S and VS groups, percentages of putative - Th1 cells or M1 macrophages (CD45+CD4+IFN-γ+) and putative - NK, NKT or cytotoxic CD8T cells (CD45+CD4-IFN-γ+) were similar in control and all treatment groups. Further interrogation revealed that the BMP-6 treatment promoted type 2 immune response by significantly increasing the numbers of CD45+CD3-CD11b/c+CD38low putative M2 macrophages, putative - Th2 cells or M2 macrophages (CD45+CD4+IL-4+) cells and putative - mast cells, eosinophils or basophils (CD45+CD4-IL-4+ cells). CD45- non-haematopoietic fractions of cells which encompass all known osteoprogenitor stem cells populations, were similar in control and treatment groups. Discussion: This study uncovers previously unidentified regulatory functions of BMP-6 and shows that BMP-6 enhances fracture healing by not only acting on osteoprogenitor stem cells but also by promoting type 2 immune response.
Assuntos
Proteína Morfogenética Óssea 6 , Fraturas Ósseas , Animais , Ratos , Consolidação da Fratura , Fraturas Ósseas/metabolismo , Proteínas Hedgehog , Imunidade , Interleucina-4 , Fator A de Crescimento do Endotélio VascularRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a pandemic of the respiratory disease coronavirus disease 2019 (COVID-19). Antibody testing is essential to identify persons exposed to the virus and potentially in predicting disease immunity. 183 COVID-19 patients (68 of whom required mechanical ventilation) and 41 controls were tested for plasma IgG, IgA and IgM against the SARS-CoV-2 S1, S2, receptor binding domain (RBD) and N proteins using the MILLIPLEX® SARS-CoV-2 Antigen Panel. Plasma cytokines were concurrently measured using the MILLIPLEX® MAP Human Cytokine/Chemokine/Growth Factor Panel A. As expected the 183 COVID-19 positive patients had high levels of IgG, IgA and IgM anti-SARS-CoV-2 antibodies against each of the viral proteins. Sensitivity of anti-S1 IgG increased from 60% to 93% one week after symptom onset. S1-IgG and S1-IgA had specificities of 98% compared to the 41 COVID-19 negative patients. The 68 ventilated COVID-19 positive patients had higher antibody levels than the 115 COVID-19 positive patients who were not ventilated. IgG antibody levels against S1 protein had the strongest positive correlation to days from symptom onset. There were no statistically significant differences in IgG, IgA and IgM antibodies against S1 based on age. We found that patients with the highest levels of anti-SARS-CoV-2 antibodies had the lowest viral load in the nasopharynx. Finally there was a correlation of high plasma IL-10 with low anti-SARS-CoV-2 antibodies. Anti-SARS-CoV-2 antibody levels, as measured by a novel antigen panel, increased within days after symptom onset, achieving > 90% sensitivity and specificity within one week, and were highest in patients who required mechanical ventilation. Antibody levels were inversely associated with viral load but did not differ as a function of age. The correlation of high IL-10 with low antibody response suggests a potentially suppressive role of this cytokine in the humoral immune response in COVID-19.
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Neutrophils are professional phagocytes that are important for innate host defenses against pathogens and resolution of inflammation. Traditionally, the phagocytic capacity of neutrophils was quantified by enumeration of cells containing either internalized or bound bacteria or other cargo from a series of microscopic images. Here we describe an imaging flow cytometry-based protocol and analysis method for quantifying the binding and uptake of Neisseria gonorrhoeae by primary adherent human neutrophils. Imaging flow cytometry combines the capacity for quantitative, high-throughput analysis of tens of thousands of cells per condition, with the imaging power of fluorescence microscopy. Here, all bacteria are labeled with Tag-it Violet™ and bound bacteria are differentially stained with a DyLight™ 650-conjugated antibody. Images are analyzed using spot count and other algorithms. Outputs include the percent of neutrophils associated with bacteria, the percent of neutrophils with internalized bacteria, and the percent of internalized bacteria. This basic protocol can be adapted to a variety of particle types and can be used for multiplex analysis in combination with staining for different neutrophil surface and intracellular markers.
Assuntos
Citometria de Fluxo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Biomarcadores , Humanos , Imunofenotipagem , Interleucina-8 , Microscopia de Fluorescência/métodos , Fagócitos/imunologia , Fagócitos/metabolismoRESUMO
Mass cytometry is a powerful tool for high-dimensional single cell characterization. Since the introduction of the first commercial CyTOF mass cytometer by DVS Sciences in 2009, mass cytometry technology has matured and become more widely utilized, with sequential platform upgrades designed to address specific limitations and to expand the capabilities of the platform. Fluidigm's third-generation Helios mass cytometer introduced a number of upgrades over the previous CyTOF2. One of these new features is a modified narrow bore sample injector that generates smaller ion clouds, which is expected to improve sensitivity and throughput. However, following rigorous testing, we find that the narrow-bore sample injector may have unintended negative consequences on data quality and result in lower median and higher coefficients of variation in many antibody-associated signal intensities. We describe an alternative Helios acquisition protocol using a wider bore injector, which largely mitigates these data quality issues. We directly compare these two protocols in a multisite study of 10 Helios instruments across 7 institutions and show that the modified protocol improves data quality and reduces interinstrument variability. These findings highlight and address an important source of technical variability in mass cytometry experiments that is of particular relevance in the setting of multicenter studies. © 2019 International Society for Advancement of Cytometry.
Assuntos
Citometria de Fluxo/métodos , Análise de Célula Única/instrumentação , Anticorpos , Citometria de Fluxo/instrumentação , Humanos , Imunofenotipagem/normas , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Reprodutibilidade dos Testes , Análise de Célula Única/métodosRESUMO
Quantifying the efficiency of particle uptake by host cells is important in the fields of infectious diseases, autoimmunity, cancer, developmental biology, and drug delivery. Here we present a protocol for high-throughput analysis of particle uptake by imaging flow cytometry, using the bacterium Neisseria gonorrhoeae attached to and internalized by neutrophils as an example. Cells are exposed to fluorescently labeled bacteria, fixed, and stained with a bacteria-specific antibody of a different fluorophore. Thus, in the absence of a permeabilizing agent, extracellular bacteria are double-labeled with two fluorophores while intracellular bacteria remain single-labeled. A spot count algorithm is used to determine the number of single- and double-labeled bacteria in individual cells, to calculate the percent of cells associated with bacteria, percent of cells with internalized bacteria, and percent of cell-associated bacteria that are internalized. These analyses quantify bacterial association and internalization across thousands of cells and can be applied to diverse experimental systems. © 2017 by John Wiley & Sons, Inc.
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Bactérias/citologia , Ensaios de Triagem em Larga Escala/métodos , Citometria por Imagem/métodos , Actinas/metabolismo , Adesão Celular , Temperatura Baixa , Fluoresceínas/metabolismo , Humanos , Polimerização , Coloração e Rotulagem , Succinimidas/metabolismo , SuspensõesRESUMO
Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type.
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Transporte Biológico/fisiologia , Diferenciação Celular/fisiologia , Citometria de Fluxo/métodos , Citometria por Imagem/métodos , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Microscopia de Fluorescência/métodos , Neutrófilos/fisiologia , Fagócitos/fisiologia , Fagocitose/fisiologiaRESUMO
Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrhoeal disease in the developing world. The parasite was named "histolytica" for its ability to destroy host tissues, which is probably driven by direct killing of human cells. The mechanism of human cell killing has been unclear, although the accepted model was that the parasites use secreted toxic effectors to kill cells before ingestion. Here we report the discovery that amoebae kill by ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of human cell fragments is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of fragments of living human cells is reminiscent of trogocytosis (from Greek trogo, nibble) observed between immune cells, but amoebic trogocytosis differs because it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms. These findings change the model for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange.
Assuntos
Morte Celular , Entamoeba histolytica/fisiologia , Entamoeba histolytica/patogenicidade , Entamebíase/patologia , Entamebíase/parasitologia , Intestinos/patologia , Intestinos/parasitologia , Evolução Biológica , Células CACO-2 , Cálcio/metabolismo , Sobrevivência Celular , Entamoeba histolytica/citologia , Eritrócitos/parasitologia , Humanos , Células Jurkat , Doenças Negligenciadas/parasitologia , Doenças Negligenciadas/patologiaRESUMO
BACKGROUND: Toxins A and B (TcdA and TcdB) are Clostridium difficile's principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure. RESULTS: We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G1/S and known G2/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis. CONCLUSIONS: This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.
Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Ceco/citologia , Ciclo Celular/efeitos dos fármacos , Enterotoxinas/toxicidade , Células Epiteliais/efeitos dos fármacos , Íleo/citologia , Transcrição Gênica/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Análise em Microsséries , Biologia de Sistemas/métodos , Fatores de TempoRESUMO
BACKGROUND: Thymic stromal lymphopoietin (TSLP) is expressed at sites of allergic inflammation, including eczematous skin. This cytokine has been reported to exert its T(H)2-inducing properties through dendritic cells. Expression of TSLP receptor on the surface of activated T(H)2 cells could amplify T(H)2 responses at inflamed sites through the direct actions of TSLP. OBJECTIVE: To test rigorously whether T(H)2 cells induced by "proallergic" factors express TSLP receptor and characterize these cells using an experimental platform that combines flow cytometry with microscopic capabilities. METHODS: CD4(+) T cells isolated from patients with atopic dermatitis or normal healthy controls were cocultured with autologous dendritic cells in the presence of T(H)2-promoting stimuli (TSLP ± allergen and staphylococcal enterotoxin B ± TSLP). Surface expression of TSLP receptor was analyzed by image-based flow cytometry, and responsiveness of purified T cells to TSLP was assessed by phosphorylation of signal transducer and activator of transcription-5 and cytokine secretion. RESULTS: T(H)2-promoting stimuli induced a robust population of activated T(H)2 cells (CD25(+)IL-4(+)). Regardless of the nature of the stimulus, flow cytometry imaging confirmed that T cells expressing TSLP receptor were rare, constituting a minor fraction of the IL-4(+) T cell pool; however, TSLP responsiveness was nonetheless detectable. Analysis of cell size and nuclear morphology revealed preferential expression of TSLP receptor on IL-4-expressing cells undergoing mitosis. Analysis of lesional skin in atopic dermatitis supported the view that rare IL-4(+) T cells expressing TSLP receptor are present at inflamed sites. CONCLUSION: In a "proallergic" milieu, TSLP receptor is preferentially expressed on rare actively dividing T(H)2 cells. The direct action of TSLP on T cells could amplify T(H)2 responses at sites of allergic inflammation.
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Dermatite Atópica/imunologia , Receptores de Citocinas/biossíntese , Células Th2/imunologia , Separação Celular , Dermatite Atópica/metabolismo , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Receptores de Citocinas/imunologia , Células Th2/metabolismoRESUMO
BACKGROUND: It is unresolved whether circulating CD25hiCD4+ T cells in patients with atopic dermatitis who have elevated IgE (IgE(high)) are regulatory or effector in nature. OBJECTIVE: To analyze the properties of CD25hi T-cell subtypes in IgE(high) atopic dermatitis. METHODS: The phenotype of circulating CD25hi T cells was analyzed by flow cytometry using PBMCs from patients with atopic dermatitis (total IgE > 250 IU/mL). Cytokines induced in CD25hi subtypes were analyzed after activation with anti-CD3 mAb (+/-IL-2) and in the presence of activated autologous effector T cells (CD25negCD4+). Reactivity to bacterial superantigen derived from the skin-colonizing organism Staphylococcus aureus was also evaluated. RESULTS: CD25(hi) T cells expressing regulatory T-cell markers (Foxp3, CCR4, cutaneous lymphocyte-associated antigen) were increased in atopic dermatitis compared with IgE(low) controls. This phenomenon was linked to disease severity. Two subtypes of CD25hi T cells were identified on the basis of differential expression of the chemokine receptor CCR6. Although the ratio of CCR6+ and CCR6neg subtypes within the CD25hi subset was unaltered in atopic dermatitis, each subtype proliferated spontaneously ex vivo, suggesting in vivo activation. Activated CCR6neg cells secreted T(H)2 cytokines, and coculture with effector T cells selectively enhanced IL-5 production. Moreover, induction of a T(H)2-dominated cytokine profile on activation with bacterial superantigen was restricted to the CCR6neg subtype. CONCLUSION: Despite a regulatory phenotype, activated CD25hi T cells that lack expression of CCR6 promote T(H)2 responses.
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Citocinas/metabolismo , Dermatite Atópica/sangue , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th2/metabolismo , Adulto , Contagem de Linfócito CD4 , Movimento Celular , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunoglobulina E/sangue , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores CCR6/deficiência , Receptores CCR6/metabolismo , Índice de Gravidade de Doença , Pele/patologia , Staphylococcus aureus/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Complement plays an important role in the immunotherapeutic action of the anti-CD20 mAb rituximab, and therefore we investigated whether complement might be the limiting factor in rituximab therapy. Our in vitro studies indicate that at high cell densities, binding of rituximab to human CD20(+) cells leads to loss of complement activity and consumption of component C2. Infusion of rituximab in chronic lymphocytic leukemia patients also depletes complement; sera of treated patients have reduced capacity to C3b opsonize and kill CD20(+) cells unless supplemented with normal serum or component C2. Initiation of rituximab infusion in chronic lymphocytic leukemia patients leads to rapid clearance of CD20(+) cells. However, substantial numbers of B cells, with significantly reduced levels of CD20, return to the bloodstream immediately after rituximab infusion. In addition, a mAb specific for the Fc region of rituximab does not bind to these recirculating cells, suggesting that the rituximab-opsonized cells were temporarily sequestered by the mononuclear phagocytic system, and then released back into the circulation after the rituximab-CD20 complexes were removed by phagocytic cells. Western blots provide additional evidence for this escape mechanism that appears to occur as a consequence of CD20 loss. Treatment paradigms to prevent this escape, such as use of engineered or alternative anti-CD20 mAbs, may allow for more effective immunotherapy of chronic lymphocytic leukemia.
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Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Proteínas Inativadoras do Complemento/administração & dosagem , Proteínas Inativadoras do Complemento/farmacologia , Leucemia Linfocítica Crônica de Células B/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Murinos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/biossíntese , Antígenos CD20/sangue , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular Tumoral , Complemento C3b/metabolismo , Via Clássica do Complemento/imunologia , Proteínas do Sistema Complemento/biossíntese , Proteínas do Sistema Complemento/metabolismo , Humanos , Infusões Intravenosas , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/terapia , Proteínas Opsonizantes/metabolismo , Rituximab , Soro/fisiologiaRESUMO
The B cell C receptor specific for C3dg (CR2) shares a number of features with the primate E C receptor (CR1). Previously, we have demonstrated, both in vitro and in animal models, that immune complexes (IC) bound to primate E CR1, either via C opsonization or by means of bispecific mAb complexes, can be transferred to acceptor macrophages in a process that also removes CR1 from the E. We have now extended this paradigm, the transfer reaction, to include B cell CR2. We used both flow cytometry and fluorescence microscopy to demonstrate that IC bound to Raji cell CR2, either via C opsonization or through the use of an anti-CR2 mAb, are transferred to acceptor THP-1 cells. This reaction, which appears to require Fc recognition of IgG bound to Raji cell CR2, also leads to transfer of CR2. Additional support for the B cell transfer reaction is provided in a prototype study in a monkey model in which IC bound to B cell CR2 are localized to the spleen. These findings may have important implications with respect to defining the role of C in IC handling during the normal immune response.
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Linfócitos B/imunologia , Linfócitos B/metabolismo , Receptores de Complemento 3d/metabolismo , Animais , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/metabolismo , Apresentação de Antígeno , Complexo Antígeno-Anticorpo/metabolismo , Linhagem Celular Transformada , Humanos , Macaca fascicularis , Proteínas Opsonizantes/metabolismo , Ligação Proteica/imunologia , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3d/imunologia , Formação de Roseta , Células Tumorais CultivadasRESUMO
We investigated deposition of the complement protein fragment C3b and its breakdown products (collectively designated as C3b(i)) on CD20-positive cells treated with rituximab (RTX) in the presence of normal human serum (NHS). Radioimmunoassay (RIA) demonstrates that about 500 000 C3b(i) molecules deposit per cell, and fluorescence microscopy reveals that C3b(i) colocalizes with bound RTX. Use of mAb 3E7, specific for C3b(i) bound to substrates, enhances C3b(i) deposition; > 1 million C3b(i) deposit when cells are incubated with NHS, RTX and mAb 3E7. Treatment of Raji cells in NHS plus RTX leads to robust cell killing (95%) after 24 to 48 hours, and mAb 3E7 significantly enhances RTX-mediated killing of Raji and DB cells. A cynomolgus monkey model based on intravenous infusion of RTX followed by mAb 3E7 demonstrated that RTX rapidly binds to B cells and promotes complement activation and C3b(i) deposition; fluorescence microscopy analyses revealed the same pattern of colocalization of C3b(i) on cell-bound RTX in vivo as observed in vitro. Preliminary in vitro studies with blood samples from patients with chronic lymphocytic leukemia lead to similar findings. These experiments suggest that complement plays a key role in the mechanism of action of RTX; moreover, the in vivo molecular form of RTX (and possibly other antitumor mAbs) in the circulation or in tissues may include C3b(i) molecules covalently bound to the therapeutic mAb, thus allowing it to interact with cells containing both Fc and complement receptors.