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1.
J Immunol ; 199(12): 4091-4102, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29127145

RESUMO

T cell expression of TIM-3 following Ag encounter has been associated with a continuum of functional states ranging from effector memory T cells to exhaustion. We have designed an in vitro culture system to specifically address the impact of anti-TIM-3/TIM-3 engagement on human Ag-specific CD8 T cells during a normal response to Ag and found that anti-TIM-3 treatment enhances T cell function. In our in vitro T cell culture system, MART1-specific CD8 T cells were expanded from healthy donors using artificial APCs. To ensure that the T cells were the only source of TIM-3, cells were rechallenged with peptide-loaded artificial APCs in the presence of anti-TIM-3 Ab. In these conditions, anti-TIM-3 treatment promotes generation of effector T cells as shown by acquisition of an activated phenotype, increased cytokine production, enhanced proliferation, and a transcription program associated with T cell differentiation. Activation of mTORC1 has been previously demonstrated to enhance CD8 T cell effector function and differentiation. Anti-TIM-3 drives CD8 T cell differentiation through activation of the mTORC1 as evidenced by increased levels of phosphorylated S6 protein and rhebl1 transcript. Altogether these findings suggest that anti-TIM-3, together with Ag, drives differentiation in favor of effector T cells via the activation of mTOR pathway. To our knowledge, this is the first report demonstrating that TIM-3 engagement during Ag stimulation directly influences T cell differentiation through mTORC1.


Assuntos
Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Memória Imunológica/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Anticorpos Monoclonais/farmacologia , Divisão Celular , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Humanos , Ativação Linfocitária , Linfocinas/biossíntese , Linfocinas/genética , Antígeno MART-1/imunologia , Fosforilação , Processamento de Proteína Pós-Traducional , Especificidade do Receptor de Antígeno de Linfócitos T , Proteínas ras/biossíntese , Proteínas ras/genética
2.
Immunity ; 29(1): 79-89, 2008 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-18571443

RESUMO

DR3 (TRAMP, LARD, WSL-1, TNFRSF25) is a death-domain-containing tumor necrosis factor (TNF)-family receptor primarily expressed on T cells. TL1A, the TNF-family ligand for DR3, can costimulate T cells, but the physiological function of TL1A-DR3 interactions in immune responses is not known. Using DR3-deficient mice, we identified DR3 as the receptor responsible for TL1A-induced T cell costimulation and dendritic cells as the likely source for TL1A during T cell activation. Despite its role in costimulation, DR3 was not required for in vivo T cell priming, for polarization into T helper 1 (Th1), Th2, or Th17 effector cell subtypes, or for effective control of infection with Toxoplasma gondii. Instead, DR3 expression was required on T cells for immunopathology, local T cell accumulation, and cytokine production in Experimental Autoimmune Encephalomyelitis (EAE) and allergic lung inflammation, disease models that depend on distinct effector T cell subsets. DR3 could be an attractive therapeutic target for T cell-mediated autoimmune and allergic diseases.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Inflamação/imunologia , Ativação Linfocitária/imunologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Hipersensibilidade Respiratória/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Toxoplasmose/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo
4.
J Biol Chem ; 288(43): 30843-54, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23986451

RESUMO

Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.


Assuntos
Anticorpos Monoclonais , Citotoxicidade Celular Dependente de Anticorpos , Sítios de Ligação de Anticorpos , Engenharia de Proteínas , Proteólise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/genética , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Linhagem Celular , Humanos , Imunoglobulina G , Receptores de IgG/genética , Receptores de IgG/imunologia
5.
Proteins ; 82(8): 1656-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24638881

RESUMO

The functional role of human antihinge (HAH) autoantibodies in normal health and disease remains elusive, but recent evidence supports their role in the host response to IgG cleavage by proteases that are prevalent in certain disorders. Characterization and potential exploitation of these HAH antibodies has been hindered by the absence of monoclonal reagents. 2095-2 is a rabbit monoclonal antibody targeting the IdeS-cleaved hinge of human IgG1. We have determined the crystal structure of the Fab of 2095-2 and its complex with a hinge analog peptide. The antibody is selective for the C-terminally cleaved hinge ending in G236 and this interaction involves an uncommon disulfide in VL CDR3. We probed the importance of the disulfide in VL CDR3 through engineering variants. We identified one variant, QAA, which does not require the disulfide for biological activity or peptide binding. The structure of this variant offers a starting point for further engineering of 2095-2 with the same specificity, but lacking the potential manufacturing liability of an additional disulfide. Proteins 2014; 82:1656-1667. © 2014 Wiley Periodicals, Inc.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Imunoglobulina G/imunologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica , Proteólise , Coelhos
6.
J Immunol ; 189(11): 5457-66, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23105143

RESUMO

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.


Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Macrófagos/imunologia , Fagocitose , Receptores de IgG/imunologia , Animais , Neoplasias da Mama/patologia , Antígeno CD11b/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Progressão da Doença , Feminino , Humanos , Imunofenotipagem , Receptores de Lipopolissacarídeos/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos SCID , Invasividade Neoplásica/imunologia , Transplante de Neoplasias , Cultura Primária de Células
7.
J Immunother Cancer ; 11(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36948506

RESUMO

BACKGROUND: The prostate tumor microenvironment (TME) is immunosuppressive, with few effector T cells and enrichment of inhibitory immune populations, leading to limited responses to treatments such as immune checkpoint therapies (ICTs). The immune composition of the prostate TME differs across soft tissue and bone, the most common site of treatment-refractory metastasis. Understanding immunosuppressive mechanisms specific to prostate TMEs will enable rational immunotherapy strategies to generate effective antitumor immune responses. Daratumumab (anti-CD38 antibody) and edicotinib (colony-stimulating factor-1 receptor (CSF-1R) inhibitor) may alter the balance within the prostate TME to promote antitumor immune responses. HYPOTHESIS: Daratumumab or edicotinib will be safe and will alter the immune TME, leading to antitumor responses in localized prostate cancer. PATIENTS AND METHODS: In this presurgical study, patients with localized prostate cancer received 4 weekly doses of daratumumab or 4 weeks of daily edicotinib prior to radical prostatectomy (RP). Treated and untreated control (Gleason score ≥8 in prostate biopsy) prostatectomy specimens and patient-matched pre- and post-treatment peripheral blood mononuclear cells (PBMCs) and bone marrow samples were evaluated. The primary endpoint was incidence of adverse events (AEs). The secondary endpoint was pathologic complete remission (pCR) rate. RESULTS: Twenty-five patients were treated (daratumumab, n=15; edicotinib, n=10). All patients underwent RP without delays. Grade 3 treatment-related AEs with daratumumab occurred in 3 patients (12%), and no ≥grade 3 treatment-related AEs occurred with edicotinib. No changes in serum prostate-specific antigen (PSA) levels or pCRs were observed. Daratumumab led to a decreased frequency of CD38+ T cells, natural killer cells, and myeloid cells in prostate tumors, bone marrow, and PBMCs. There were no consistent changes in CSF-1R+ immune cells in prostate, bone marrow, or PBMCs with edicotinib. Neither treatment induced T cell infiltration into the prostate TME. CONCLUSIONS: Daratumumab and edicotinib treatment was safe and well-tolerated in patients with localized prostate cancer but did not induce pCRs. Decreases in CD38+ immune cells were observed in prostate tumors, bone marrow, and PBMCs with daratumumab, but changes in CSF-1R+ immune cells were not consistently observed with edicotinib. Neither myeloid-targeted agent alone was sufficient to generate antitumor responses in prostate cancer; thus, combinations with agents to induce T cell infiltration (eg, ICTs) will be needed to overcome the immunosuppressive prostate TME.


Assuntos
Antineoplásicos , Neoplasias da Próstata , Masculino , Humanos , Leucócitos Mononucleares/patologia , Antineoplásicos/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Imunossupressores , Microambiente Tumoral
8.
Blood ; 115(24): 5012-22, 2010 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-20357242

RESUMO

Fatty acid metabolism governs multiple intracellular signaling pathways in many cell types, but its role in hematopoietic stem cells (HSCs) is largely unknown. Herein, we establish a critical role for 12/15-lipoxygenase (12/15-LOX)-mediated unsaturated fatty acid metabolism in HSC function. HSCs from 12/15-LOX-deficient mice are severely compromised in their capacity to reconstitute the hematopoietic compartment in competitive and serial reconstitution assays. Furthermore, we demonstrate that 12/15-LOX is required for the maintenance of long-term HSC quiescence and number. The defect in HSCs is cell-autonomous and associated with a selective reduction in 12/15-LOX-mediated generation of bioactive lipid mediators and reactive oxygen species and with a decrease in canonical Wnt signaling as measured by nuclear beta-catenin staining. These results have implications for development, aging, and transformation of the hematopoietic compartment.


Assuntos
Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Ácidos Graxos/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/enzimologia , Animais , Contagem de Células , Citometria de Fluxo , Expressão Gênica/fisiologia , Hematopoese/fisiologia , Fatores Reguladores de Interferon/genética , Leucopenia/genética , Leucopenia/metabolismo , Leucopenia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo
9.
Infect Immun ; 77(12): 5690-700, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19822654

RESUMO

Interleukin-12 (IL-12) is critical for resistance to Toxoplasma gondii during both the acute and chronic stages of infection. However, the cellular and molecular pathways that regulate IL-12 production during chronic toxoplasmosis are incompletely defined. We recently discovered that 12/15-lipoxygenase (12/15-LOX), which oxidizes unsaturated lipids in macrophages, is a novel and selective regulator of IL-12 production. We now demonstrate the essential role of this enzyme in the chronic phase of toxoplasmosis. Although 12/15-LOX-deficient mice were resistant to acute T. gondii infection, 80% of 12/15-LOX-deficient mice died during chronic toxoplasmosis, compared to no deaths in wild-type controls. The morbidity of chronically infected 12/15-LOX mice was associated with an increase in brain inflammation and parasite burden. These data suggest that the evolution of the immune response to T. gondii is accompanied by an increasing requirement for 12/15-LOX-mediated signaling. Consistent with this conclusion, 12/15-LOX activity was enhanced during chronic, but not acute, toxoplasmosis. Furthermore, the enhanced susceptibility of 12/15-LOX-deficient mice to chronic toxoplasmosis was associated with reduced production of IL-12 and gamma interferon (IFN-gamma) that was not evident during acute infection. Importantly, ex vivo IFN-gamma production by 12/15-LOX-deficient splenocytes could be rescued by the addition of recombinant IL-12. These data establish that 12/15-LOX is a critical mediator of the chronic type 1 inflammatory response and that immune mediators can be subject to distinct cellular and/or molecular mechanisms of regulation at different stages of inflammation.


Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Interleucina-12/biossíntese , Interleucina-12/imunologia , Toxoplasma/imunologia , Toxoplasmose Animal/imunologia , Animais , Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 15-Lipoxigenase/deficiência , Peso Corporal , Encéfalo/parasitologia , Encéfalo/patologia , Células Cultivadas , Feminino , Inflamação/patologia , Interferon gama/biossíntese , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Análise de Sobrevida
10.
Arterioscler Thromb Vasc Biol ; 28(7): 1283-9, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18420998

RESUMO

OBJECTIVE: The purpose of this study was to distinguish the contributions of CD44 expressed on bone marrow-derived and non-bone marrow-derived cells to atherosclerosis. METHODS AND RESULTS: Using bone marrow chimeras, we compared the contributions of CD44 expressed on bone marrow-derived cells versus non-bone marrow-derived cells to the vascular inflammation underlying atherosclerosis. We show that CD44 in both bone marrow-derived and non-bone marrow-derived compartments promotes atherosclerosis in apoE-/- mice and mediates macrophage and T cell recruitment to lesions in vivo. We also demonstrate that CD44 on endothelial cells (ECs) as well as on macrophages and T cells enhances leukocyte-endothelial cell adhesion and transendothelial migration in vitro. Furthermore, CD44 on vascular smooth muscle cells (VSMCs) regulates their hyaluronan (HA)-dependent migration. Interestingly, in mice lacking CD44 in both compartments, where we observed the least inflammation, we also observed enhanced fibrous cap formation. CONCLUSIONS: CD44 expressed on bone marrow-derived and non-bone marrow-derived cells both promote atherosclerosis in apoE-deficient mice. Furthermore, CD44 plays a pivotal role in determining the balance between inflammation and fibrosis in atherosclerotic lesions which can impact clinical outcome in humans.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/imunologia , Células da Medula Óssea/imunologia , Células Endoteliais/imunologia , Receptores de Hialuronatos/metabolismo , Macrófagos/imunologia , Músculo Liso Vascular/imunologia , Linfócitos T/imunologia , Animais , Aorta/imunologia , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Movimento Celular , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Migração e Rolagem de Leucócitos , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/imunologia , Fatores de Tempo , Quimeras de Transplante
11.
Gastroenterol Nurs ; 32(6): 401-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20010233

RESUMO

The purpose of this qualitative study was to explore and describe male patients' experiences of undergoing and completing treatment for the hepatitis C virus. A qualitative, descriptive, and phenomenological methodology was used. Eight male patients diagnosed with the hepatitis C virus, aged 41-60 years and enrolled in a health maintenance organization, were interviewed in their private residences in a community setting. In depth, semistructured, tape-recorded, and transcribed interviews were analyzed by using theme coding. Six themes emerged that described the lived experience of male patients after undergoing treatment for the hepatitis C virus: acquisition of the disease, the diagnosis, the treatment decision making, the "horror stories" regarding treatment, what helped, and feelings now.Patients who are diagnosed with the hepatitis C virus and undergo treatment face a difficult therapy regimen. All participants knew how they acquired the disease and had no sense of apology for how they attracted it. Their only goal was to achieve viral clearance of this disease. Treatment was their option. Nurses play a key role in the management of treatment for patients undergoing therapy for hepatitis C. The management of the adverse effects of therapy has a great impact on patients' quality of life and compliance with the therapy. Nurses are able to help patients by recognizing and managing the adverse effects of this therapy.


Assuntos
Hepatite C/enfermagem , Adaptação Psicológica , Adulto , Antivirais/administração & dosagem , Antivirais/efeitos adversos , Quimioterapia Combinada , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Hepatite C/tratamento farmacológico , Humanos , Interferon-alfa/administração & dosagem , Interferon-alfa/efeitos adversos , Masculino , Pessoa de Meia-Idade , Pesquisa Metodológica em Enfermagem , Pesquisa Qualitativa , Qualidade de Vida , Ribavirina/administração & dosagem , Ribavirina/efeitos adversos , Fatores de Risco , Inquéritos e Questionários , Resultado do Tratamento
12.
MAbs ; 7(3): 494-504, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25933349

RESUMO

Cytotoxic therapeutic monoclonal antibodies (mAbs) often mediate target cell-killing by eliciting immune effector functions via Fc region interactions with cellular and humoral components of the immune system. Key functions include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). However, there has been increased appreciation that along with cell-killing functions, the induction of antibody-dependent cytokine release (ADCR) can also influence disease microenvironments and therapeutic outcomes. Historically, most Fc engineering approaches have been aimed toward modulating ADCC, ADCP, or CDC. In the present study, we describe an Fc engineering approach that, while not resulting in impaired ADCC or ADCP, profoundly affects ADCR. As such, when peripheral blood mononuclear cells are used as effector cells against mAb-opsonized tumor cells, the described mAb variants elicit a similar profile and quantity of cytokines as IgG1. In contrast, although the variants elicit similar levels of tumor cell-killing as IgG1 with macrophage effector cells, the variants do not elicit macrophage-mediated ADCR against mAb-opsonized tumor cells. This study demonstrates that Fc engineering approaches can be employed to uncouple macrophage-mediated phagocytic and subsequent cell-killing functions from cytokine release.


Assuntos
Anticorpos Monoclonais , Anticorpos Antineoplásicos , Citotoxicidade Celular Dependente de Anticorpos , Citocinas/imunologia , Fragmentos Fc das Imunoglobulinas , Macrófagos/imunologia , Neoplasias/tratamento farmacológico , Engenharia de Proteínas , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/genética , Linhagem Celular Tumoral , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Neoplasias/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
13.
MAbs ; 6(5): 1265-73, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25517311

RESUMO

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')2 fragments, but not for full-length IgG or for closely-related F(ab')2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')2 fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')2 fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.


Assuntos
Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/metabolismo , Anticorpos Monoclonais Murinos/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Proteínas de Bactérias/metabolismo , Plaquetas/imunologia , Plaquetas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Imunoglobulina G/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Contagem de Plaquetas , Proteólise , Ratos , Rituximab
14.
J Exp Med ; 208(10): 1989-2003, 2011 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-21930769

RESUMO

Cancer immunoediting is the process whereby the immune system suppresses neoplastic growth and shapes tumor immunogenicity. We previously reported that type I interferon (IFN-α/ß) plays a central role in this process and that hematopoietic cells represent critical targets of type I IFN's actions. However, the specific cells affected by IFN-α/ß and the functional processes that type I IFN induces remain undefined. Herein, we show that type I IFN is required to initiate the antitumor response and that its actions are temporally distinct from IFN-γ during cancer immunoediting. Using mixed bone marrow chimeric mice, we demonstrate that type I IFN sensitivity selectively within the innate immune compartment is essential for tumor-specific T cell priming and tumor elimination. We further show that mice lacking IFNAR1 (IFN-α/ß receptor 1) in dendritic cells (DCs; Itgax-Cre(+)Ifnar1(f/f) mice) cannot reject highly immunogenic tumor cells and that CD8α(+) DCs from these mice display defects in antigen cross-presentation to CD8(+) T cells. In contrast, mice depleted of NK cells or mice that lack IFNAR1 in granulocytes and macrophage populations reject these tumors normally. Thus, DCs and specifically CD8α(+) DCs are functionally relevant targets of endogenous type I IFN during lymphocyte-mediated tumor rejection.


Assuntos
Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Neoplasias/imunologia , Transferência Adotiva , Animais , Quimera , Apresentação Cruzada/imunologia , Células Dendríticas/citologia , Granulócitos/imunologia , Imunidade Inata/imunologia , Interferon gama/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/patologia , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia
15.
Exp Hematol ; 38(11): 1036-1046.e1-4, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20647030

RESUMO

OBJECTIVE: Several transcription factors determine the cell fate decision between granulocytes and monocytes, but the upstream signal transduction pathways that govern myelopoiesis are largely unknown. Based on our observation of aberrant myeloid cell representation in hematopoietic tissues of 12/15-lipoxygenase (12/15-LOX)-deficient (Alox15) mice, we tested the hypothesis that polyunsaturated fatty acid metabolism regulates myelopoiesis. MATERIALS AND METHODS: Multicolor flow cytometric analysis and methylcellulose assays were used to compare myelopoiesis and the differentiative capacity of progenitors from Alox15 and wild-type mice. Furthermore, we elucidated the mechanism by which 12/15-LOX is involved in regulation of myelopoiesis. RESULTS: Granulopoiesis in Alox15 mice is increased while monopoiesis is reduced. Moreover, there is an accumulation of granulocyte-macrophage progenitors that exhibit defective differentiation. Mechanistically, we demonstrate that transcriptional activity of interferon regulatory factor-8 (Irf8), which regulates myelopoiesis, is impaired in Alox15 progenitors and bone marrow-derived macrophages due to loss of 12/15-LOX-mediated redox regulation of Irf8 nuclear accumulation. Restoration of redox signaling in Alox15 bone marrow cells and granulocyte-macrophage progenitors reversed the defect in myeloid differentiation. CONCLUSIONS: These data establish 12/15-LOX-mediated redox signaling as a novel regulator of myelopoiesis and Irf8.


Assuntos
Araquidonato 12-Lipoxigenase/deficiência , Araquidonato 15-Lipoxigenase/deficiência , Fatores Reguladores de Interferon/metabolismo , Células Progenitoras Mieloides/metabolismo , Transdução de Sinais , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Eritropoetina/farmacologia , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células Progenitoras de Granulócitos e Macrófagos/citologia , Células Progenitoras de Granulócitos e Macrófagos/metabolismo , Granulócitos/citologia , Granulócitos/metabolismo , Immunoblotting , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Monócitos/metabolismo , Células Progenitoras Mieloides/citologia , Mielopoese/efeitos dos fármacos , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
16.
Exp Cell Res ; 314(1): 173-83, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17976581

RESUMO

Breast cancer preferentially metastasizes to the skeleton, a hospitable environment that attracts and allows breast cancer cells to thrive. Growth factors released as bone is degraded support tumor cell growth, and establish a cycle favoring continued bone degradation. While the osteoclasts are the direct effectors of bone degradation, we found that osteoblasts also contribute to bone loss. Osteoblasts are more than intermediaries between tumor cells and osteoclasts. We have presented evidence that osteoblasts contribute through loss of function induced by metastatic breast cancer cells. Metastatic breast cancer cells suppress osteoblast differentiation, alter morphology, and increase apoptosis. In this study we show that osteoblasts undergo an inflammatory stress response in the presence of human metastatic breast cancer cells. When conditioned medium from cancer cells was added to human osteoblasts, the osteoblasts were induced to express increased levels of IL-6, IL-8, and MCP-1; cytokines known to attract, differentiate, and activate osteoclasts. Similar findings were seen with murine osteoblasts and primary murine calvarial osteoblasts. Osteoblasts are co-opted into creating a microenvironment that exacerbates bone loss and are prevented from producing matrix proteins for mineralization. This is the first study implicating osteoblast produced IL-6, IL-8 (human; MIP-2 and KC mouse), and MCP-1 as key mediators in the osteoblast response to metastatic breast cancer cells.


Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Carcinoma/secundário , Metástase Neoplásica/fisiopatologia , Osteíte/fisiopatologia , Osteoblastos/metabolismo , Animais , Neoplasias Ósseas/imunologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/fisiopatologia , Neoplasias da Mama/imunologia , Calcificação Fisiológica/fisiologia , Carcinoma/imunologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Feminino , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos , Metástase Neoplásica/imunologia , Osteíte/etiologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/fisiologia , Estresse Fisiológico/etiologia , Estresse Fisiológico/metabolismo , Estresse Fisiológico/fisiopatologia
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