RESUMO
Activating autoantibodies to the angiotensin type 1 receptor (AT1R) are associated with hypertensive disorders. The angiotensin type 2 receptor (AT2R) is known to counter-regulate the actions of AT1R. We investigated whether AT2R autoantibodies produced in immunized rabbits will activate AT2R and suppress the vasopressor responses to angiotensin II and AT1R-activating autoantibodies. Five rabbits immunized with a peptide corresponding to the second extracellular loop of AT2R developed high AT2R antibody titers. Rabbit anti-AT2R sera failed to directly dilate isolated rat cremaster arterioles; however, when co-perfused with angiotensin II or AT1R-activating autoantibodies, the anti-AT2R sera significantly inhibited their contractile effects. Rabbit anti-AT2R sera recognized a predominant sequence near the N-terminus of the AT2R second extracellular loop. A decoy peptide based on this sequence effectively reversed the opposing effect of the anti-AT2R sera on angiotensin II-induced contraction of rat cremaster arterioles. A similar blockade of the anti-AT2R sera effect was observed with the AT2R antagonist PD 123319 and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Rabbit anti-AT2R sera reacted specifically with AT2R. No cross-reactivity with AT1R was observed. Blood pressure did not change in immunized animals. However, the pressor responses to incremental angiotensin II infusions were blunted in immunized animals. Thirteen subjects with primary aldosteronism demonstrated increased AT2R autoantibody levels compared with normal controls. In conclusion, AT2R autoantibodies produced in immunized rabbits have the ability to activate AT2R and counteract the AT1R-mediated vasoconstriction. These autoantibodies provide useful and selective tools for the study of their roles in blood pressure regulation and possible therapeutic intervention.
Assuntos
Angiotensina II/imunologia , Anticorpos Bloqueadores/fisiologia , Autoanticorpos/imunologia , Hipertensão/imunologia , Receptor Tipo 1 de Angiotensina/imunologia , Receptor Tipo 2 de Angiotensina/imunologia , Vasoconstrição/imunologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiopatologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Masculino , Coelhos , Ratos , Vasoconstrição/efeitos dos fármacosRESUMO
T cell Ig and mucin domain 3 (Tim3) is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis in the mouse model of infection. In this study, we sought to determine whether the Tim3-Gal9 pathway plays a similar role in human pulmonary TB. We identified that pulmonary TB patients have reduced expression of Tim3 on CD14(+) monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1ß. Our results establish that Tim3-Gal9 interaction activates human M. tuberculosis -infected macrophages and leads to the control of bacterial growth through the production of the proinflammatory cytokine IL-1ß. Data presented in this study suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1ß, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.
Assuntos
Anticorpos Bloqueadores/fisiologia , Galectinas/fisiologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/fisiologia , Mycobacterium tuberculosis/imunologia , Transdução de Sinais/imunologia , Adulto , Idoso , Anticorpos Bloqueadores/biossíntese , Feminino , Galectinas/antagonistas & inibidores , Galectinas/imunologia , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mapeamento de Interação de ProteínasRESUMO
VCAM-1 plays a key role in leukocyte trafficking during inflammatory responses. However, molecular mechanisms underlying this function have not been clearly elucidated. In this study, using phage display technology, we developed a rabbit/human chimeric VCAM-1 Ab, termed VCAM-1 domain 6 (VCAM-1-D6), which specifically recognizes aa 511-599 within the sixth Ig-like domain. We report that the VCAM-1-D6 Ab blocked U937 cell transmigration across activated HUVECs but did not alter adhesion of U937 cells to the HUVECs. We also demonstrate that VCAM-1-D6 does not alter TNF-α-stimulated endothelial cell chemokine or cytokine production. Furthermore, through in vivo efficacy testing using a mouse islet allograft model, we demonstrate that VCAM-1-D6 significantly alleviates allograft rejection by blocking leukocyte infiltration to the grafted islets. Taken together, our results suggest that the VCAM-1-D6 Ab may block VCAM-1-mediated inflammation and could be a useful tool in treating inflammatory diseases.
Assuntos
Anticorpos Bloqueadores/fisiologia , Adesão Celular/imunologia , Inibição de Migração Celular/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Imunoglobulina G/fisiologia , Leucócitos/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Anticorpos Bloqueadores/genética , Adesão Celular/genética , Inibição de Migração Celular/genética , Endotélio Vascular/química , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Fragmentos Fc das Imunoglobulinas/biossíntese , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/fisiologia , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Leucócitos/citologia , Camundongos , Estrutura Terciária de Proteína/genética , Coelhos , Células U937 , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Epithelial membrane protein 2 (EMP2) regulates collagen gel contraction by the retinal pigment epithelium cell line ARPE-19 by modulating FAK activation. Collagen gel contraction is one in vitro model for an aberrant wound healing response, proliferative vitreoretinopathy (PVR), which occurs as a complication of severe ocular trauma. The purpose of this study is to investigate whether EMP2 specific recombinant diabody decreases activation of FAK and collagen gel contraction in ARPE-19. Anti-EMP2 diabody was recombinantly constructed from a human phage library-derived clone selected for reactivity against an extracellular domain of human EMP2. ARPE-19 cells were exposed to an anti-EMP2 or control diabody, and toxicity, adhesion, and migration were assessed respectively through toluidine blue exclusion, binding to collagen type 1, and a migration assay. Collagen gel contraction was assessed using an in vitro assay. FAK activation was evaluated using Western blot. Exposure to anti-EMP2 diabody, resulted in a 75% reduction in EMP2 protein levels at 4 h. No significant toxicity was observed with anti-EMP2 diabody at levels that maximally reduced EMP2. Anti-EMP2 diabody, but not control diabody, significantly reduced collagen gel contraction (p < 0.001), without changes in adhesion or migration. Concordantly, anti-EMP2 diabody as compared to a control diabody reduced collagen stimulated FAK activation (p = 0.01). Anti-EMP2 diabody decreases EMP2 protein levels, FAK activation, and collagen gel contraction by ARPE-19 cells without an adverse effect on cell survival. Modulation of EMP2 using anti-EMP2 diabody could be a new approach for targeting EMP2 and pathologic consequences associated with EMP2.
Assuntos
Anticorpos Bloqueadores/fisiologia , Colágeno Tipo I/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glicoproteínas de Membrana/imunologia , Epitélio Pigmentado da Retina/metabolismo , Reações Antígeno-Anticorpo , Apoptose/efeitos dos fármacos , Western Blotting , Camptotecina/toxicidade , Linhagem Celular , Movimento Celular , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Fragmentos de Imunoglobulinas , Microscopia Confocal , Biblioteca de Peptídeos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Anticorpos de Cadeia ÚnicaRESUMO
Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate ß-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble ß-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.
Assuntos
Complemento C3/fisiologia , Lectinas Tipo C/fisiologia , beta-Glucanas/imunologia , Animais , Anticorpos Bloqueadores/fisiologia , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Complemento C3/antagonistas & inibidores , Complemento C3/deficiência , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Lectinas Tipo C/administração & dosagem , Lectinas Tipo C/metabolismo , Ligantes , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fagocitose/imunologia , beta-Glucanas/administração & dosagem , beta-Glucanas/metabolismoRESUMO
Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Transplante de Células-Tronco Hematopoéticas , Receptores Imunológicos/fisiologia , Anticorpos Bloqueadores/fisiologia , Anticorpos Bloqueadores/uso terapêutico , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Epitopos de Linfócito T/metabolismo , Marcação de Genes/métodos , Humanos , Memória Imunológica , Antígenos de Histocompatibilidade Menor/metabolismo , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/terapia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral/fisiologia , Células Tumorais CultivadasRESUMO
Despite promising results in the use of anti-epidermal growth factor receptor (EGFR) Abs for cancer therapy, several issues remain to be addressed. An increasing emphasis is being placed on immune effector mechanisms. It has become clear for other Abs directed to tumor targets that their effects involve the adaptive immunity, mainly by the contribution of Fc region-mediated mechanisms. Given the relevance of EGFR signaling for tumor biology, we wonder whether the oncogene inhibition could contribute to Ab-induced vaccine effect. In a mouse model in which 7A7 (an anti-murine EGFR Ab) and AG1478 (an EGFR-tyrosine kinase inhibitor) displayed potent antimetastatic activities, depletion experiments revealed that only in the case of the Ab, the effect was dependent on CD4(+) and CD8(+) T cells. Correspondingly, 7A7 administration elicited a remarkable tumor-specific CTL response in hosts. Importantly, experiments using 7A7 F(ab')(2) suggested that in vivo Ab-mediated EGFR blockade may play an important role in the linkage with adaptive immunity. Addressing the possible mechanism involved in this effect, we found quantitative and qualitative differences between 7A7 and AG1478-induced apoptosis. EGFR blocking by 7A7 not only prompted a higher proapoptotic effect on tumor metastases compared with AG1478, but also was able to induce apoptosis with immunogenic potential in an Fc-independent manner. As expected, 7A7 but not AG1478 stimulated exposure of danger signals on tumor cells. Subcutaneous injection of 7A7-treated tumor cells induced an antitumor immune response. This is the first report, to our knowledge, of a tumor-specific CTL response generated by Ab-mediated EGFR inhibition, suggesting an important contribution of immunogenic apoptosis to this effect.
Assuntos
Anticorpos Monoclonais/fisiologia , Especificidade de Anticorpos/fisiologia , Apoptose/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Animais , Anticorpos Bloqueadores/fisiologia , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/secundário , Linhagem Celular Tumoral , Células Cultivadas , Receptores ErbB/metabolismo , Feminino , Fragmentos Fc das Imunoglobulinas/fisiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/patologiaRESUMO
The challenge of controlling HIV infection involves an understanding of the heterogeneity of the virus, its wide cellular host range, its primary routes of transmission, and the immunologic and intrinsic cellular factors that can prevent its transmission and replication. Identification of HIV-infected individuals who have survived more than 10 years without signs of the infection and without therapy encourages studies examining the natural mechanisms for resistance to infection and disease. Within the immune system, emphasis should be given to the innate or natural response that appears within minutes of the infection and offers the optimal time for controlling HIV. All these parameters in HIV pathogenesis underline the information needed to develop optimal anti-HIV therapies and an effective AIDS vaccine.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno/imunologia , Anticorpos Bloqueadores/fisiologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/fisiologia , Infecções por HIV/tratamento farmacológico , Sobreviventes de Longo Prazo ao HIV , HIV-1/imunologia , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata/fisiologia , Macrófagos/virologia , Internalização do Vírus , Replicação ViralRESUMO
The blockade of immune suppression against antitumor responses is a particularly attractive strategy when combined with agents that promote tumor-specific CTLs. In this study, we have attempted to further improve the CTL induction and potent antitumor efficacy of a combination mAb-based therapy (termed "trimAb therapy") that comprises tumor cell death-inducing anti-death receptor 5 mAb and immune activating anti-CD40 and anti-CD137 mAbs. Among trimAb-treated tumors, the infiltration of CD4(+) Foxp3(+) cells was greater in progressing tumors compared with stable tumors. Blockade of CTLA-4 (CD152)-mediated signals by an antagonistic mAb substantially increased the tumor rejection rate of trimAb therapy, although the immune responses of draining lymph node cells were not augmented. Interestingly, by comparison, additional treatment with agonistic anti-glucocorticoid-induced TNF receptor mAb, antagonistic anti-programmed death-1 (CD279) mAb, or agonistic anti-OX40 (CD134) mAb significantly augmented immune responses of draining lymph node cells, but did not augment the therapeutic effect of trimAb. CD4 T cell depletion reduced the antitumor effect of anti-CTLA-4 mAb treatment alone, but did not reduce the tumor rejection rate of trimAb in conjunction with anti-CTLA-4 mAb. Thus, the blockade of the CTLA-4-mediated inhibitory signal in tumor infiltrating CTL may be the most effective strategy to augment the effect of immune therapies that generate tumor-specific CTL.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/terapia , Animais , Anticorpos Bloqueadores/fisiologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/fisiologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Superfície/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Antígeno CTLA-4 , Citotoxicidade Imunológica , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Ativação Linfocitária/imunologia , Depleção Linfocítica , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Morte Celular Programada 1 , Transporte Proteico/imunologia , Receptores de Fator de Crescimento Neural/imunologia , Receptores OX40/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/patologiaRESUMO
CD1d is expressed on APCs and presents glycolipids to CD1d-restricted NKT cells. For the first time, we demonstrate the ability of anti-CD1d mAbs to inhibit the growth of different CD1d-negative experimental carcinomas in mice. Anti-CD1d mAbs systemically activated CD1d(+) APC, as measured by production of IFN-gamma and IL-12. Tumor growth inhibition was found to be completely dependent on IFN-gamma and IL-12 and variably dependent on CD8(+) T cells and NK cells, depending upon the tumor model examined. Anti-CD1d mAb induced greater CD8(+) T cell-dependent tumor suppression where regulatory CD1d-restricted type II NKT cells have been implicated, and were less effective in a NK cell-dependent manner against tumors where T regulatory cells were immunosuppressive. The ability of anti-CD1d mAbs to coincidently activate CD1d(+) APCs to release IL-12 and inhibit CD1d-restricted type II NKT cells makes CD1d an exciting new target for immunotherapy of cancer based on tumor immunoregulation.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Antineoplásicos/uso terapêutico , Inibidores do Crescimento/uso terapêutico , Animais , Anticorpos Bloqueadores/fisiologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1d/biossíntese , Antineoplásicos/imunologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Inibidores do Crescimento/biossíntese , Inibidores do Crescimento/imunologia , Interferon gama/biossíntese , Interferon gama/fisiologia , Interleucina-12/biossíntese , Interleucina-12/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos SCID , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Células T Matadoras Naturais/patologiaRESUMO
Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.
Assuntos
Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/fisiologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/fisiologia , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Antígeno B7-H1 , Linhagem Celular , Doença Crônica , Humanos , Imunoglobulina G/fisiologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Morte Celular Programada 1 , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Carga ViralRESUMO
A CD4(+) T cell clone (HC/2G-1) was established by stimulating peripheral blood T cells from a patient with renal cell carcinoma (RCC) with dendritic cells preincubated with the autologous apoptotic renal tumor line in the presence of IFN-alpha. It recognizes the autologous RCC and most allogeneic RCC lines by IFN-gamma release (10 of 11 lines) and lysis (9 of 10 lines), but does not recognize multiple EBV B cells or fibroblasts. It shows little or no recognition of a panel of melanomas, breast cancers and non-small-cell lung cancers. Phenotypically, HC/2G-1 is CD3(+)CD4(+) TCR alphabeta(+), but CD161(-)CD16(-)NKG2D(-). Tumor recognition by clone HC/2G-1 was not blocked by Abs to HLA class I or class II, but was significantly reduced by anti-TCR alphabeta Ab. Furthermore, tumor recognition was beta(2)-microglobulin-independent. HC/2G-1 does not use a Valpha or Vbeta described for classical NKT cells, but rather Valpha14 and Vbeta2.1. Allogeneic T cells cotransfected with mRNAs encoding the alpha and beta chains of the HC/2G-1 TCR recognized renal tumor lines, demonstrating that tumor recognition is TCR-mediated. Interestingly, TRAIL appears to play a role in tumor recognition by HC/2G-1 in that reactivity was blocked by anti-TRAIL Ab, and soluble TRAIL could enhance IFN-gamma secretion by HC/2G-1 in response to renal tumors. Our findings suggest that clone HC/2G-1 represents a novel type of CD4(+) cell that has broad TCR-mediated recognition of a determinant widely expressed by RCC.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Subpopulações de Linfócitos T/imunologia , Anticorpos Bloqueadores/fisiologia , Apoptose/imunologia , Carcinoma de Células Renais/terapia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Células Clonais , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Epitopos de Linfócito T/imunologia , Humanos , Neoplasias Renais/terapia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Células Tumorais CultivadasRESUMO
The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.
Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Fímbrias Bacterianas/imunologia , Mediadores da Inflamação/metabolismo , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Anticorpos Bloqueadores/fisiologia , Linhagem Celular , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Proteínas de Fímbrias/metabolismo , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/metabolismo , Humanos , Interleucina-8/antagonistas & inibidores , Interleucina-8/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Porphyromonas gingivalis/química , Porphyromonas gingivalis/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , TransfecçãoRESUMO
Polymyositis (PM) is an acquired, systemic, connective tissue disease characterized by the proximal muscle weakness and infiltration of mononuclear cells into the affected muscles. To understand its etiology and immunopathogenesis, appropriate animal model is required. It has been demonstrated that immunization with native human skeletal C protein induces severe and reproducible experimental autoimmune myositis (EAM) in Lewis rats, and that the muscle inflammatory lesions in the EAM mimic those of human PM. In the present study, we prepared recombinant skeletal C protein fragment and succeeded in inducing as severe EAM as that by native C protein. We found ICOS expression on muscle fiber-infiltrating T cells in the EAM rats, but not in normal rats. Treatment with anti-ICOS mAb reduced incidence and severity of myositis; decreased the number of muscle-infiltrating CD11b/c+, TCR+, and CD8a+ cells; and inhibited the expression of IL-1alpha and CCL2 in the hamstring muscles of the EAM rats. However, the treatment neither inhibited serum anti-C protein IgG level, C protein-induced proliferation of lymph node (LN) cells, or LN T cells, nor production of IFN-gamma by C protein-stimulated LN cells in EAM rats. These data indicate that analysis of C protein-induced EAM provides not only insights into pathogenesis of PM, but also useful information regarding development of effective immunotherapy against the disease. ICOS-ICOS ligand interaction would be a novel therapeutic target for PM.
Assuntos
Anticorpos Bloqueadores/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Doenças Autoimunes/metabolismo , Polimiosite/imunologia , Polimiosite/metabolismo , Animais , Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/fisiologia , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Doenças Autoimunes/patologia , Doenças Autoimunes/terapia , Proteínas de Transporte/imunologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Imunidade Celular , Imunoglobulina G/biossíntese , Proteína Coestimuladora de Linfócitos T Induzíveis , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Ligantes , Polimiosite/patologia , Polimiosite/terapia , Ratos , Ratos Endogâmicos Lew , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
Functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4(+) and CD8(+) T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4(+) T cells. To investigate the role of PD-1 in HIV-associated CD4(+) T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-gamma-producing HIV-specific CD4(+) T cells compared with total or CMV-specific CD4(+) T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4(+) T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4(+) T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4(+) T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4(+) T cells.
Assuntos
Antígenos CD/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , HIV/imunologia , Replicação Viral/imunologia , Adulto , Anticorpos Bloqueadores/fisiologia , Antígenos CD/sangue , Antígenos CD/imunologia , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/sangue , Proteínas Reguladoras de Apoptose/metabolismo , Antígeno B7-H1 , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Doença Crônica , Epitopos de Linfócito T/imunologia , Produtos do Gene gag/imunologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Ligantes , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Receptor de Morte Celular Programada 1 , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Regulação para Cima/imunologia , Carga ViralRESUMO
Nonneutralizing Abs may play a role in protecting animals and humans from lentiviral infections. We explored the Ab-dependent, cell-mediated virus inhibition (ADCVI) Ab response to recombinant gp120 (rgp120) vaccination in sera from 530 participants in the Vax 004 trial. Serum ADCVI activity was measured against a clinical R5 strain of HIV-1 using peripheral blood mononuclear effector cells from healthy donors. The level of vaccine-induced ADCVI activity correlated inversely with the rate of acquiring HIV infection following vaccination, such that for every 10% increase in ADCVI activity, there was a 6.3% decrease in the hazard rate of infection (p=0.019). Some vaccinated individuals also mounted an ADCVI response against two other clinical R5 strains of HIV-1. However, ADCVI activity correlated poorly with neutralizing or CD4-gp120-blocking Ab activity measured against laboratory strains. Finally, the degree to which the ADCVI Ab response predicted the rate of infection was influenced by polymorphisms at the FcgammaRIIa and FcgammaRIIIa gene loci. These data indicate that rgp120 vaccination can elicit Abs with antiviral activity against clinical strains of HIV-1. However, such activity requires the presence of FcR-bearing effector cells. Our results provide further evidence that ADCVI may play a role in preventing HIV infection.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Bloqueadores/biossíntese , Citotoxicidade Celular Dependente de Anticorpos , Anticorpos Anti-HIV/biossíntese , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/crescimento & desenvolvimento , Receptores Fc/metabolismo , Vacinas contra a AIDS/administração & dosagem , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/fisiologia , Citotoxicidade Celular Dependente de Anticorpos/genética , Antígenos CD/genética , Anticorpos Anti-HIV/metabolismo , Anticorpos Anti-HIV/fisiologia , Proteína gp120 do Envelope de HIV/administração & dosagem , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Imunidade Celular/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Masculino , Testes de Neutralização , Receptores Fc/genética , Receptores de IgG/genética , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Programmed death-1 ligand-1 (PD-L1), a member of the B7 family of costimulatory molecules, plays an important role in the regulations of the cellular and humoral immune responses. In this study, two mouse anti-human PD-L1 monoclonal antibodies named 10E10 and 2H11 were successfully generated and further characterized. Monoclonal antibody 10E10 bound to distinct PD-L1 epitope comparing an available anti-PD-L1 monoclonal antibody on a series of malignant cell lines, activated T lymphocytes, B lymphocytes and dendritic cells. Then, by using immunohistochemistry staining with monoclonal antibody 2H11, the expression of PD-L1 was found in human gastric carcinoma specimens but not in normal or gastric adenoma tissues. Additional data show that PD-L1 can be regarded as a decisive factor in evaluating gastric carcinoma prognosis and anti-human PD-L1 monoclonal antibody 10E10 could inhibit T-cell apoptosis induced by tumor-associated PD-L1. Taken together, these results showed that the two functional mouse anti-human PD-L1 monoclonal antibodies we generated might be of great value for further exploration of the costimulatory molecule regulating network and immunointervention for tumor immunotherapy.
Assuntos
Anticorpos Bloqueadores/fisiologia , Anticorpos Monoclonais/fisiologia , Antígenos CD/genética , Antígenos CD/imunologia , Carcinoma/metabolismo , Transdução de Sinais/imunologia , Neoplasias Gástricas/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos CD/fisiologia , Antígeno B7-H1 , Carcinoma/imunologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Gástricas/imunologiaRESUMO
Activating mutations in the kinase domain of the EGF receptor have been reported in non-small cell lung cancer. The majority of tumors expressing these mutants are sensitive to ATP mimetics that inhibit the EGFR tyrosine kinase. The effect of antibodies that bind to the ectodomain of the receptor is less clear. We report herein the effects and mechanisms of action of the antibody cetuximab in lung cancer cells that naturally express receptor mutations and in ErbB-null 32D hematopoietic cells transfected with mutant EGFR. Treatment with cetuximab down-regulated EGFR levels and inhibited cell growth both in vitro and in vivo. This was associated with inhibition of ligand-independent EGFR signaling. These effects were seen in 32D cells arguing the growth inhibitory action was not because of the blockade of autocrine ligand action. Both antibody-induced EGFR down-regulation and inhibition of growth required receptor dimerization as monovalent Fab fragments only eliminated receptor levels or reduced cell proliferation in the presence of antihuman IgG. Finally, cetuximab inhibited growth of H1975 lung cancer cells and xenografts, which expressed L858R/T790M EGFR and were resistant to EGFR tyrosine kinase inhibitors. These data suggest that cetuximab is an effective therapy against mutant EGFR-expressing cancer cells and thus can be considered in combination with other anti-EGFR molecules.
Assuntos
Anticorpos Monoclonais/fisiologia , Antineoplásicos/farmacologia , Regulação para Baixo/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Inibidores do Crescimento/fisiologia , Mutagênese , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Bloqueadores/fisiologia , Anticorpos Monoclonais Humanizados , Sítios de Ligação de Anticorpos/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Cetuximab , Regulação para Baixo/imunologia , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Humanos , Ligantes , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Camundongos , Camundongos Nus , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
In HIV infection, dendritic cells (DCs) may play multiple roles, probably including initial HIV uptake in the anogenital mucosa, transport to lymph nodes, and subsequent transfer to T cells. The effects of HIV-1 on DC maturation are controversial, with several recent conflicting reports in the literature. In this study, microarray studies, confirmed by real-time PCR, demonstrated that the genes encoding DC surface maturation markers were among the most differentially expressed in monocyte-derived dendritic cells (MDDCs), derived from human blood, treated with live or aldrithriol-2-inactivated HIV-1(BaL). These effects translated to enhanced cell surface expression of these proteins but differential expression of maturation markers was only partial compared with the effects of a conventional potent maturation stimulus. Such partially mature MDDCs can be converted to fully mature cells by this same potent stimulus. Furthermore, live HIV-1 stimulated greater changes in maturation marker surface expression than aldrithriol-2-inactivated HIV-1 and this enhanced stimulation by live HIV-1 was mediated via CCR5, thus suggesting both viral replication-dependent and -independent mechanisms. These partially mature MDDCs demonstrated enhanced CCR7-mediated migration and are also able to stimulate interacting T cells in a MLR, suggesting DCs harboring HIV-1 might prepare CD4 lymphocytes for transfer of HIV-1. Increased maturation marker surface expression was also demonstrated in native DCs, ex vivo Langerhans cells derived from human skin. Thus, HIV initiates maturation of DCs which could facilitate subsequent enhanced transfer to T cells.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/virologia , HIV-1/imunologia , Células de Langerhans/citologia , Células de Langerhans/virologia , Anticorpos Bloqueadores/fisiologia , Diferenciação Celular/genética , Quimiocina CCL21 , Quimiocinas CC/química , Quimiocinas CC/fisiologia , Quimiotaxia de Leucócito/imunologia , Regulação Viral da Expressão Gênica , Marcadores Genéticos , Inibidores do Crescimento/fisiologia , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Monócitos/imunologia , Monócitos/virologia , Receptores CCR5/imunologia , Ativação Viral/genética , Ativação Viral/imunologiaRESUMO
Several human mucosal fluids are known to possess an innate ability to inhibit human immunodeficiency virus type 1 (HIV-1) infection and replication in vitro. This study compared the HIV-1 inhibitory activities of several mucosal fluids, whole, submandibular/sublingual (sm/sl), and parotid saliva, breast milk, colostrum, seminal plasma, and cervicovaginal secretions, from HIV-1-seronegative donors by using a 3-day microtiter infection assay. A wide range of HIV-1 inhibitory activity was exhibited in all mucosal fluids tested, with some donors exhibiting high levels of activity while others showed significantly lower levels. Colostrum, whole milk, and whole saliva possessed the highest levels of anti-HIV-1 activity, seminal fluid, cervicovaginal secretions, and sm/sl exhibited moderate levels, and parotid saliva consistently demonstrated the lowest levels of HIV-1 inhibition. Fast protein liquid chromatography gel filtration studies revealed the presence of at least three distinct peaks of inhibitory activity against HIV-1 in saliva and breast milk. Incubation of unfractionated and fractionated whole saliva with antibodies raised against human lactoferrin (hLf), secretory leukocyte protease inhibitor (SLPI), and, to a lesser extent, MG2 (high-molecular-weight mucinous glycoprotein) reduced the HIV-1 inhibitory activity significantly. The results suggest that hLf and SLPI are two key components responsible for HIV-1 inhibitory activity in different mucosal secretions. The variation in HIV inhibitory activity between the fluids and between individuals suggests that there may be major differences in susceptibility to HIV infection depending both on the individual and on the mucosal fluid involved.