RESUMO
NK cells are critical innate immune cells that target the tumor cells and cancer-initiating cells and clear viruses by producing cytokines and cytotoxic granules. However, the role of the purinergic receptor P2Y6 in the NK cells remains largely unknown. In this study, we discovered that the expression of P2Y6 was decreased upon the activation of the NK cells. Moreover, in the P2Y6-deficient mice, we found that the deficiency of P2Y6 promoted the development of the NK precursor cells into immature NK and mature NK cells. We also found that the P2Y6 deficiency increased, but the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP decreased the production of IFN-γ in the activated NK cells. Furthermore, we demonstrated that the P2Y6-deficient NK cells exhibited stronger cytotoxicity in vitro and antimetastatic effects in vivo. Mechanistically, P2Y6 deletion promoted the expression of T-bet (encoded by Tbx21), with or without the stimulation of IL-15. In the absence of P2Y6, the levels of phospho-serine/threonine kinase and pS6 in the NK cells were significantly increased upon the stimulation of IL-15. Collectively, we demonstrated that the P2Y6 receptor acted as a negative regulator of the NK cell function and inhibited the maturation and antitumor activities of the NK cells. Therefore, inhibition of the P2Y6 receptor increases the antitumor activities of the NK cells, which may aid in the design of innovative strategies to improve NK cell-based cancer therapy.
Assuntos
Diferenciação Celular/genética , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Melanoma/patologia , Receptores Purinérgicos P2/deficiência , Transdução de Sinais/genética , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Interleucina-15/farmacologia , Neoplasias Pulmonares/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimera por Radiação/imunologia , Receptores Purinérgicos P2/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T/metabolismo , Carga Tumoral/genética , Carga Tumoral/imunologiaRESUMO
Murine bone marrow (BM) chimeras are a versatile and valuable research tool in stem cell and immunology research. Engraftment of donor BM requires myeloablative conditioning of recipients. The most common method used for mice is ionizing radiation, and Cesium-137 gamma irradiators have been preferred. However, radioactive sources are being out-phased worldwide due to safety concerns, and are most commonly replaced by X-ray sources, creating a need to compare these sources regarding efficiency and potential side effects. Prior research has proven both methods capable of efficiently ablating BM cells and splenocytes in mice, but with moderate differences in resultant donor chimerism across tissues. Here, we compared Cesium-137 to 350 keV X-ray irradiation with respect to immune reconstitution, assaying complete, syngeneic BM chimeras and a mixed chimera model of autoimmune disease. Based on dose titration, we find that both gamma and X-ray irradiation can facilitate a near-complete donor chimerism. Mice subjected to 13 Gy Cesium-137 irradiation and reconstituted with syngeneic donor marrow were viable and displayed high donor chimerism, whereas X-ray irradiated mice all succumbed at 13 Gy. However, a similar degree of chimerism as that obtained following 13 Gy gamma irradiation could be achieved by 11 Gy X-ray irradiation, about 85% relative to the gamma dose. In the mixed chimera model of autoimmune disease, we found that a similar autoimmune phenotype could be achieved irrespective of irradiation source used. It is thus possible to compare data generated, regardless of the irradiation source, but every setup and application likely needs individual optimization.
Assuntos
Doenças Autoimunes/imunologia , Células da Medula Óssea/efeitos da radiação , Transplante de Medula Óssea/métodos , Medula Óssea/efeitos da radiação , Radioisótopos de Césio , Raios gama , Quimera por Radiação/imunologia , Animais , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Irradiação Corporal Total , Raios XRESUMO
T cells increase cholesterol biosynthesis upon activation to generate substrates for cellular growth and proliferation. The ubiquitously expressed liver X receptor ß (LXRß) encoded by the Nr1h2 gene is a critical regulator of cholesterol homeostasis in mammalian cells; however, its cell-intrinsic role in T cell biology remains poorly understood. We report that ablation of LXRß in T cells leads to spontaneous T cell activation and T lymphocytopenia. Unexpectedly, analysis of mixed bone marrow chimeric mice revealed a cell-autonomous survival defect that reduced the fitness of LXRß-deficient effector T cells, suggesting that the heightened immune activation in mice harboring LXRß-deficient T cells was due to impaired regulatory T (T reg) cell functionality. Indeed, we found that single-copy deletion of Nr1h2 in T reg cells disrupted activated T reg cell metabolism and fitness and resulted in early-onset fatal autoimmune disease. Our study demonstrated an indispensable requirement for T reg cell-intrinsic LXRß function in immune homeostasis and provides a basis for immunological therapies through targeting of this receptor.
Assuntos
Doenças Autoimunes/imunologia , Homeostase/imunologia , Receptores X do Fígado/fisiologia , Ativação Linfocitária/genética , Linfócitos T Reguladores/imunologia , T-Linfocitopenia Idiopática CD4-Positiva/imunologia , Animais , Doenças Autoimunes/genética , Células Cultivadas , Colesterol/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Homeostase/genética , Receptores X do Fígado/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação/imunologia , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismo , T-Linfocitopenia Idiopática CD4-Positiva/genéticaAssuntos
Coxiella burnetii , Imunidade Inata , Antígeno 96 de Linfócito/metabolismo , Febre Q/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Coxiella burnetii/crescimento & desenvolvimento , Citocinas/genética , Citocinas/metabolismo , Humanos , Imunidade Inata/genética , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Antígeno 96 de Linfócito/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Febre Q/genética , Febre Q/microbiologia , Febre Q/patologia , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Quimera por Radiação/microbiologia , Baço/imunologia , Baço/microbiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
The interferon (IFN) response to viral pathogens is critical for host survival. In humans and mouse models, defects in IFN responses can result in lethal herpes simplex virus 1 (HSV-1) infections, usually from encephalitis. Although rare, HSV-1 can also cause fulminant hepatic failure, which is often fatal. Although herpes simplex encephalitis has been extensively studied, HSV-1 generalized infections and subsequent acute liver failure are less well understood. We previously demonstrated that IFN-αßγR-/- mice are exquisitely susceptible to liver infection following corneal infection with HSV-1. In this study, we used bone marrow chimeras of IFN-αßγR-/- (AG129) and wild-type (WT; 129SvEv) mice to probe the underlying IFN-dependent mechanisms that control HSV-1 pathogenesis. After infection, WT mice with either IFN-αßγR-/- or WT marrow exhibited comparable survival, while IFN-αßγR-/- mice with WT marrow had a significant survival advantage over their counterparts with IFN-αßγR-/- marrow. Furthermore, using bioluminescent imaging to maximize data acquisition, we showed that the transfer of IFN-competent hematopoietic cells controlled HSV-1 replication and damage in the livers of IFN-αßγR-/- mice. Consistent with this, the inability of IFN-αßγR-/- immune cells to control liver infection in IFN-αßγR-/- mice manifested as profoundly elevated aspartate transaminase (AST) and alanine transaminase (ALT) levels, indicative of severe liver damage. In contrast, IFN-αßγR-/- mice receiving WT marrow exhibited only modest elevations of AST and ALT levels. These studies indicate that IFN responsiveness of the immune system is a major determinant of viral tropism and damage during visceral HSV infections. IMPORTANCE: Herpes simplex virus 1 (HSV-1) infection is an incurable viral infection with the most significant morbidity and mortality occurring in neonates and patients with compromised immune systems. Severe pathologies from HSV include the blindness-inducing herpetic stromal keratitis, highly debilitating and lethal herpes simplex encephalitis, and generalized infections that can lead to herpes simplex virus-induced acute liver failure. While immune compromise is a known factor, the precise mechanisms that lead to generalized HSV infections are unknown. In this study, we used and developed a mouse model system in combination with real-time bioluminescence imaging to demonstrate the relative importance of the immune and nonimmune compartments for containing viral spread and promoting host survival after corneal infection. Our results shed light on the pathogenesis of HSV infections that lead to generalized infection and acute liver failure.
Assuntos
Herpes Simples/imunologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Interferons/metabolismo , Falência Hepática Aguda/imunologia , Animais , Modelos Animais de Doenças , Feminino , Herpes Simples/etiologia , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Humanos , Hospedeiro Imunocomprometido , Interferons/deficiência , Interferons/genética , Ceratite Herpética/etiologia , Ceratite Herpética/imunologia , Ceratite Herpética/virologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/etiologia , Falência Hepática Aguda/virologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Quimera por Radiação/imunologia , Receptores de Interferon/deficiência , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Replicação Viral/imunologiaRESUMO
Inflammation in the priming host environment has critical effects on the graft-versus-host (GVH) responses mediated by naive donor T cells. However, it is unclear how a quiescent or inflammatory environment impacts the activity of GVH-reactive primed T and memory cells. We show in this article that GVH-reactive primed donor T cells generated in irradiated recipients had diminished ability compared with naive T cells to increase donor chimerism when transferred to quiescent mixed allogeneic chimeras. GVH-reactive primed T cells showed marked loss of cytotoxic function and activation, and delayed but not decreased proliferation or accumulation in lymphoid tissues when transferred to quiescent mixed chimeras compared with freshly irradiated secondary recipients. Primed CD4 and CD8 T cells provided mutual help to sustain these functions in both subsets. CD8 help for CD4 cells was largely IFN-γ dependent. TLR stimulation after transfer of GVH-reactive primed T cells to mixed chimeras restored their cytotoxic effector function and permitted the generation of more effective T cell memory in association with reduced PD-1 expression on CD4 memory cells. Our data indicate that an inflammatory host environment is required for the maintenance of GVH-reactive primed T cell functions and the generation of memory T cells that can rapidly acquire effector functions. These findings have important implications for graft-versus-host disease and T cell-mediated immunotherapies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Reação Enxerto-Hospedeiro/imunologia , Inflamação/imunologia , Ativação Linfocitária/imunologia , Animais , Apoptose/imunologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD8-Positivos/transplante , Proliferação de Células , Feminino , Memória Imunológica/imunologia , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1/biossíntese , Quimera por Radiação/imunologiaRESUMO
We generated a new humanized mouse model to study HLA-restricted immune responses. For this purpose, we created unique murine hosts by enforcing the expression of human SIRPα by murine phagocytes in murine MHC-deficient HLA-transgenic alymphoid hosts, an approach that allowed the immune reconstitution of nonpermissive mice following injection of human hematopoietic stem cells. We showed that these mouse/human chimeras were able to generate HLA-restricted responses to immunization. These new humanized mice may offer attractive models to study immune responses to human diseases, such as HIV and EBV infections, as well as to assay new vaccine strategies.
Assuntos
Antígenos HLA/administração & dosagem , Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Quimera por Radiação/imunologia , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/administração & dosagem , Antígenos de Diferenciação/sangue , Antígenos de Diferenciação/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Modelos Animais de Doenças , Feminino , Antígenos HLA/genética , Humanos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Quimera por Radiação/genética , Receptores Imunológicos/administração & dosagem , Receptores Imunológicos/sangue , Receptores Imunológicos/genéticaRESUMO
IL12B is required for resistance to Mycobacterium tuberculosis (Mtb) infection, promoting the initiation and maintenance of Mtb-specific effector responses. While this makes the IL12-pathway an attractive target for experimental tuberculosis (TB) therapies, data regarding what lineages express IL12B after infection is established are limited. This is not obvious in the lung, an organ in which both hematopoietic and non-hematopoietic lineages produce IL12p40 upon pathogen encounter. Here, we use radiation bone marrow chimeras and Yet40 reporter mice to determine what lineages produce IL12p40 during experimental TB. We observed that hematopoietic IL12p40-production was sufficient to control Mtb, with no contribution by non-hematopoietic lineages. Furthermore, rather than being produced by a single subset, IL12p40 was produced by cells that were heterogenous in their size, granularity, autofluorescence and expression of CD11c, CD11b and CD8α. While depending on the timepoint and tissue examined, the surface phenotype of IL12p40-producers most closely resembled macrophages based on previous surveys of lung myeloid lineages. Importantly, depletion of CD11c(hi) cells during infection had no affect on lung IL12p40-concentrations. Collectively, our data demonstrate that IL12p40 production is sustained by a heterogenous population of myeloid lineages during experimental TB, and that redundant mechanisms of IL12p40-production exist when CD11c(hi) lineages are absent.
Assuntos
Células da Medula Óssea/imunologia , Subunidade p40 da Interleucina-12/biossíntese , Tuberculose/imunologia , Animais , Carga Bacteriana , Antígeno CD11c/análise , Imunidade Inata , Subunidade p40 da Interleucina-12/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Linfonodos/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/isolamento & purificação , Quimera por Radiação/imunologia , Baço/imunologia , Baço/microbiologia , Tuberculose/microbiologiaRESUMO
Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αß T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Antígenos H-2/fisiologia , Homeostase/imunologia , Imunidade Inata , Memória Imunológica , Ativação Linfocitária/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Antígenos H-2/genética , Hematopoese/genética , Hematopoese/imunologia , Imunofenotipagem , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Quimera por Radiação/imunologia , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
HSV-1 is the leading cause of sporadic viral encephalitis, with mortality rates approaching 30% despite treatment with the antiviral drug of choice, acyclovir. Permanent neurologic deficits are common in patients that survive, but the mechanism leading to this pathology is poorly understood, impeding clinical advancements in treatment to reduce CNS morbidity. Using magnetic resonance imaging and type I IFN receptor-deficient mouse chimeras, we demonstrate HSV-1 gains access to the murine brain stem and subsequently brain ependymal cells, leading to enlargement of the cerebral lateral ventricle and infection of the brain parenchyma. A similar enlargement in the lateral ventricles is found in a subpopulation of herpes simplex encephalitic patients. Associated with encephalitis is an increase in CXCL1 and CXCL10 levels in the cerebral spinal fluid, TNF-α expression in the ependymal region, and the influx of neutrophils of encephalitic mouse brains. Reduction in lateral ventricle enlargement using anti-secretory factor peptide 16 reduces mortality significantly in HSV-1-infected mice without any effect on expression of inflammatory mediators, infiltration of leukocytes, or changes in viral titer. Microglial cells but not infiltrating leukocytes or other resident glial cells or neurons are the principal source of resistance in the CNS during the first 5 d postinfection through a Toll/IL-1R domain-containing adapter inducing IFN-ß-dependent, type I IFN pathway. Our results implicate lateral ventricle enlargement as a major cause of mortality in mice and speculate such an event transpires in a subpopulation of human HSV encephalitic patients.
Assuntos
Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/patologia , Interferon Tipo I/fisiologia , Ventrículos Laterais/imunologia , Ventrículos Laterais/patologia , Microglia/imunologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Encefalite por Herpes Simples/virologia , Predisposição Genética para Doença , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Ventrículos Laterais/virologia , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Microglia/virologia , Dados de Sequência Molecular , Quimera por Radiação/imunologia , Estudos Retrospectivos , Transdução de Sinais/genéticaRESUMO
CD8 T cells expressing memory markers exist in naive mice and are thought to be of heterogeneous origin. It was recently reported that among such memory-phenotype (MP) CD8 T cells in naive mice, those expressing programmed death-1 (PD-1) had immune regulatory activity, but their origin and relationship with other regulatory CD8 T cell subsets remain unclear. In the current study, we examined detailed characteristics and functions of PD-1(+) MP CD8 T cells in naive mice. Their expression pattern of surface molecules resembled that of exhausted CD8 T cells seen in chronic viral infection. However, PD-1(+) MP CD8 T cells were detected from neonatal periods, even in the thymus; thus, they are naturally occurring. By analyzing bone marrow chimera mice in which ß(2)-microglobulin-deficient mice were used as the recipients, it was revealed that PD-1(+) MP CD8 T cells were positively selected by hematopoietic cells, indicating that they belong to nonconventional CD8 T cells. However, in contrast to majority of MP CD8 T cells, PD-1(+) MP CD8 T cells were IL-15 independent. PD-1(+) MP CD8 T cells showed the fastest cell cycling among various T cell subsets in naive mice, which was consistent with the highest sensitivity to cyclophosphamide (CP) treatment. Importantly, PD-1(+) MP CD8 T cells were able to suppress delayed-type hypersensitivity response that was augmented by CP treatment. Taken together, our data indicate that the naturally occurring PD-1(+) MP CD8 T cells in naive mice are a unique subset of nonconventional CD8 T cells and represent the CP-sensitive suppressor CD8 T cells.
Assuntos
Antígenos de Diferenciação/biossíntese , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Ciclofosfamida/farmacologia , Memória Imunológica , Imunofenotipagem , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Animais Recém-Nascidos , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/fisiologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1 , Quimera por Radiação/imunologia , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Nucleic acid-reactive B cells frequently arise in the bone marrow but are tolerized by mechanisms including receptor editing, functional anergy, and/or deletion. TLR9, a sensor of endosomal dsDNA, both promotes and regulates systemic autoimmunity in vivo, but the precise nature of its apparently contradictory roles in autoimmunity remained unclear. In this study, using the 3H9 anti-DNA BCR transgene in the autoimmune-prone MRL.Fas(lpr) mouse model of systemic lupus erythematosus, we identify the stages at which TLR9 contributes to establishing and breaking B cell tolerance. Although TLR9 is dispensable for L chain editing during B cell development in the bone marrow, TLR9 limits anti-DNA B cell life span in the periphery and is thus tolerogenic. In the absence of TLR9, anti-DNA B cells have much longer life spans and accumulate in the follicle, neither activated nor deleted. These cells retain some characteristics of anergic cells, in that they have elevated basal BCR signaling but impaired induced responses and downregulate their cell-surface BCR expression. In contrast, whereas TLR9-intact anergic B cells accumulate near the T/B border, TLR9-deficient anti-DNA B cells are somewhat more dispersed throughout the follicle. Nonetheless, in older autoimmune-prone animals, TLR9 expression specifically within the B cell compartment is required for spontaneous peripheral activation of anti-DNA B cells and their differentiation into Ab-forming cells via an extrafollicular pathway. Thus, TLR9 has paradoxical roles in regulating anti-DNA B cells: it helps purge the peripheral repertoire of autoreactive cells, yet is also required for their activation.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Anergia Clonal/imunologia , DNA/imunologia , Inibidores do Crescimento/fisiologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Receptor Toll-Like 9/fisiologia , Animais , Subpopulações de Linfócitos B/citologia , Anergia Clonal/genética , Inibidores do Crescimento/genética , Humanos , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Camundongos Transgênicos , Quimera por Radiação/imunologia , Receptor Toll-Like 9/genéticaRESUMO
PURPOSE: Severe combined immunodeficiency (SCID) is a syndrome of diverse genetic cause characterized by profound deficiencies of T, B and sometimes NK cell function. Non-ablative HLA-identical or rigorously T cell-depleted haploidentical parental bone marrow transplantation (BMT) results in thymus-dependent genetically donor T cell development in the recipients, leading to a high rate of long-term survival. However, the development of B cell function has been more problematic. We report here results of analyses of B cell function in 125 SCID recipients prior to and long-term after non-ablative BMT, according to their molecular type. METHODS: Studies included blood immunoglobulin measurements; antibody titers to standard vaccines, blood group antigens and bacteriophage Φ X 174; flow cytometry to examine for markers of immaturity, memory, switched memory B cells and BAFF receptor expression; B cell chimerism; B cell spectratyping; and B cell proliferation. RESULTS: The results showed that B cell chimerism was not required for normal B cell function in IL7Rα-Def, ADA-Def and CD3-Def SCIDs. In X-linked-SCID, Jak3-Def SCID and those with V-D-J recombination defects, donor B cell chimerism was necessary for B cell function to develop. CONCLUSION: The most important factor determining whether B cell function develops in SCID T cell chimeras is the underlying molecular defect. In some types, host B cells function normally. In those molecular types where host B cell function did not develop, donor B cell chimerism was necessary to achieve B cell function. 236 words.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/transplante , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/patologia , Adulto , Subpopulações de Linfócitos B/patologia , Transplante de Medula Óssea/métodos , Transformação Celular Viral/imunologia , Células Cultivadas , Criança , Feminino , Humanos , Imunofenotipagem , Lactente , Células Jurkat , Depleção Linfocítica , Transfusão de Linfócitos/métodos , Masculino , Período Pós-Operatório , Quimera por Radiação/imunologia , Imunodeficiência Combinada Severa/cirurgia , Cariotipagem Espectral , Subpopulações de Linfócitos T/patologia , Quimeras de Transplante/imunologia , Células Tumorais CultivadasRESUMO
FcγRIIb is the sole inhibitory FcR for IgG in humans and mice, where it is involved in the negative regulation of Ab production and cellular activation. FcγRIIb-deficient mice show exacerbated disease following the induction of nephrotoxic nephritis (NTN). In this study, we determined the cellular origin of the FcγRIIb-knockout phenotype by inducing NTN in mice with a deficiency of FcγRIIb on either B cells alone (FcγRIIB(fl/fl)/CD19Cre(+)) or myeloid cells (FcγRIIB(fl/fl)/CEBPαCre(+)). Deletion of FcγRIIb from B cells did not increase susceptibility to NTN, compared with wild-type (WT) mice, despite higher Ab titers in the FcγRIIB(fl/fl)/CD19Cre(+) mice compared with the WT littermate controls. In contrast, mice lacking FcγRIIb on myeloid cells had exacerbated disease as measured by increased glomerular thrombosis, glomerular crescents, albuminuria, serum urea, and glomerular neutrophil infiltration when compared with WT littermate controls. The role for FcγRIIb expression on radioresistant intrinsic renal cells in the protection from NTN was then investigated using bone marrow chimeric mice. FcγRIIb(-/-) mice transplanted with FcγRIIb(-/-) bone marrow were more susceptible to NTN than WT mice transplanted with FcγRIIb(-/-) bone marrow, indicating that the presence of WT intrinsic renal cells protects from NTN. These results demonstrate that FcγRIIb on myeloid cells plays a major role in protection from NTN, and therefore, augmentation of FcγRIIb on these cells could be a therapeutic target in human Ab-mediated glomerulonephritis. Where there was a lack of FcγRIIb on circulating myeloid cells, expression of FcγRIIb on intrinsic renal cells provided an additional level of protection from Ab-mediated glomerulonephritis.
Assuntos
Subpopulações de Linfócitos B/imunologia , Glomerulonefrite/imunologia , Glomerulonefrite/prevenção & controle , Rim/imunologia , Células Mieloides/imunologia , Receptores de IgG/fisiologia , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Predisposição Genética para Doença , Glomerulonefrite/patologia , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismo , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Receptores de IgG/deficiência , Receptores de IgG/genéticaRESUMO
Epidermal Langerhans cells (LCs) of the skin represent the prototype migratory dendritic cell (DC) subtype. In the skin, they take up Ag, migrate to the draining lymph nodes, and contribute to Ag transport and immunity. Different depletion models for LCs have revealed contrasting roles and contributions of this cell type. To target the migratory properties of DCs, we generated mice lacking the Rho-family GTPase Cdc42 specifically in DCs. In these animals, the initial seeding of the epidermis with LCs is functional, resulting in slightly reduced Langerhans cell numbers. However, Cdc42-deficient LCs fail to leave the skin in steady state as well as upon stimulation, as they do not enter the skin-draining afferent lymph vessels. Similarly, also other Cdc42-deficient migratory DC subsets fail to home properly to the corresponding draining lymph nodes. We used this novel mouse model, in which LCs are locked out, to demonstrate that these cells contribute substantially to priming of Ag-specific CD4 and CD8 T cell responses upon epicutaneous immunization, but could not detect a role in the induction of contact hypersensitivity to various doses of hapten.
Assuntos
Inibição de Migração Celular/imunologia , Movimento Celular/imunologia , Células de Langerhans/imunologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Animais , Inibição de Migração Celular/genética , Movimento Celular/genética , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Modelos Animais de Doenças , Epiderme/enzimologia , Epiderme/imunologia , Epiderme/patologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Células de Langerhans/enzimologia , Células de Langerhans/patologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Proteína cdc42 de Ligação ao GTP/deficiência , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
C57BL/6 (B6) mice carrying the Sle1b sublocus (named B6.Sle1b), which harbors the lupus-associated NZM2410/NZW SLAM family genes, produce antinuclear Abs (ANAs). However, the role and mechanism(s) involved in the alteration of the germinal center (GC) tolerance checkpoint in the development of ANAs in these mice is not defined. In this study, we show significantly higher spontaneously formed GCs (Spt-GCs) in B6.Sle1b female mice compared with B6 controls. We also found a significant increase in CD4(+)CXCR5(hi)PD-1(hi) spontaneously activated follicular Th cells in B6.Sle1b female mice. Compared with B6 controls, B6.Sle1b female mice had increased numbers of proliferating B cells predominantly located in Spt-GCs. The elevated Spt-GCs in B6.Sle1b female mice were strongly associated with increased ANA-specific Ab-forming cells and ANA titers. The increased numbers of Spt-GCs and spontaneously activated follicular Th cells in B6.Sle1b mice were not the result of a generalized defect in B cells expressing Sle1b. Consistent with the elevated spontaneous response in B6.Sle1b mice, the attenuated GC response characteristic of DNA and p-azophenylarsonate reactive B cells from Ig V(H) knock-in mice (termed HKIR) were relieved in adoptively transferred recipients in the presence of Sle1b. Finally, by generating mixed bone marrow chimeras, we showed that the effect of Sle1b on Spt-GC, follicular Th cell, and autoantibody responses in B6.Sle1b mice was B cell autonomous. These data indicate that the NZM2410/NZW-derived Sle1b sublocus in conjunction with the female sex primarily affects B cells, leading to the alteration of the GC tolerance checkpoint and the generation of ANA-specific Ab-forming cells.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Predisposição Genética para Doença , Centro Germinativo/imunologia , Centro Germinativo/patologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Animais , Anticorpos Antinucleares/biossíntese , Especificidade de Anticorpos/genética , Subpopulações de Linfócitos B/metabolismo , Feminino , Técnicas de Introdução de Genes , Loci Gênicos/imunologia , Centro Germinativo/metabolismo , Tolerância Imunológica/genética , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NZB , Camundongos Knockout , Quimera por Radiação/imunologiaRESUMO
Essential help for long-lived alloantibody responses is theoretically provided only by CD4 T cells that recognize target alloantigen, processed and presented by the allospecific B cell. We demonstrate that in an alloresponse to multiple MHC disparities, cognate help for class-switched alloantibody may also be provided by CD4 T cells specific for a second "helper" alloantigen. This response was much shorter-lived than when help was provided conventionally, by Th cell recognition of target alloantigen. Nevertheless, long-lasting humoral alloimmunity developed when T cell memory against the helper alloantigen was first generated. Costimulatory blockade abrogated alloantibody produced through naive Th cell recognition of target alloantigen but, crucially, blockade was ineffective when help was provided by memory responses to the accessory helper alloantigen. These results suggest that memory Th cell responses against previously encountered graft alloantigen may be the dominant mechanism for providing help to generate new specificities of alloantibody in transplant patients receiving immunosuppression.
Assuntos
Memória Imunológica/imunologia , Isoanticorpos/biossíntese , Linfócitos T Auxiliares-Indutores/imunologia , Transferência Adotiva , Animais , Feminino , Transplante de Coração/imunologia , Transplante de Coração/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Quimera por Radiação/imunologia , Transplante de Pele/imunologia , Transplante de Pele/patologia , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de TempoRESUMO
Thymus-derived CD4(+)Foxp3(+) regulatory T lymphocytes (Tregs) play a central role in the suppression of immune responses to self-antigens and thus avoid autoimmune disorders. It remains unclear if the specialized thymic niche controls the number of differentiating Tregs. We investigated development of murine Tregs from precursors expressing the naturally very large repertoire of TCRs. By analyzing their developmental kinetics, we observed that differentiating Tregs dwell in the thymus â¼1 d longer than their conventional T cell counterparts. By generating hematopoietic chimeras with very low proportions of trackable precursors, we could follow individual waves of developing T cells in the thymus. We observed strongly increased proportions of Tregs at the end of the waves, confirming that these cells are the last to leave the thymus. To assess whether the thymic niche limits Treg development, we generated hematopoietic chimeras in which very few T cell precursors could develop. The substantial increase in the proportion of Tregs we found in these mice suggested a limiting role of the thymic niche; however, this increase was accounted for entirely by the prolonged thymic dwell time of Tregs. We conclude that, when precursors express a naturally diverse TCR repertoire, the thymic niche does not limit differentiation of Tregs.
Assuntos
Diferenciação Celular/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Timo/citologia , Timo/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Corantes Fluorescentes , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Hematopoese/genética , Hematopoese/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Quimera por Radiação/imunologia , Linfócitos T Reguladores/metabolismo , Timo/metabolismoRESUMO
CD4(+) memory T cells are generated in response to infection or vaccination, provide protection to the host against reinfection, and persist through a combination of enhanced survival and slow homeostatic turnover. We used timed deletion of the TCR-signaling adaptor molecule Src homology 2 domain-containing phosphoprotein of 76 kDa (SLP-76) with MHC:peptide tetramers to study the requirements for tonic TCR signals in the maintenance of polyclonal Ag-specific CD4(+) memory T cells. SLP-76-deficient I-A(b):gp61 cells are unable to rapidly generate effector cytokines or proliferate in response to secondary infection. In mice infected with lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes expressing the LCMV gp61-80 peptide, SLP-76-deficient I-A(b):gp61(+) cells exhibit reduced division, similar to that seen in in vitro-generated CD44(hi) and endogenous CD4(+)CD44(hi) cells. Competitive bone marrow chimera experiments demonstrated that the decrease in homeostatic turnover in the absence of SLP-76 is a cell-intrinsic process. Surprisingly, despite the reduction in turnover, I-A(b):gp61(+) Ag-specific memory cells persist in normal numbers for >30 wk after LCMV infection in the absence of SLP-76. These data suggest the independent maintenance of a population of Ag-specific CD4(+) memory T cells in the absence of SLP-76 and normal levels of homeostatic division.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Divisão Celular/imunologia , Epitopos de Linfócito T/imunologia , Homeostase/imunologia , Memória Imunológica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Epitopos de Linfócito T/metabolismo , Homeostase/genética , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Quimera por Radiação/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Donor-specific tolerance induced by mixed chimerism is one approach that may eliminate the need for long-term immunosuppressive therapy, while preventing chronic rejection of an islet transplant. However, even in the presence of chimerism it is possible for certain donor tissues or cells to be rejected whereas others from the same donor are accepted (split tolerance). We previously developed a nonmyeloablative protocol that generated mixed chimerism across full major histocompatability complex plus minor mismatches in NOD (nonobese diabetic) mice, however, these chimeras demonstrated split tolerance. In this study, we used radiation chimeras and found that the radiosensitive component of NOD has a greater role in the split tolerance NOD mice develop. We then show that split tolerance is mediated primarily by preexisting NOD lymphocytes and have identified T cells, but not NK cells or B cells, as cells that both resist chimerism induction and mediate split tolerance. Finally, after recognizing the barrier that preexisting T cells impose on the generation of fully tolerant chimeras, the chimerism induction protocol was refined to include nonmyeloablative recipient NOD T cell depletion which generated long-term mixed chimerism across fully allogeneic barriers. Furthermore, these chimeric NOD mice are immunocompetent, diabetes free and accept donor islet allografts.