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1.
Proc Natl Acad Sci U S A ; 120(50): e2314429120, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38055739

RESUMO

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.


Assuntos
Proteínas de Ligação ao GTP , Linfopenia , Humanos , Proteínas de Ligação ao GTP/metabolismo , Proteômica , Fígado/metabolismo , Linfócitos/metabolismo , Linfopenia/genética , Homeostase
2.
Proc Natl Acad Sci U S A ; 119(18): e2200128119, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35482923

RESUMO

Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cell­intrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)­dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.


Assuntos
Linfócitos T CD8-Positivos , Ribonucleoproteínas , Animais , Linfócitos T CD8-Positivos/metabolismo , Hematopoese/genética , Homozigoto , Mamíferos/metabolismo , Camundongos , Receptores do Fator de Necrose Tumoral/metabolismo , Ribonucleoproteínas/metabolismo , Deleção de Sequência , Fatores de Necrose Tumoral/metabolismo
3.
Nat Commun ; 12(1): 1379, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654074

RESUMO

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Herpesvirus Humano 1/fisiologia , Humanos , Interleucina-6/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Inibidor de NF-kappaB alfa/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo
4.
EMBO J ; 40(9): e104888, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33630350

RESUMO

Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.


Assuntos
Retículo Endoplasmático/metabolismo , Deleção de Genes , Linfopenia/genética , Transtornos Linfoproliferativos/genética , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/genética , Animais , Linfócitos B/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Linfopenia/metabolismo , Transtornos Linfoproliferativos/metabolismo , Masculino , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismo
5.
Elife ; 92020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33350388

RESUMO

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach selecting for defective vocalization in mice, we identified Kdm5a as a candidate ASD gene. To validate our discovery, we generated a Kdm5a knockout mouse model (Kdm5a-/-) and confirmed that inactivating Kdm5a disrupts vocalization. In addition, Kdm5a-/- mice displayed repetitive behaviors, sociability deficits, cognitive dysfunction, and abnormal dendritic morphogenesis. Loss of KDM5A also resulted in dysregulation of the hippocampal transcriptome. To determine if KDM5A mutations cause ASD in humans, we screened whole exome sequencing and microarray data from a clinical cohort. We identified pathogenic KDM5A variants in nine patients with ASD and lack of speech. Our findings illustrate the power and efficacy of forward genetics in identifying ASD genes and highlight the importance of KDM5A in normal brain development and function.


Assuntos
Transtorno do Espectro Autista/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Adolescente , Animais , Pré-Escolar , Feminino , Predisposição Genética para Doença/genética , Técnicas Genéticas , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação
6.
Blood Adv ; 4(15): 3594-3605, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32761233

RESUMO

Adenosine monophosphate deaminase 3 (Ampd3) encodes the erythrocyte isoform of the adenosine monophosphate (AMP) deaminase gene family. Mutations in this gene have been reported in humans, leading to autosomal-recessive erythrocyte AMP deaminase deficiency. However, the mutation is considered clinically asymptomatic. Using N-ethyl-N-nitrosourea mutagenesis to find mutations that affect peripheral lymphocyte populations, we identified 5 Ampd3 mutations (Ampd3guangdong, Ampd3carson, Ampd3penasco, Ampd3taos, and Ampd3commanche) that strongly correlated with a reduction in naive CD4+ T and naive CD8+ T-cell populations. Causation was confirmed by targeted ablation of Ampd3. Knockout mice had reduced frequencies of CD62LhiCD44lo CD4+ naive and CD8+ naive T cells. Interestingly, these phenotypes were restricted to T cells circulating in peripheral blood and were not seen in T cells from secondary lymphoid organs (lymph nodes and spleen). We found that reduction of naive T cells in the peripheral blood of Ampd3-/- mice was caused by T-cell-extrinsic factor(s), which we hypothesize to be elevated levels of adenosine triphosphate released by Ampd3-deficient erythrocytes. These findings provide an example in which disruption of an erythrocyte-specific protein can affect the physiological status of lymphocytes in peripheral blood.


Assuntos
AMP Desaminase , Mutação com Perda de Função , AMP Desaminase/genética , Monofosfato de Adenosina , Animais , Camundongos , Camundongos Knockout , Linfócitos T
7.
Proc Natl Acad Sci U S A ; 117(9): 4894-4901, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32071239

RESUMO

γ-secretase is an intramembrane protease complex that catalyzes the proteolytic cleavage of amyloid precursor protein and Notch. Impaired γ-secretase function is associated with the development of Alzheimer's disease and familial acne inversa in humans. In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for defects in adaptive immunity, we identified animals within a single pedigree exhibiting both hypopigmentation of the fur and diminished T cell-independent (TI) antibody responses. The causative mutation was in Ncstn, an essential gene encoding the protein nicastrin (NCSTN), a member of the γ-secretase complex that functions to recruit substrates for proteolysis. The missense mutation severely limits the glycosylation of NCSTN to its mature form and impairs the integrity of the γ-secretase complex as well as its catalytic activity toward its substrate Notch, a critical regulator of B cell and T cell development. Strikingly, however, this missense mutation affects B cell development but not thymocyte or T cell development. The Ncstn allele uncovered in these studies reveals an essential requirement for NCSTN during the type 2 transitional-marginal zone precursor stage and peritoneal B-1 B cell development, the TI antibody response, fur pigmentation, and intestinal homeostasis in mice.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Subpopulações de Linfócitos B/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Imunidade Adaptativa , Doença de Alzheimer/metabolismo , Animais , Membrana Celular/metabolismo , Etilnitrosoureia/efeitos adversos , Feminino , Hidradenite Supurativa/metabolismo , Humanos , Hipopigmentação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Linhagem , Linfócitos T/metabolismo , Transcriptoma
8.
J Exp Med ; 217(4)2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-31985756

RESUMO

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.


Assuntos
Linfócitos/imunologia , Células Mieloides/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Animais , Fator Ativador de Células B/imunologia , Linhagem Celular , Feminino , Células HEK293 , Hematopoese/imunologia , Humanos , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Proteína Wnt3A/imunologia
9.
Sci Immunol ; 5(43)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980486

RESUMO

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell-dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2-/- spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2-/- CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Animais , Linfócitos B/imunologia , Transplante de Medula Óssea , Diferenciação Celular , Feminino , Centro Germinativo/imunologia , Células HEK293 , Humanos , Imunoglobulinas/sangue , Imunoterapia Adotiva , Masculino , Camundongos Transgênicos , Mutação , Proteína Quinase D2 , Proteínas Quinases/genética
10.
Dis Model Mech ; 11(9)2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30279225

RESUMO

Myosin ID (MYO1D) is a member of the class I myosin family. We screened 48,649 third generation (G3) germline mutant mice derived from N-ethyl-N-nitrosourea-mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). We found and validated mutations in Myo1d as a cause of increased susceptibility to DSS-induced colitis. MYO1D is produced in the intestinal epithelium, and the colitis phenotype is dependent on the nonhematopoietic compartment of the mouse. Moreover, MYO1D appears to couple cytoskeletal elements to lipid in an ATP-dependent manner. These findings demonstrate that MYO1D is needed to maintain epithelial integrity and protect against DSS-induced colitis.


Assuntos
Colite/metabolismo , Colite/prevenção & controle , Lipídeos de Membrana/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Diferenciação Celular , Colite/genética , Colite/patologia , Sulfato de Dextrana , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Genes Recessivos , Hematopoese , Mucosa Intestinal/patologia , Intestinos/patologia , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , Reprodutibilidade dos Testes
11.
Proc Natl Acad Sci U S A ; 115(37): E8698-E8706, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30150374

RESUMO

Successful cancer immunotherapy entails activation of innate immune receptors to promote dendritic cell (DC) maturation, antigen presentation, up-regulation of costimulatory molecules, and cytokine secretion, leading to activation of tumor antigen-specific cytotoxic T lymphocytes (CTLs). Here we screened a synthetic library of 100,000 compounds for innate immune activators using TNF production by THP-1 cells as a readout. We identified and optimized a potent human and mouse Toll-like receptor (TLR)1/TLR2 agonist, Diprovocim, which exhibited an EC50 of 110 pM in human THP-1 cells and 1.3 nM in primary mouse peritoneal macrophages. In mice, Diprovocim-adjuvanted ovalbumin immunization promoted antigen-specific humoral and CTL responses and synergized with anti-PD-L1 treatment to inhibit tumor growth, generating long-term antitumor memory, curing or prolonging survival of mice engrafted with the murine melanoma B16-OVA. Diprovocim induced greater frequencies of tumor-infiltrating leukocytes than alum, of which CD8 T cells were necessary for the antitumor effect of immunization plus anti-PD-L1 treatment.


Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Melanoma Experimental/terapia , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Sinergismo Farmacológico , Humanos , Imunoterapia/métodos , Estimativa de Kaplan-Meier , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Células THP-1 , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo
12.
Nat Commun ; 9(1): 441, 2018 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-29382827

RESUMO

Computational inference of mutation effects is necessary for genetic studies in which many mutations must be considered as etiologic candidates. Programs such as PolyPhen-2 predict the relative severity of damage caused by missense mutations, but not the actual probability that a mutation will reduce/eliminate protein function. Based on genotype and phenotype data for 116,330 ENU-induced mutations in the Mutagenetix database, we calculate that putative null mutations, and PolyPhen-2-classified "probably damaging", "possibly damaging", or "probably benign" mutations have, respectively, 61%, 17%, 9.8%, and 4.5% probabilities of causing phenotypically detectable damage in the homozygous state. We use these probabilities in the estimation of genome saturation and the probability that individual proteins have been adequately tested for function in specific genetic screens. We estimate the proportion of essential autosomal genes in Mus musculus (C57BL/6J) and show that viable mutations in essential genes are more likely to induce phenotype than mutations in non-essential genes.


Assuntos
Algoritmos , Bases de Dados Genéticas , Etilnitrosoureia/toxicidade , Mutação , Proteínas/genética , Alelos , Animais , Genes Essenciais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/genética , Probabilidade
13.
Exp Parasitol ; 134(3): 389-99, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23541881

RESUMO

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.


Assuntos
Interferon gama/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Óxido Nítrico/metabolismo , Toxoplasma/enzimologia , Toxoplasmose Animal/parasitologia , Animais , Linhagem Celular , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Fígado/parasitologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Baço/parasitologia , Toxoplasma/genética , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose Animal/imunologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/fisiologia
14.
Cancer Res ; 72(8): 2089-99, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22496463

RESUMO

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.


Assuntos
Envelhecimento/imunologia , Imunoterapia/métodos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Toxina Diftérica/uso terapêutico , Modelos Animais de Doenças , Citometria de Fluxo , Interleucina-2/uso terapêutico , Depleção Linfocítica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T Reguladores/imunologia
15.
J Immunol ; 185(5): 2747-53, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20686128

RESUMO

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.


Assuntos
Antígeno B7-1/fisiologia , Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Glicoproteínas de Membrana/fisiologia , Peptídeos/fisiologia , Caracteres Sexuais , Animais , Antígenos de Diferenciação/fisiologia , Antígeno B7-1/genética , Antígeno B7-H1 , Linhagem Celular Tumoral , Feminino , Imunidade Inata/genética , Masculino , Melanoma Experimental/fisiopatologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/biossíntese , Ovalbumina/genética , Ovalbumina/imunologia , Peptídeos/deficiência , Peptídeos/genética , Receptor de Morte Celular Programada 1 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia
16.
J Immunol ; 184(11): 6151-60, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20427766

RESUMO

Both innate and adaptive immune systems are considered important for cancer prevention, immunosurveillance, and control of cancer progression. It is known that, although both systems initially eliminate emerging tumor cells efficiently, tumors eventually escape immune attack by a variety of mechanisms, including differentiation and recruitment of immunosuppressive CD11b(+)Gr-1(+) myeloid suppressor cells into the tumor microenvironment. However, we show that CD11b(+)Gr-1(+) cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover, like dendritic cells, immunostimulatory CD11b(+)Gr-1(+) cells can strongly cross-prime, augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b(+)Gr-1(+) cells from ascites of ovarian cancer-bearing mice results in the significant regression of s.c. tumors even without being pulsed with exogenous tumor Ag prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b(+)Gr-1(+)) cells, thereby mediating highly effective antitumor immunity in an adoptive transfer model of immunity.


Assuntos
Antígeno CD11b/imunologia , Apresentação Cruzada/imunologia , Células Mieloides/imunologia , Neoplasias Ovarianas/imunologia , Receptores de Quimiocinas/imunologia , Transferência Adotiva , Animais , Antígenos/imunologia , Separação Celular , Feminino , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia
17.
Mol Cell Biol ; 26(23): 8976-83, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17000766

RESUMO

The functions of molecular chaperones have been extensively investigated biochemically in vitro and genetically in bacteria and yeast. We have embarked on a functional genomic analysis of the Hsp90 chaperone machine in the mouse by disrupting the p23 gene using a gene trap approach. p23 is an Hsp90 cochaperone that is thought to stabilize Hsp90-substrate complexes and, independently, to act as the cytosolic prostaglandin E2 synthase. Gene deletions in budding and fission yeasts and knock-down experiments with the worm have not revealed any clear in vivo requirements for p23. We find that p23 is not essential for overall prenatal development and morphogenesis of the mouse, which parallels the observation that it is dispensable for proliferation in yeast. In contrast, p23 is absolutely necessary for perinatal survival. Apart from an incompletely formed skin barrier, the lungs of p23 null embryos display underdeveloped airspaces and substantially reduced expression of surfactant genes. Correlating with the known function of glucocorticoids in promoting lung maturation and the role of p23 in the assembly of a hormone-responsive glucocorticoid receptor-Hsp90 complex, p23 null fibroblast cells have a defective glucocorticoid response. Thus, p23 contributes a nonredundant, temporally restricted, and tissue-specific function during mouse development.


Assuntos
Animais Recém-Nascidos , Proteínas de Choque Térmico HSP90/fisiologia , Chaperonas Moleculares/fisiologia , Fosfoproteínas/fisiologia , Animais , Linhagem Celular Transformada , Transformação Celular Viral , Fibroblastos/metabolismo , Deleção de Genes , Proteínas de Choque Térmico HSP90/metabolismo , Oxirredutases Intramoleculares , Pulmão/embriologia , Pulmão/ultraestrutura , Camundongos , Camundongos Knockout , Camundongos Mutantes , Chaperonas Moleculares/genética , Mutagênese Insercional , Fosfoproteínas/genética , Prostaglandina-E Sintases , Receptores de Glucocorticoides/análise , Pele/embriologia , Pele/ultraestrutura
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