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1.
Mol Biol Cell ; : mbcE20020111, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33206585

RESUMO

We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding extracellular matrix (ECM) proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed upon silencing this gene. Aberrant ciliation could also be suppressed through hyper-activation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the extracellular matrix and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.

2.
Cancers (Basel) ; 12(8)2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32823814

RESUMO

T-cell exhaustion is a phenomenon that represents the dysfunctional state of T cells in chronic infections and cancer and is closely associated with poor prognosis in many cancers. The endogenous T-cell immunity and genetically edited cell therapies (CAR-T) failed to prevent tumor immune evasion. The effector T-cell activity is perturbed by an imbalance between inhibitory and stimulatory signals causing a reprogramming in metabolism and the high levels of multiple inhibitory receptors like programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and Lymphocyte-activation gene 3 (Lag-3). Despite the efforts to neutralize inhibitory receptors by a single agent or combinatorial immune checkpoint inhibitors to boost effector function, PDAC remains unresponsive to these therapies, suggesting that multiple molecular mechanisms play a role in stimulating the exhaustion state of tumor-infiltrating T cells. Recent studies utilizing transcriptomics, mass cytometry, and epigenomics revealed a critical role of Thymocyte selection-associated high mobility group box protein (TOX) genes and TOX-associated pathways, driving T-cell exhaustion in chronic infection and cancer. Here, we will review recently defined molecular, genetic, and cellular factors that drive T-cell exhaustion in PDAC. We will also discuss the effects of available immune checkpoint inhibitors and the latest clinical trials targeting various molecular factors mediating T-cell exhaustion in PDAC.

3.
J Transl Med ; 18(1): 314, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32799889

RESUMO

BACKGROUND: Lightheadedness, fatigue, weakness, heart palpitations, cognitive dysfunction, muscle pain, and exercise intolerance are some of the symptoms of orthostatic intolerance (OI). There is substantial comorbidity of OI in ME/CFS (Myalgic Encephalomyelitis/Chronic Fatigue Syndrome). The 10-minute NASA Lean Test (NLT) is a simple, point-of-care method that can aid ME/CFS diagnosis and guide management and treatment of OI. The objective of this study was to understand the hemodynamic changes that occur in ME/CFS patients during the 10-minute NLT. METHODS: A total of 150 ME/CFS patients and 75 age, gender and race matched healthy controls (HCs) were enrolled. We recruited 75 ME/CFS patients who had been sick for less than 4 years (< 4 ME/CFS) and 75 ME/CFS patients sick for more than 10 years (> 10 ME/CFS). The 10-minute NLT involves measurement of blood pressure and heart rate while resting supine and every minute for 10 min while standing with shoulder-blades on the wall for a relaxed stance. Spontaneously reported symptoms are recorded during the test. ANOVA and regression analysis were used to test for differences and relationships in hemodynamics, symptoms and upright activity between groups. RESULTS: At least 5 min of the 10-minute NLT were required to detect hemodynamic changes. The < 4 ME/CFS group had significantly higher heart rate and abnormally narrowed pulse pressure compared to > 10 ME/CFS and HCs. The < 4 ME/CFS group experienced significantly more OI symptoms compared to > 10 ME/CFS and HCs. The circulatory decompensation observed in the < 4 ME/CFS group was not related to age or medication use. CONCLUSIONS: Circulatory decompensation characterized by increased heart rate and abnormally narrow pulse pressure was identified in a subgroup of ME/CFS patients who have been sick for < 4 years. This suggests inadequate ventricular filling from low venous pressure. The 10-minute NLT can be used to diagnose and treat the circulatory decompensation in this newly recognized subgroup of ME/CFS patients. The > 10 ME/CFS group had less pronounced hemodynamic changes during the NLT possibly from adaptation and compensation that occurs over time. The 10-minute NLT is a simple and clinically useful point-of-care method that can be used for early diagnosis of ME/CFS and help guide OI treatment.

4.
EMBO Mol Med ; 12(8): e11592, 2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609955

RESUMO

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell-specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases.

5.
Nature ; 583(7816): 447-452, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499651

RESUMO

Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52-7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.


Assuntos
Cromossomos Humanos Par 11/genética , Colite/genética , Colite/imunologia , Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença/genética , Linfócitos T Reguladores/imunologia , Acetilação , Alelos , Animais , Cromossomos de Mamíferos/genética , Feminino , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Sintenia/genética
6.
Mol Cell Biol ; 40(12)2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32229578

RESUMO

The LMO2/LDB1 macromolecular complex is critical in hematopoietic stem and progenitor cell specification and in the development of acute leukemia. This complex is comprised of core subunits of LMO2 and LDB1 as well as single-stranded DNA-binding protein (SSBP) cofactors and DNA-binding basic helix-loop-helix (bHLH) and GATA transcription factors. We analyzed the steady-state abundance and kinetic stability of LMO2 and its partners via Halo protein tagging in conjunction with variant proteins deficient in binding their respective direct protein partners. We discovered a hierarchy of protein stabilities (with half-lives in descending order) as follows: LDB1 > SSBP > LMO2 > TAL1. Importantly, LDB1 is a remarkably stable protein that confers enhanced stability upon direct and indirect partners, thereby nucleating the formation of the multisubunit protein complex. The data imply that free subunits are more rapidly degraded than those incorporated within the LMO2/LDB1 complex. Our studies provided significant insights into LMO2/LDB1 macromolecular protein complex assembly and stability, which has implications for understanding its role in blood cell formation and for therapeutically targeting this complex in human leukemias.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas com Domínio LIM/metabolismo , Leucemia/metabolismo , Proteínas Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Ubiquitina/metabolismo
7.
Nature ; 579(7798): 260-264, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132711

RESUMO

The production of pore-forming toxins that disrupt the plasma membrane of host cells is a common virulence strategy for bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA)1-3. It is unclear, however, whether host species possess innate immune mechanisms that can neutralize pore-forming toxins during infection. We previously showed that the autophagy protein ATG16L1 is necessary for protection against MRSA strains encoding α-toxin4-a pore-forming toxin that binds the metalloprotease ADAM10 on the surface of a broad range of target cells and tissues2,5,6. Autophagy typically involves the targeting of cytosolic material to the lysosome for degradation. Here we demonstrate that ATG16L1 and other ATG proteins mediate protection against α-toxin through the release of ADAM10 on exosomes-extracellular vesicles of endosomal origin. Bacterial DNA and CpG DNA induce the secretion of ADAM10-bearing exosomes from human cells as well as in mice. Transferred exosomes protect host cells in vitro by serving as scavengers that can bind multiple toxins, and improve the survival of mice infected with MRSA in vivo. These findings indicate that ATG proteins mediate a previously unknown form of defence in response to infection, facilitating the release of exosomes that serve as decoys for bacterially produced toxins.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Toxinas Bacterianas/metabolismo , Exossomos/metabolismo , Células A549 , Proteína ADAM10/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Sobrevivência Celular/efeitos dos fármacos , DNA Bacteriano/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estafilocócicas/mortalidade
8.
J Biol Chem ; 295(18): 5944-5959, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32179646

RESUMO

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton-Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

10.
Immunity ; 49(5): 873-885.e7, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30366765

RESUMO

Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin-17A-mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on RORα transcription factor and interleukin-1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell-permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell-intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses.


Assuntos
Diferenciação Celular/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Células Th17/citologia , Células Th17/metabolismo , Animais , Aterosclerose , Biomarcadores , Domínio de Ativação e Recrutamento de Caspases , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/mortalidade , Expressão Gênica , Imunofenotipagem , Inflamação/genética , Inflamação/metabolismo , Interleucina-17/biossíntese , Interleucina-1beta , Camundongos , Camundongos Knockout , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
11.
Front Immunol ; 9: 1901, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30197641

RESUMO

Background: T follicular helper (Tfh) cells are crucial for B cell differentiation and antigen-specific antibody production. Dysregulation of Tfh-mediated B cell help weakens B cell responses in HIV infection. Moreover, Tfh cells in the lymph node and peripheral blood comprise a significant portion of the latent HIV reservoir. There is limited data on the effects of perinatal HIV infection on Tfh cells in children. We examined peripheral Tfh (pTfh) cell frequencies and phenotype in HIV-infected children and their associations with disease progression, immune activation, and B cell differentiation. Methods: In a Kenyan cohort of 76 perinatally HIV-infected children, comprised of 43 treatment-naïve (ART-) and 33 on antiretroviral therapy (ART+), and 42 healthy controls (HIV-), we identified memory pTfh cells, T cell activation markers, and B cell differentiation states using multi-parameter flow cytometry. Soluble CD163 and intestinal fatty acid-binding protein plasma levels were quantified by ELISA. Results: ART- children had reduced levels of pTfh cells compared with HIV- children that increased with antiretroviral therapy. HIV+ children had higher programmed cell death protein 1 (PD-1) expression on pTfh cells, regardless of treatment status. Low memory pTfh cells with elevated PD-1 levels correlated with advancing HIV disease status, indicated by increasing HIV viral loads and T cell and monocyte activation, and decreasing %CD4 and CD4:CD8 ratios. Antiretroviral treatment, particularly when started at younger ages, restored pTfh cell frequency and eliminated correlations with disease progression, but failed to lower PD-1 levels on pTfh cells and their associations with CD4 T cell percentages and activation. Altered B cell subsets, with decreased naïve and resting memory B cells and increased activated and tissue-like memory B cells in HIV+ children, correlated with low memory pTfh cell frequencies. Last, HIV+ children had decreased proportions of CXCR5+ CD8 T cells that associated with low %CD4 and CD4:CD8 ratios. Conclusion: Low memory pTfh cell frequencies with high PD-1 expression in HIV+ children correlate with worsening disease status and an activated and differentiated B cell profile. This perturbed memory pTfh cell population may contribute to weak vaccine and HIV-specific antibody responses in HIV+ children. Restoring Tfh cell capacity may be important for novel pediatric HIV cure and vaccine strategies.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , Linfócitos T Auxiliares-Indutores/imunologia , Adolescente , Terapia Antirretroviral de Alta Atividade , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Biomarcadores , Contagem de Linfócito CD4 , Criança , Pré-Escolar , Progressão da Doença , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Humanos , Memória Imunológica , Imunofenotipagem , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Ativação Linfocitária/imunologia , Masculino , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Carga Viral
12.
Mucosal Immunol ; 11(6): 1591-1605, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30115998

RESUMO

Human mucosal-associated invariant T (MAIT) cell receptors (TCRs) recognize bacterial riboflavin pathway metabolites through the MHC class 1-related molecule MR1. However, it is unclear whether MAIT cells discriminate between many species of the human microbiota. To address this, we developed an in vitro functional assay through human T cells engineered for MAIT-TCRs (eMAIT-TCRs) stimulated by MR1-expressing antigen-presenting cells (APCs). We then screened 47 microbiota-associated bacterial species from different phyla for their eMAIT-TCR stimulatory capacities. Only bacterial species that encoded the riboflavin pathway were stimulatory for MAIT-TCRs. Most species that were high stimulators belonged to Bacteroidetes and Proteobacteria phyla, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. Activation of MAIT cells by high- vs low-stimulating bacteria also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. Remarkably, we found that human T-cell subsets can also present riboflavin metabolites to MAIT cells in a MR1-restricted fashion. This T-T cell-mediated signaling also induced IFNγ, TNF and granzyme B from MAIT cells, albeit at lower level than professional APC. These findings suggest that MAIT cells can discriminate and categorize complex human microbiota through computation of TCR signals depending on antigen load and presenting cells, and fine-tune their functional responses.


Assuntos
Bacteroidetes/imunologia , Macrófagos/imunologia , Microbiota/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Proteobactérias/imunologia , Riboflavina/metabolismo , Apresentação do Antígeno , Antígenos de Bactérias/imunologia , Células Cultivadas , Engenharia Genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Macrófagos/microbiologia , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariáveis Associadas à Mucosa/microbiologia , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
13.
JCO Precis Oncol ; 20182018.
Artigo em Inglês | MEDLINE | ID: mdl-30079384

RESUMO

Purpose: The promise of precision oncology is that identification of genomic alterations will direct the rational use of molecularly targeted therapy. This approach is particularly applicable to neoplasms that are resistant to standard cytotoxic chemotherapy, like T-cell leukemias and lymphomas. In this study, we tested the feasibility of targeted next-generation sequencing in profiles of diverse T-cell neoplasms and focused on the therapeutic utility of targeting activated JAK1 and JAK3 in an index case. Patients and Methods: Using Foundation One and Foundation One Heme assays, we performed genomic profiling on 91 consecutive T-cell neoplasms for alterations in 405 genes. The samples were sequenced to high uniform coverage with an Illumina HiSeq and averaged a coverage depth of greater than 500× for DNA and more than 8M total pairs for RNA. An index case of T-cell prolymphocytic leukemia (T-PLL), which was analyzed by targeted next-generation sequencing, is presented. T-PLL cells were analyzed by RNA-seq, in vitro drug testing, mass cytometry, and phospho-flow. Results: One third of the samples had genomic aberrations in the JAK-STAT pathway, most often composed of JAK1 and JAK3 gain-of-function mutations. We present an index case of a patient with T-PLL with a clonal JAK1 V658F mutation that responded to ruxolitinib therapy. After relapse developed, an expanded clone that harbored mutant JAK3 M511I and downregulation of the phosphatase, CD45, was identified. We demonstrate that the JAK missense mutations were activating, caused pathway hyperactivation, and conferred cytokine hypersensitivity. Conclusion: These results underscore the utility of profiling occurrences of resistance to standard regimens and support JAK enzymes as rational therapeutic targets for T-cell leukemias and lymphomas.

14.
J Immunol ; 201(5): 1586-1598, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30021769

RESUMO

Developing precise and efficient gene editing approaches using CRISPR in primary human T cell subsets would provide an effective tool in decoding their functions. Toward this goal, we used lentiviral CRISPR/Cas9 systems to transduce primary human T cells to stably express the Cas9 gene and guide RNAs that targeted either coding or noncoding regions of genes of interest. We showed that multiple genes (CD4, CD45, CD95) could be simultaneously and stably deleted in naive, memory, effector, or regulatory T cell (Treg) subsets at very high efficiency. Additionally, nuclease-deficient Cas9, associated with a transcriptional activator or repressor, can downregulate or increase expression of genes in T cells. For example, expression of glycoprotein A repetitions predominant (GARP), a gene that is normally and exclusively expressed on activated Tregs, could be induced on non-Treg effector T cells by nuclease-deficient Cas9 fused to transcriptional activators. Further analysis determined that this approach could be used in mapping promoter sequences involved in gene transcription. Through this CRISPR/Cas9-mediated genetic editing we also demonstrated the feasibility of human T cell functional analysis in several examples: 1) CD95 deletion inhibited T cell apoptosis upon reactivation; 2) deletion of ORAI1, a Ca2+ release-activated channel, abolished Ca2+ influx and cytokine secretion, mimicking natural genetic mutations in immune-deficient patients; and 3) transcriptional activation of CD25 or CD127 expression enhanced cytokine signaling by IL-2 or IL-7, respectively. Taken together, application of the CRISPR toolbox to human T cell subsets has important implications for decoding the mechanisms of their functional outputs.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Linfócitos T Reguladores/imunologia , Feminino , Humanos , Masculino
15.
J Allergy Clin Immunol ; 142(4): 1297-1310.e11, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29155098

RESUMO

BACKGROUND: Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels is an essential signaling pathway in many cell types. Ca2+ release-activated Ca2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. OBJECTIVE: We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. METHODS: We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. RESULTS: We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. CONCLUSION: ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling pathway (IKBKG and NFKBIA).


Assuntos
Displasia Ectodérmica/genética , Síndromes de Imunodeficiência/genética , Proteína ORAI1/genética , Cálcio/metabolismo , Células Cultivadas , Pré-Escolar , Evolução Fatal , Feminino , Humanos , Lactente , Masculino , Modelos Moleculares , Mutação
16.
Breast Cancer Res Treat ; 168(1): 57-67, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29124456

RESUMO

BACKGROUND AND PURPOSE: Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant challenge. Prior studies have shown that low-dose oral cyclophosphamide can transiently deplete regulatory T cells (Tregs) and improve anti-tumor immunity. We investigated the combination of exemestane with cyclophosphamide in patients with advanced HR-positive breast cancer and assessed changes in circulating immune cell subsets. METHODS: This was a single-arm phase II trial of exemestane with cyclophosphamide in patients with metastatic HR-positive/HER2-negative breast cancer who had progressed on prior endocrine therapy (ClinicalTrials.gov: NCT01963481). Primary endpoint was progression-free survival (PFS) at 3 months (RECIST 1.1). Secondary objectives included median PFS, objective response rate, duration of response, and safety. Circulating Tregs (FOXP3+Helios+) and other immune cell subsets were monitored during treatment and compared with healthy controls. RESULTS: Twenty-three patients were enrolled. Treatment was well tolerated, without grade 4/5 toxicities. Objective responses were seen in 6/23 patients (26.1%; 95% CI 10.2-48.4%) and were durable (median 11.6 months). Three-month PFS rate was 50.1% (95% CI 33.0-76.0%); median PFS was 4.23 months (95% CI 2.8-11.7). No treatment-related decrease in Tregs was observed. However, elevated baseline levels of Naïve Tregs [greater than 2.5 (the median of the naïve Tregs)] were associated with relative risk of disease progression or death [hazard ratio 11.46 (95% CI 2.32-56.5)]. In addition, the baseline levels of Naïve Tregs (adj-p = 0.04), Memory Tregs (adj-p = 0.003), CD4 + Central Memory T cells (adj-p = 0.0004), PD-1 + CD4 + Central Memory T cells (adj-p = 0.008), and PD-1 + CD4 + Effector Memory T cells (adj-p = 0.009) were significantly greater in the patients than in the healthy controls; the baseline levels of  %CD4 + Naïve T cells (adj-p = 0.0004) were significantly lower in patients compared with healthy controls (n = 40). CONCLUSION: Treg depletion was not observed with low-dose cyclophosphamide when assessed by the specific marker FOXP3 + Helios +; however, baseline naïve Tregs were associated with 3-month PFS. Exemestane/cyclophosphamide combination had favorable safety profile with evidence of clinical activity in heavily pretreated patients.


Assuntos
Androstadienos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Ciclofosfamida/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Administração Oral , Adulto , Idoso , Androstadienos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/imunologia , Mama/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Ciclofosfamida/uso terapêutico , Progressão da Doença , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Humanos , Pessoa de Meia-Idade , Pós-Menopausa , Intervalo Livre de Progressão , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Linfócitos T Reguladores/imunologia
17.
J Infect Dis ; 216(6): 641-650, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28934428

RESUMO

Background: During human immunodeficiency virus (HIV) disease, chronic immune activation leads to T-cell exhaustion. PD-1 identifies "exhausted" CD8 T cells with impaired HIV-specific effector functions, but its role on CD4 T cells and in HIV-infected children is poorly understood. Methods: In a Kenyan cohort of vertically HIV-infected children, we measured PD-1+ CD4 T-cell frequencies and phenotype by flow cytometry and their correlation with HIV disease progression and immune activation. Second, in vitro CD4 T-cell proliferative and cytokine responses to HIV-specific and -nonspecific stimuli were assessed with and without PD-1 blockade. Results: HIV-infected children have increased frequencies of PD-1+ memory CD4 T cells that fail to normalize with antiretroviral treatment. These cells are comprised of central and effector memory subsets and correlate with HIV disease progression, measured by viral load, CD4 percentage, CD4:CD8 T-cell ratio, and immune activation. Last, PD-1+ CD4 T cells predict impaired proliferative potential yet preferentially secrete the Th1 and Th17 cytokines interferon-γ and interleukin 17A, and are unresponsive to in vitro PD-1 blockade. Conclusions: This study highlights differences in PD-1+ CD4 T-cell memory phenotype and response to blockade between HIV-infected children and adults, with implications for potential immune checkpoint therapies.


Assuntos
Linfócitos T CD4-Positivos/citologia , Citocinas/imunologia , Infecções por HIV/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Adolescente , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Proliferação de Células , Criança , Pré-Escolar , Estudos de Coortes , Progressão da Doença , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Transmissão Vertical de Doença Infecciosa , Quênia , Masculino , RNA Viral/genética , Carga Viral
18.
PLoS One ; 12(3): e0173329, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28278197

RESUMO

BACKGROUND: Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. OBJECTIVES: To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. METHODS: Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. RESULTS: Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. CONCLUSIONS: Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.


Assuntos
Plaquetas/metabolismo , Células Endoteliais/metabolismo , Hemostasia , Proteínas de Membrana/metabolismo , Trombose/metabolismo , Animais , Colágeno/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Ativação Plaquetária , Adesividade Plaquetária , Agregação Plaquetária , Receptor TIE-2/metabolismo , Trombose/genética , Trombose/patologia , Trombose/fisiopatologia
19.
PLoS One ; 11(12): e0168135, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27942037

RESUMO

Gaucher disease (GD) patients often present with abnormalities in immune response that may be the result of alterations in cellular and/or humoral immunity. However, how the treatment and clinical features of patients impact the perturbation of their immunological status remains unclear. To address this, we assessed the immune profile of 26 GD patients who were part of an enzyme replacement therapy (ERT) study. Patients were evaluated clinically for onset of GD symptoms, duration of therapy and validated outcome measures for ERT. According to DS3 disease severity scoring system criteria, they were assigned to have mild, moderate or severe GD. Flow cytometry based immunophenotyping was performed to analyze subsets of T, B, NK, NKT and dendritic cells. GD patients showed multiple types of immune abnormalities associated to T and B lymphocytes with respect to their subpopulations as well as memory and activation markers. Skewing of CD4 and CD8 T cell numbers resulting in lower CD4/CD8 ratio and an increase in overall T cell activation were observed. A decrease in the overall B cells and an increase in NK and NKT cells were noted in the GD patients compared to controls. These immune alterations do not correlate with GD clinical type or level of biomarkers. However, subjects with persistent immune alterations, especially in B cells and DCs correlate with longer delay in initiation of ERT (ΔTX). Thus, while ERT may reverse some of these immune abnormalities, the immune cell alterations become persistent if therapy is further delayed. These findings have important implications in understanding the immune disruptions before and after treatment of GD patients.


Assuntos
Terapia de Reposição de Enzimas , Doença de Gaucher/imunologia , Doença de Gaucher/terapia , Tempo para o Tratamento , Adolescente , Adulto , Idoso , Criança , Feminino , Citometria de Fluxo , Doença de Gaucher/fisiopatologia , Humanos , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Adulto Jovem
20.
PLoS One ; 11(8): e0161786, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27560150

RESUMO

Mucosal-associated invariant T cells (MAIT) are innate T cells restricted by major histocompatibility related molecule 1 (MR1) presenting riboflavin metabolite ligands derived from microbes. Specificity to riboflavin metabolites confers MAIT cells a broad array of host-protective activity against gram-negative and -positive bacteria, mycobacteria, and fungal pathogens. MAIT cells are present at low levels in the peripheral blood of neonates and gradually expand to relatively abundant levels during childhood. Despite no anti-viral activity, MAIT cells are depleted early and irreversibly in HIV infected adults. Such loss or impaired expansion of MAIT cells in HIV-positive children may render them more susceptible to common childhood illnesses and opportunistic infections. In this study we evaluated the frequency of MAIT cells in perinatally HIV-infected children, their response to antiretroviral treatment and their associations with HIV clinical status and related innate and adaptive immune cell subsets with potent antibacterial effector functions. We found HIV+ children between ages 3 to 18 years have significantly decreased CD8+ MAIT cell frequencies compared to uninfected healthy children. Remarkably, CD8 MAIT levels gradually increased with antiretroviral therapy, with greater recovery when treatment is initiated at a young age. Moreover, diminished CD8+ MAIT cell frequencies are associated with low CD4:CD8 ratios and elevated sCD14, suggesting a link with HIV disease progression. Last, CD8+ MAIT cell levels tightly correlate with other antibacterial and mucosa-protective immune subsets, namely, neutrophils, innate-like T cells, and Th17 and Th22 cells. Together these findings suggest that low frequencies of MAIT cells in HIV positive children are part of a concerted disruption to the innate and adaptive immune compartments specialized in sensing and responding to pathogenic or commensal bacteria.


Assuntos
Linfócitos T CD8-Positivos/citologia , Infecções por HIV/sangue , Células T Invariáveis Associadas à Mucosa/citologia , Células Th17/citologia , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Contagem de Linfócitos , Masculino
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