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2.
Immunity ; 49(5): 873-885.e7, 2018 Nov 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.

3.
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.

4.
Mucosal Immunol ; 11(6): 1591-1605, 2018 Nov.
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.

5.
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.

6.
J Immunol ; 201(5): 1586-1598, 2018 Sep 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.

7.
J Allergy Clin Immunol ; 142(4): 1297-1310.e11, 2018 Oct.
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).

8.
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.

9.
J Infect Dis ; 216(6): 641-650, 2017 Sep 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
10.
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
11.
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
12.
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
13.
Immunity ; 44(6): 1350-64, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27261277

RESUMO

T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.


Assuntos
Autoimunidade , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Humoral , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Linfócitos T/imunologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
14.
Trends Biotechnol ; 34(9): 722-732, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27296078

RESUMO

Improving the ability to predict the efficacy and toxicity of drug candidates earlier in the drug discovery process will speed up the introduction of new drugs into clinics. 3D in vitro systems have significantly advanced the drug screening process as 3D tissue models can closely mimic native tissues and, in some cases, the physiological response to drugs. Among various in vitro systems, bioprinting is a highly promising technology possessing several advantages such as tailored microarchitecture, high-throughput capability, coculture ability, and low risk of cross-contamination. In this opinion article, we discuss the currently available tissue models in pharmaceutics along with their limitations and highlight the possibilities of bioprinting physiologically relevant tissue models, which hold great potential in drug testing, high-throughput screening, and disease modeling.


Assuntos
Bioimpressão , Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Modelos Biológicos , Análise Serial de Tecidos , Animais , Humanos
15.
J Acquir Immune Defic Syndr ; 72(5): 474-84, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-27003495

RESUMO

Regulatory T cells (Tregs) are functionally suppressive CD4 T cells, critical for establishing peripheral tolerance and controlling inflammatory responses. Previous reports of Tregs during chronic HIV disease have conflicting results with higher or lower levels compared with controls. Identifying true Tregs with suppressive activity proves challenging during HIV infection, as traditional Treg markers, CD25 and FOXP3, may transiently upregulate expression as a result of immune activation (IA). Helios is an Ikaros family transcription factor that marks natural Tregs with suppressive activity and does not upregulate expression after activation. Coexpression of FOXP3 and Helios has been suggested as a highly specific marker of "bona fide" Tregs. We evaluated Treg subsets by FOXP3 coexpressed with either CD25 or Helios and their association with HIV disease progression in perinatally infected HIV-positive children. Identifying Tregs by FOXP3 coexpression with Helios rather than CD25 revealed markedly higher Treg frequencies, particularly in HIV+ children. Regardless of antiretroviral therapy, HIV-infected children had a selective expansion of memory FOXP3+Helios+ Tregs. The rise in memory Tregs correlated with declining HIV clinical status, indicated by falling CD4 percentages and CD4:CD8 ratios and increasing HIV plasma viremia and IA. In addition, untreated HIV+ children exhibited an imbalance between the levels of Tregs and activated T cells. Finally, memory Tregs expressed IA markers CD38 and Ki67 and exhaustion marker, PD-1, that tightly correlated with a similar phenotype in memory CD4 T cells. Overall, HIV-infected children had significant disruptions of memory Tregs that associated with advancing HIV disease.


Assuntos
Progressão da Doença , Fatores de Transcrição Forkhead/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/patologia , Fator de Transcrição Ikaros/metabolismo , Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , ADP-Ribosil Ciclase 1/metabolismo , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Criança , Citometria de Fluxo , Fatores de Transcrição Forkhead/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Memória Imunológica , Inflamação/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Antígeno Ki-67/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T Reguladores/citologia
16.
J Immunol ; 196(2): 573-85, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26673135

RESUMO

The function of CD4(+) T cells is dependent on Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca(2+) influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4(+) T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell-mediated autoimmune diseases.


Assuntos
Canais de Cálcio/imunologia , Encefalomielite Autoimune Experimental/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Separação Celular , Cromatografia Líquida , Encefalomielite Autoimune Experimental/patologia , Citometria de Fluxo , Humanos , Camundongos , Camundongos Transgênicos , Proteína ORAI1 , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/imunologia , Medula Espinal/patologia , Linfócitos T Reguladores/imunologia , Espectrometria de Massas em Tandem
17.
Peptides ; 71: 296-303, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26026377

RESUMO

The major mode of transmission of the human immunodeficiency virus (HIV) is by sexual intercourse. In the effort to halt the spread of HIV, one measure that holds great promise is the development of effective microbicides that can prevent transmission. Previously we showed that several amphibian antimicrobial peptides (AMPs) completely inhibit HIV infection of T cells while maintaining good viability of the T cell targets. These peptides also inhibited the transfer of HIV by dendritic cells (DCs) to T cells when added up to 8h after virus exposure. Here we report on the anti-HIV activity of 18 additional structurally related caerin 1 family peptides in comparison with our previous best candidate caerin 1.9. Nine peptides were equally effective or more effective in the inhibition of T cell infection and disruption of the HIV envelope as caerin 1.9. Of those nine peptides, three peptides (caerin 1.2, caerin 1.10, and caerin 1.20) exhibited excellent inhibition of HIV infectivity at low concentrations (12-25µM) and limited toxicity against target T cells and endocervical epithelial cells. There was a direct correlation between the effectiveness of the peptides in disruption of the viral envelope and their capacity to inhibit infection. Thus, several additional caerin 1 family peptides inhibit HIV infection have limited toxicity for vaginal epithelial cells, and would be good candidates for inclusion in microbicide formulations.


Assuntos
Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1 , Linfócitos T/virologia , Relação Dose-Resposta a Droga , Humanos , Linfócitos T/metabolismo
18.
J Immunol ; 195(3): 1202-17, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26109647

RESUMO

Store-operated Ca(2+) entry (SOCE) through Ca(2+) release-activated Ca(2+) (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodeficiency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune system such as macrophages and dendritic cells (DCs) and may provide Ca(2+) signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca(2+) signaling strongly impairs many effector functions of bone marrow-derived macrophages and bone marrow-derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Células Dendríticas/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/metabolismo , Animais , Apresentação do Antígeno/genética , Apresentação do Antígeno/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Retículo Endoplasmático/metabolismo , Humanos , Imunidade Inata/imunologia , Inflamassomos/imunologia , Ativação Linfocitária/imunologia , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína ORAI1 , Fagocitose/imunologia , Imunodeficiência Combinada Severa/genética , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Linfócitos T/imunologia
19.
J Clin Invest ; 125(6): 2347-62, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25938788

RESUMO

Chronic infections induce a complex immune response that controls pathogen replication, but also causes pathology due to sustained inflammation. Ca2+ influx mediates T cell function and immunity to infection, and patients with inherited mutations in the gene encoding the Ca2+ channel ORAI1 or its activator stromal interaction molecule 1 (STIM1) are immunodeficient and prone to chronic infection by various pathogens, including Mycobacterium tuberculosis (Mtb). Here, we demonstrate that STIM1 is required for T cell-mediated immune regulation during chronic Mtb infection. Compared with WT animals, mice with T cell-specific Stim1 deletion died prematurely during the chronic phase of infection and had increased bacterial burdens and severe pulmonary inflammation, with increased myeloid and lymphoid cell infiltration. Although STIM1-deficient T cells exhibited markedly reduced IFN-γ production during the early phase of Mtb infection, bacterial growth was not immediately exacerbated. During the chronic phase, however, STIM1-deficient T cells displayed enhanced IFN-γ production in response to elevated levels of IL-12 and IL-18. The lack of STIM1 in T cells was associated with impaired activation-induced cell death upon repeated TCR engagement and pulmonary lymphocytosis and hyperinflammation in Mtb-infected mice. Chronically Mtb-infected, STIM1-deficient mice had reduced levels of inducible regulatory T cells (iTregs) due to a T cell-intrinsic requirement for STIM1 in iTreg differentiation and excessive production of IFN-γ and IL-12, which suppress iTreg differentiation and maintenance. Thus, STIM1 controls multiple aspects of T cell-mediated immune regulation to limit injurious inflammation during chronic infection.


Assuntos
Canais de Cálcio/imunologia , Imunidade Celular , Mycobacterium tuberculosis/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose Pulmonar/imunologia , Animais , Canais de Cálcio/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Doença Crônica , Citocinas/genética , Citocinas/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/genética , Molécula 1 de Interação Estromal , Linfócitos T Reguladores/patologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/patologia
20.
PLoS One ; 10(5): e0126917, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25993666

RESUMO

Establishment of long-lived cellular reservoirs of HIV-1 represents a major therapeutic challenge to virus eradication. In this study, we utilized a human primary cell model of HIV-1 latency to evaluate the requirements for efficient virus reactivation from, and the selective elimination of, latently infected human T cells. Ectopic expression of BCL2 supported the replication and spread of R5-tropic HIV-1 in activated CD4(+) T cells. After IL-2 withdrawal, the HIV-1-infected T cells survived as resting cells for several months. Unexpectedly, these resting T cells continue to produce detectable levels of infectious virus, albeit at a lower frequency than cells maintained in IL-2. In the presence of HIV-1 inhibitors, reactivation of the resting T cells with γc-cytokines and allogeneic dendritic cells completely extinguished HIV-1 infectivity. We also evaluated the ability of the bacterial LukED cytotoxin to target and kill CCR5-expressing cells. After γc-cytokine stimulation, LukED treatment eliminated both HIV-1-infected resting cells and the non-infected CCR5+ cells. Importantly, complete clearance of in vitro HIV-1 reservoirs by LukED required a lower threshold of cytokine signals relative to HIV-1 inhibitors. Thus, the primary T cell-based HIV-1 latency model could facilitate the development of novel agents and therapeutic strategies that could effectively eradicate HIV-1.


Assuntos
Linfócitos T CD4-Positivos/virologia , Reservatórios de Doenças , HIV-1/fisiologia , Latência Viral , Humanos , Técnicas In Vitro , Replicação Viral
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