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1.
Immunogenetics ; 71(8-9): 545-559, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31384962

RESUMO

Butyrophilins (BTN), specifically BTN3A, play a central role in the modulation of γδ T cells, which are mainly present in gut and mucosal tissues. BTN3A1 is known, for example, to activate Vγ9Vδ2 T cells by means of a phosphoantigen interaction. In the extended HLA region, three genes are located, designated BTN3A1, BTN3A2 and BTN3A3, which were also defined in rhesus macaques. In contrast to humans, rhesus monkeys have an additional gene, BTN3A3Like, which has the features of a pseudogene. cDNA analysis of 32 Indian rhesus and 16 cynomolgus macaques originating from multiple-generation families revealed that all three genes are oligomorphic, and the deduced amino acids display limited variation. The macaque BTN3A alleles segregated together with MHC alleles, proving their location in the extended (Major Histocompatibility Complex) MHC. BTN3A nearly full-length transcripts of macaques and humans cluster tightly together in the phylogenetic tree, suggesting that the genes represent true orthologs of each other. Despite the limited level of polymorphism, 15 Mamu- and 14 Mafa-BTN3A haplotypes were defined, and, as in humans, all three BTN3A genes are transcribed in PBMCs and colon tissues. In addition to regular full-length transcripts, a high number of various alternative splicing (AS) products were observed for all BTN3A alleles, which may result in different isoforms. The comparable function of certain subsets of γδ T cells in human and non-human primates in concert with high levels of sequence conservation observed for the BTN3A transcripts presents the opportunity to study these not yet well understood molecules in macaques as a model species.


Assuntos
Antígenos CD/genética , Butirofilinas/genética , Antígenos de Histocompatibilidade/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Sequência de Aminoácidos , Animais , Butirofilinas/metabolismo , Sequência Conservada , Feminino , Haplótipos , Humanos , Macaca mulatta , Masculino , Filogenia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Homologia de Sequência , Linfócitos T/metabolismo
2.
Vet Immunol Immunopathol ; 214: 109893, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31378220

RESUMO

Differentiation between canine chronic enteropathy (CCE) and intestinal lymphoma is a diagnostic challenge as histopathology might fail to yield unequivocal results. Detection of clonal rearrangements of the T-cell-receptor gamma (TCRG) chain and IG heavy chain (IGH) V-J genes offer a useful solution. In this retrospective study, histopathology samples of 35 CCE patients and 7 healthy Beagle dogs underwent clonality testing. Patients suffered either from inflammatory bowel disease (IBD), food responsive diarrhea (FRD) or protein loosing enteropathy secondary to IBD (PLE/IBD). Healthy Beagles served as controls (CO). Canine IBD activity index (CIBDAI) and histopathological WSAVA-grading differed significantly (p<0.001) between groups. CIBDAI improved significantly after appropriate therapy (p < 0.0001). Intestinal biopsies of all CO showed polyclonal patterns for B- and T-cell primers. All samples from CCE patients showed polyclonal patterns for the B-cell primers. Targeting TCRG, 4 patients showed a monoclonal or oligoclonal pattern of the lymphocytic infiltrates in the duodenum and/or colon. Clinical improvement was observed in all dogs. Although a small cell lymphoma cannot be excluded in view of the short follow up duration, a false positive result, in the sense of a canonical rearrangement or unspecific amplification due to a antigenic stimulation in a non-neoplastic inflammatory process is possible.


Assuntos
Doenças do Cão/diagnóstico , Doenças do Cão/genética , Enteropatias Perdedoras de Proteínas/genética , Enteropatias Perdedoras de Proteínas/veterinária , Animais , Biópsia , Estudos de Casos e Controles , Doença Crônica , Diagnóstico Diferencial , Cães , Feminino , Rearranjo Gênico do Linfócito T , Doenças Inflamatórias Intestinais/imunologia , Intestinos/patologia , Linfoma/diagnóstico , Linfoma/veterinária , Masculino , Enteropatias Perdedoras de Proteínas/diagnóstico , Receptores de Antígenos de Linfócitos T gama-delta/genética , Estudos Retrospectivos
3.
J Immunol Res ; 2019: 3836942, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236420

RESUMO

Pregnancy is an immunological enigma where paternal antigens are present at the fetomaternal interface. What regulates the local immunotolerance, which is necessary to prevent rejection of the conceptus, is still under strong investigation. Gamma/delta T cells are believed to play a role in the local regulation of this immunotolerance towards the semiallogenic fetus. Gamma/delta T cells from the uterus and spleen of pregnant and nonpregnant mice were analyzed by flow cytometry. We confirmed that the rate of γδT cells in the decidua increases during murine pregnancy and half of decidual γδT cells are CD4+. Furthermore, we found a unique association of CD4 or CD8 coreceptor expression with their γδTCR intensity, where in all investigated groups CD4- or CD8-positive γδT cells seemed principally to be γδTCRdim. In addition, compared to peripheral γδT lymphocytes, a greater proportion of decidual γδT cells expressed the cytotoxic marker CD107a and markers of Th1 or Th2 polarization (TIM-3, TIM-1), where decidual γδTCRbright cells were characterized by high TIM-3 and TIM-1 receptor expression. On the other hand, no difference in the expression of CD160, a receptor with dual function affecting cytotoxicity and T cell inhibition, was detected. Within lymphocytes expressing CD107a, TIM-1, or CD160, the rate of γδT cells was significantly higher in the decidua. According to our results, cytotoxic potential of decidual γδTCRbright cells could be regulated by TIM-3 ligation, while the TIM-1 receptor seems to be able to influence the Th1-Th2 balance at the fetomaternal interface. These mechanisms could play a part in the active maternal immunotolerance towards the fetus, allowing an efficient protection against pathogens during healthy murine pregnancy.


Assuntos
Decídua/imunologia , Decídua/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Imunomodulação , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Feminino , Expressão Gênica , Receptor Celular 1 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/genética , Imunofenotipagem , Camundongos , Especificidade de Órgãos , Gravidez , Receptores de Antígenos de Linfócitos T gama-delta/genética , Baço/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
6.
J Immunol ; 202(8): 2460-2472, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30877169

RESUMO

Tcrd and Tcrg display identical developmental programs that depend on the activity of the enhancers Eδ and Eγ being "on" in pre-ß-selection thymocytes to activate transcription and V(D)J recombination of the unrearranged genes and "off" in post-ß-selection CD4+CD8+ double-positive thymocytes to inhibit transcription of the rearranged genes and avoid the expression of TCR δ- and TCR γ-chains in αß T lymphocytes. Eδ and Eγ activity depends on transcription factor binding to essential Runx and Myb sites and parallels that of Notch signaling. We performed Notch gain- and loss-of-function experiments and found that Notch signaling activates Tcrd and Tcrg transcription by favoring the recruitment of RUNX1 and MYB to the enhancers. Our results suggest that the dissociation of RUNX1 and MYB from Eδ and Eγ chromatin in double-positive thymocytes, which results in enhancer inactivation, is caused by decreased Notch signaling triggered by pre-TCR signaling, thereby deciphering the molecular mechanism of Tcrd and Tcrg silencing during ß-selection. These findings reveal a novel molecular mechanism for gene regulation via Notch signaling through the recruitment of RUNX1 and MYB to enhancer chromatin during thymocyte development.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Elementos Facilitadores Genéticos/imunologia , Proteínas Proto-Oncogênicas c-myb/imunologia , Receptores Notch/imunologia , Transdução de Sinais/imunologia , Timócitos/imunologia , Transcrição Genética/imunologia , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Humanos , Células Jurkat , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myb/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores Notch/genética , Transdução de Sinais/genética
7.
Cell Physiol Biochem ; 52(2): 354-367, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816679

RESUMO

BACKGROUND/AIMS: Although a cross-talk between immune and endocrine systems has been well established, the precise pathways by which these signals co-regulate pro- and antiinflammatory responses on antigen-presenting cells remain poorly understood. In this work we investigated the mechanisms by which triiodothyronine (T3) controls T cell activity via dendritic cell (DC) modulation. METHODS: DCs from wild-type (WT) and IL-6-deficient mice were pulsed with T3. Cytokine production and programmed death protein ligands (PD-L) 1 and 2 expression were assayed by flow cytometry and ELISA. Interferon-regulatory factor-4 (IRF4) expression was evaluated by RT-qPCR and flow cytometry. The ability of DCs to stimulate allogenic splenocytes was assessed in a mixed lymphocyte reaction and the different profile markers were analyzed by flow cytometry and ELISA. For in vivo experiments, DCs treated with ovalbumin and T3 were injected into OTII mice. Proliferation, cytokine production, frequency of FoxP3+ regulatory T (Treg) cells and PD-1+ cells were determined by MTT assay, ELISA and flow cytometry, respectively. RESULTS: T3 endows DCs with pro-inflammatory potential capable of generating IL-17-dominant responses and down-modulating expression of PD-L1 and 2. T3-stimulated WT-DCs increased the proportion of IL-17-producing splenocytes, an effect which was eliminated when splenocytes were incubated with T3-treated DCs derived from IL-6-deficient mice. Enhanced IL-17 expression was recorded in both, CD4- and CD4+ populations and involved the IRF-4 pathway. Particularly, γδ-T cells but not natural killer (NK), NKT, B lymphocytes nor CD8+ T cells were the major source of IL-17-production from CD4- cells. Moreover, T3-conditioned DCs promoted a decrease of the FoxP3+ Treg population. Furthermore, T3 down-modulated PD-1 expression on CD4- cells thereby limiting inhibitory signals driven by this co-inhibitory pathway. Thus, T3 acts at the DC level to drive proinflammatory responses in vitro. Accordingly, we found that T3 induces IL-17 and IFNγ-dominant antigen-specific responses in vivo. CONCLUSION: These results emphasize the relevance of T3 as an additional immune-endocrine checkpoint and a novel therapeutic target to modulate IL-17-mediated pro-inflammatory responses.


Assuntos
Células Dendríticas/imunologia , Interleucina-17/imunologia , Transdução de Sinais/efeitos dos fármacos , Tri-Iodotironina/farmacologia , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Dendríticas/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interleucina-17/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Knockout , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais/imunologia
8.
Dev Comp Immunol ; 96: 78-82, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30738793

RESUMO

In order to better understand the development and function of γδ T cells in Xenopus frogs, it is necessary to determine where and when γδ T cells are found in Xenopus tissues. This study examined the expression of TCR genes, focused primarily on TCR γ, in tissues of adult and larval Xenopus laevis and provide new data about the expression pattern of these different TCR genes in this anuran amphibian. TCR gene expression was detected by RT-PCR in adult frog tissues including the thymus, spleen, skin, intestine, lung, and liver, but not the testes. TCR γ and ß genes were detected in the larval (tadpole) tail and intestine. The absence of RAG-1 expression in these larval tissues is consistent with differentiation of the T cells in the thymus. Together, these data provide evidence that migration of these cells from the thymus likely occurs relatively early in larval development. These studies provide a necessary foundation for future studies of the functions of γδ T cells in amphibians, which are placed at an intermediate position flanked by fishes on one end and mammals and chickens on the other.


Assuntos
Larva/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Xenopus laevis/imunologia , Animais , Diferenciação Celular/imunologia , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T , Genes Codificadores da Cadeia gama de Receptores de Linfócitos T , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Larva/genética , Larva/metabolismo , Metamorfose Biológica , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/imunologia , Xenopus laevis/genética , Xenopus laevis/metabolismo
9.
Trends Mol Med ; 25(3): 171-184, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30713007

RESUMO

Staphylococcus aureus is an opportunistic pathogen, which can readily develop antibiotic resistance and result in severe disease. To combat antibiotic resistance, new treatment strategies are being developed with a particular focus on vaccine development. S. aureus vaccines that target humoral immunity alone do not provide sufficient protection from all disease phenotypes associated with this pathogen. Recent studies have identified the requirement for cellular immunity to provide a robust immune response to this infection. Driving conventional T cell responses has therefore become the focus of intense research in this area. Recently described 'alternative' T cells could provide a novel strategy for improving therapeutic efficacy and success in next-generation anti-S. aureus vaccine design.


Assuntos
Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Suscetibilidade a Doenças/imunologia , Humanos , Imunidade Celular , Imunidade Inata , Memória Imunológica , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Infecções Estafilocócicas/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
10.
Proc Natl Acad Sci U S A ; 116(7): 2634-2639, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30683721

RESUMO

Random amino acid copolymers used in the treatment of multiple sclerosis in man or experimental autoimmune encephalomyelitis (EAE) in mice [poly(Y,E,A,K)n, known as Copaxone, and poly(Y,F,A,K)n] function at least in part by generation of IL-10-secreting regulatory T cells that mediate bystander immunosuppression. The mechanism through which these copolymers induce Tregs is unknown. To investigate this question, four previously described Vα3.2 Vß14 T cell receptor (TCR) cDNAs, the dominant clonotype generated in splenocytes after immunization of SJL mice, that differed only in their CDR3 sequences were utilized to generate retrogenic mice. The high-level production of IL-10 as well as IL-5 and small amounts of the related cytokines IL-4 and IL-13 by CD4+ T cells isolated from the splenocytes of these mice strongly suggests that the TCR itself encodes information for specific cytokine secretion. The proliferation and production of IL-10 by these Tregs was costimulated by activation of glucocorticoid-induced TNF receptor (GITR) (expressed at high levels by these cells) through its ligand GITRL. A mechanism for generation of cells with this specificity is proposed. Moreover, retrogenic mice expressing these Tregs were protected from induction of EAE by the appropriate autoantigen.


Assuntos
Células-Tronco Hematopoéticas/citologia , Interleucina-10/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Animais , DNA Complementar , Encefalomielite Autoimune Experimental/imunologia , Feminino , Vetores Genéticos , Tolerância Imunológica , Interleucinas/metabolismo , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Fatores de Necrose Tumoral/metabolismo
11.
Mol Med Rep ; 19(3): 1471-1480, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30628681

RESUMO

γδ T cells are a subset of unconventional T cells that serve a critical role in infectious diseases and various types of cancer. Cell therapy with genetically­modified γδ T cells is regarded as a promising tool for tumor treatment. However, since γδ T cells constitute a minority of T cells, their large­scale expansion is difficult to realize in an efficient and cost­effective manner. In the present study, based on previous studies, culture protocols for γδ T cells were tested using different combinations of isopentenyl pyrophosphate and interleukin 2 in order to satisfy different experimental purposes. One protocol was demonstrated to be the most suitable for lentiviral transduction. These results greatly reinforce the promising prospects of using γδ T cells in basic research and for clinical applications.


Assuntos
Técnicas de Cultura de Células/métodos , Proliferação de Células , Subpopulações de Linfócitos T/citologia , Linfócitos T/citologia , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Lentivirus/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução Genética
12.
EBioMedicine ; 39: 44-58, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30528453

RESUMO

BACKGROUND: Immune adaptation with aging is a major of health outcomes. Studies in humans have mainly focus on αß T cells while γδ T cells have been neglected despite their role in immunosurveillance. We investigated the impact of aging on γδ T cell subsets phenotypes, functions, senescence and their molecular response to stress. METHODS: Peripheral blood of young and old donors in Singapore have been used to assess the phenotype, functional capacity, proliferation capacity and gene expression of the various γδ T cell subsets. Peripheral blood mononuclear cells from apheresis cones and young donors have been used to characterize the telomere length, epigenetics profile and DNA damage response of the various γδ T cell subsets phenotype. FINDINGS: Our data shows that peripheral Vδ2+ phenotype, functional capacity (cytokines, cytotoxicity, proliferation) and gene expression profile are specific when compared against all other αß and γδ T cells in aging. Hallmarks of senescence including telomere length, epigenetic profile and DNA damage response of Vδ2+ also differs against all other αß and γδ T cells. INTERPRETATION: Our results highlight the differential impact of lifelong stress on γδ T cells subsets, and highlight possible mechanisms that enable Vδ2+ to be resistant to cellular aging. The new findings reinforce the concept that Vδ2+ have an "innate-like" behavior and are more resilient to the environment as compared to "adaptive-like" Vδ1+ T cells.


Assuntos
Envelhecimento/genética , Citocinas/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Subpopulações de Linfócitos T/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/imunologia , Proliferação de Células , Senescência Celular , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Singapura , Subpopulações de Linfócitos T/imunologia , Encurtamento do Telômero , Adulto Jovem
13.
Ann Lab Med ; 39(2): 125-132, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30430774

RESUMO

BACKGROUND: Chromosomal abnormalities and common genetic rearrangements related to T-acute lymphoblastic leukemia (T-ALL) are not clear. We investigated T-cell receptor (TCR) rearrangement in Korean T-ALL patients by fragment analysis, examining frequency, association between clinicopathologic characteristics and TCR clonality, and feasibility for detecting minimal residual disease (MRD). METHODS: In 51 Korean patients diagnosed as having T-ALL, TCR rearrangement was analyzed using the IdentiClone TCR gene clonality assay (InVivoScribe Technologies, San Diego, CA, USA) from archived bone marrow specimens. Limit of detection (LOD) and clonal stability at relapse were evaluated. The association between clinical prognosis and TCR clonality was examind by age and immunophenotypic classification. RESULTS: Thirty-eight patients (74.5%) had 62 clonal products of TCRß, TCRγ, and/or TCRδ rearrangements at diagnosis. Children with T-ALL (<12 years) showed a higher frequency of clonality (93.8%) than adolescents/adults (65.7%; ≥12 years). Patients with a mature immunophenotype (84.4%) showed a relatively higher frequency of clonality than those with the immature immunophenotype (57.9%). Survival and event-free survival were not influenced by immunophenotype or TCR clonality. The LOD was 1%. Clonal evolution at the relapse period was noted. CONCLUSIONS: The overall detection rate of TCR clonality was 74.5%. Survival did not differ by TCR clonality or immunophenotype and age group. Fragment analysis of TCR rearrangement cannot be used to assess MRD due to low sensitivity. Further research on the relationship between prognosis and frequency of TCR rearrangements is needed, using more sensitive methods to detect clonality and monitor MRD.


Assuntos
Rearranjo Gênico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Adolescente , Adulto , Fatores Etários , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/metabolismo , Criança , Feminino , Humanos , Imunofenotipagem , Masculino , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Intervalo Livre de Progressão , Recidiva , Indução de Remissão , Taxa de Sobrevida , Adulto Jovem
14.
Fish Shellfish Immunol ; 86: 641-652, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30485793

RESUMO

In mammalian, T-cell receptors (TCRs) play a key role in recognizing the presented antigen from external to protect organisms against environmental pathogens. To understand the potential roles of TCRγ and TCRδ in dojo loach (Misgurnus anguillicaudatus), Ma-TCRγ and Ma-TCRδ cDNAs were cloned and their gene expression profiles were investigated after bacterial, parasitic and fungal challenge. The open reading frame (ORF) of Ma-TCRγ and Ma-TCRδ cDNAs contained 948 and 867 bp, encoding 316 and 288 amino acid residues, respectively. Structurally, Ma-TCRγ and Ma-TCRδ were consisted of a signal peptide, a variable region, a constant region (IgC), a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic domain (CYT), which were similar to those of other vertebrates. Multiple sequence alignment and phylogenetic analysis showed Ma-TCRγ and Ma-TCRδ were closely related to fish of Cyprinidae family. Ma-TCRγ and Ma-TCRδ were widely expressed in all tested organs/tissues, as the highest expressions of Ma-TCRγ and Ma-TCRδ were detected in kidney and gill, respectively. In addition, three infection models of dojo loach with bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia sp.) were constructed. The morphological changes of gills and skin after challenged with F. columnare G4 and Ichthyophthirius multifiliis were investigated. Compared to F. columnare G4 infection, mRNA expression of both TCRγ and TCRδ showed higher sensitivity in classical immune organs (kidney and spleen) and mucosal tissues (skin and gill) after challenge with Ichthyophthirius multifiliis and Saprolegnia sp. Our results first indicated that TCRγ and TCRδ of dojo loach might function differently in response to challenge with different pathogens.


Assuntos
Bactérias/imunologia , Cyprinidae/imunologia , Doenças dos Peixes/imunologia , Fungos/imunologia , Parasitos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Animais , Clonagem Molecular , Cyprinidae/genética , DNA Complementar/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Flavobacterium/imunologia , Regulação da Expressão Gênica , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Saprolegnia/imunologia , Transcriptoma
15.
Methods Mol Biol ; 1884: 1-14, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465192

RESUMO

T cells fulfill a central role in cell-mediated immunity and can be found in the circulation and lymphoid organs upon maturation. For clinical applications, it can be important to quantify (infiltrated) T cells accurately in a variety of body fluids and tissues of benign, inflammatory, or malignant origin. For decades, flow cytometry and immunohistochemistry have been the accustomed methods to quantify T cells. Although these methods are widely used, they depend on the accessibility of T-cell epitopes and therefore require fresh, frozen, or fixated material of a certain quality. Whenever samples are low in quantity or quality, an accurate quantification can be impeded. By shifting the focus from epitopes to DNA, quantification of T cells remains achievable.Mature T cells differ genetically from other cell types as a result of T-cell receptor (TCR) gene rearrangements. This genetic dissimilarity can be exploited to quantify the T-cell fraction in DNA specimens. Conventionally, multiplex PCR and droplet digital PCR (ddPCR), combined with deep-sequencing techniques, can be applied to determine T-cell content. However, these approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. Considering this, a simple T-cell quantification, unwantedly, turns into a complex, expensive, and time-consuming procedure. We have developed two generic single duplex ddPCR assays as alternative methods to quantify T cells in a relatively simple, cheap, and fast manner by targeting sequences located between the Dδ2 and Dδ3 genes (TRD locus) and Dß1 and Jß1.1 genes (TRB locus). These specific TCR loci become deleted systematically early during lymphoid differentiation and therefore will serve as biomarkers for the quantification of mature T cells. Here, we describe a simple and sensitive ddPCR-based method to quantify T cells relatively fast, accurately and independently of the cellular context.


Assuntos
Loci Gênicos/genética , Neoplasias/diagnóstico , Reação em Cadeia da Polimerase/métodos , Linfócitos T/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Neoplasias/genética , Neoplasias/imunologia , Reação em Cadeia da Polimerase/instrumentação , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia
16.
Int J Lab Hematol ; 41(2): 242-249, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30537135

RESUMO

INTRODUCTION: T-cell receptor gene (TRG) rearrangement profiling is an essential component of the workup at diagnosis of T-cell malignancies. TRG amplification by polymerase chain reaction (PCR) and analysis by capillary electrophoresis (PCR-CE) is mostly widely used but is hampered by a subjective interpretation of its results and possible false-positive interpretation of clonality. Several studies evaluated the advantage of TRG rearrangement analysis by Next Generation Sequencing (TRG-NGS), however few have proposed an adequate data interpretation algorithm. METHODS: Eighty five fresh and 36 formalin-fixed paraffin embedded (FFPE) diagnostic samples suspected for a lymphoproliferative disorder were analyzed by PCR-CE and TRG NGS. Final clinical diagnosis was available for all fresh samples. Reproducibility, analytical specificity and sensitivity of the TRG NGS analysis was evaluated. RESULTS: We propose a new interpretation algorithm for TRG NGS data analysis. PCR-CE and TRG NGS showed identical results in 66/85 (78%) of fresh samples. Sensitivities to detect T-cell malignancies were comparable (96% versus 92%, respectively). The analysis of FFPE material was significantly more successful by TRG NGS (34/36 cases) in respect to PCR-CE (16/36 cases), most likely due to the small size of the amplicons. CONCLUSION: Assessment of T-cell clonality by TRG NGS has a significant added value in the diagnosis of T-cell disorders as an adjunct to PCR-CE, particularly in difficult to interpret cases or when analyzing FFPE samples.


Assuntos
Algoritmos , Rearranjo Gênico , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Receptores de Antígenos de Linfócitos T gama-delta/genética , Análise de Sequência de DNA , Feminino , Humanos , Masculino
19.
Mucosal Immunol ; 12(1): 247-257, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279514

RESUMO

Gram-positive pathogens, including Staphylococcus aureus, cause necrotizing pneumonia. The central feature of S. aureus pneumonia is toxin-induced necroptosis of immune and resident cells, which impedes host defense. However, the role of the NLRC4 in the lung following S. aureus infection remains elusive. Here, we demonstrate that S. aureus activates the NLRC4 to drive necroptosis and IL-18 production, which impaired IL-17A-dependent neutrophil-mediated host susceptibility. In particular, Nlrc4-/- mice exhibit reduced necroptosis, enhanced neutrophil influx into the lungs, decreased bacterial burden, and improved host survival. Loss of NLRC4 signaling in both hematopoietic and non-hematopoietic cells contributes to the host protection against S. aureus pneumonia. Secretion of IL-17A by γδ T cells is essential for neutrophil recruitment into the lungs of Nlrc4-/- mice following infection. Moreover, treatment of wild-type mice with necroptosis inhibitors or genetic ablation of MLKL and IL-18 improves host defense against S. aureus infection, which is associated with increased IL-17A+γδ T cells and neutrophils. Taken together, these novel findings reveal that S. aureus activates the NLRC4 to dampen IL-17A-dependent neutrophil accumulation through induction of necroptosis and IL-18. Thus, modulating the function of the NLRC4 may be an attractive therapeutic approach for treating S. aureus infections.


Assuntos
Pulmão/imunologia , Neutrófilos/imunologia , Pneumonia Estafilocócica/imunologia , Staphylococcus aureus/imunologia , Linfócitos T/imunologia , Animais , Apoptose , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Humanos , Doenças do Sistema Imunitário , Interleucina-18/genética , Interleucina-18/metabolismo , Transtornos Leucocíticos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Proteínas Quinases/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais , Regulação para Cima
20.
Blood Adv ; 2(23): 3506-3514, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30530777

RESUMO

Mature T-cell lymphomas consisting of an expanded clonal population of T cells that possess common rearrangements of the T-cell receptor (TCR) encoding genes can be identified and monitored using molecular methods of T-cell repertoire analysis. We have developed a hybrid-capture method that enriches DNA sequencing libraries for fragments encoding rearranged TCR genes from all 4 loci in a single reaction. We use this method to describe the TCR repertoires of 63 putative lymphoma clinical isolates, 7 peripheral blood mononuclear cell (PBMC) populations, and a collection of tumor infiltrating lymphocytes. Dominant Variable (V) and Joining (J) gene pair rearrangements in cancer cells were confirmed by polymerase chain reaction (PCR) amplification and Sanger sequencing; clonality assessment of clinical isolates using BIOMED-2 methods showed agreement for 73% and 77% of samples at the ß and γ loci, respectively, whereas ß locus V and J allele prevalence in PBMCs were well correlated with results from commercial PCR-based DNA sequencing assays (r 2 = 0.94 with Adaptive ImmunoSEQ, 0.77-0.83 with Invivoscribe LymphoTrack TRB Assay). CapTCR-seq allows for rapid, high-throughput and flexible characterization of dominant clones within TCR repertoire that will facilitate quantitative analysis of patient samples and enhance sensitivity of tumor surveillance over time.


Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Análise de Sequência de DNA/métodos , Biblioteca Gênica , Rearranjo Gênico do Linfócito T/genética , Loci Gênicos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Linfoma de Células T/diagnóstico , Linfoma de Células T/genética , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/química , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo
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