RESUMO
Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.
Assuntos
ADP-Ribosil Ciclase 1/análise , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Hepacivirus , Hepatite C , Lectinas Tipo C/análise , Ativação Linfocitária/imunologia , Células T Matadoras Naturais , Doença Aguda , Alanina Transaminase/sangue , Estudos Transversais , Hepacivirus/isolamento & purificação , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Hepatite C/sangue , Hepatite C/fisiopatologia , Hepatite C/virologia , Humanos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/virologia , Infecção Persistente/imunologia , Infecção Persistente/virologia , Remissão Espontânea , Carga Viral/imunologiaRESUMO
OBJECTIVE: The extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves' orbitopathy (GO) is not completely defined. Here we investigated the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. METHODS: We conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. RESULTS: Having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI - 0.33 to - 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. CONCLUSIONS: Mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis.
Assuntos
Oftalmopatia de Graves , Leucócitos Mononucleares , Plasmócitos , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/classificação , Descompressão Cirúrgica/métodos , Feminino , Oftalmopatia de Graves/epidemiologia , Oftalmopatia de Graves/imunologia , Oftalmopatia de Graves/patologia , Oftalmopatia de Graves/cirurgia , Humanos , Imuno-Histoquímica , Itália/epidemiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos/imunologia , Procedimentos Cirúrgicos Oftalmológicos/métodos , Plasmócitos/imunologia , Plasmócitos/patologia , Subpopulações de Linfócitos T/imunologiaRESUMO
The ectocervix is part of the lower female reproductive tract (FRT), which is susceptible to sexually transmitted infections (STIs). Comprehensive knowledge of the phenotypes and T cell receptor (TCR) repertoire of tissue-resident memory T cells (TRMs) in the human FRT is lacking. We took single-cell RNA-Seq approaches to simultaneously define gene expression and TCR clonotypes of the human ectocervix. There were significantly more CD8+ than CD4+ T cells. Unsupervised clustering and trajectory analysis identified distinct populations of CD8+ T cells with IFNGhiGZMBloCD69hiCD103lo or IFNGloGZMBhiCD69medCD103hi phenotypes. Little overlap was seen between their TCR repertoires. Immunofluorescence staining showed that CD103+CD8+ TRMs were preferentially localized in the epithelium, whereas CD69+CD8+ TRMs were distributed evenly in the epithelium and stroma. Ex vivo assays indicated that up to 14% of cervical CD8+ TRM clonotypes were HSV-2 reactive in HSV-2-seropositive persons, reflecting physiologically relevant localization. Our studies identified subgroups of CD8+ TRMs in the human ectocervix that exhibited distinct expression of antiviral defense and tissue residency markers, anatomic locations, and TCR repertoires that target anatomically relevant viral antigens. Optimization of the location, number, and function of FRT TRMs is an important approach for improving host defenses to STIs.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos T CD8-Positivos/imunologia , Colo do Útero , Herpesvirus Humano 2 , Cadeias alfa de Integrinas/análise , Lectinas Tipo C/análise , Imunidade Adaptativa , Linfócitos T CD4-Positivos/imunologia , Colo do Útero/imunologia , Colo do Útero/patologia , Colo do Útero/virologia , Feminino , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Herpesvirus Humano 2/imunologia , Herpesvirus Humano 2/isolamento & purificação , Humanos , Memória Imunológica , Imunofenotipagem/métodos , Células T de Memória/imunologia , Mucosa/imunologia , Mucosa/patologia , Mucosa/virologiaRESUMO
Precise analyses of alloreactive T cell phenotype and function can inform both the nature and intensity of adaptive responses to transplant antigens. However, alloreactive T cells are sparse and difficult to detect, particularly in cryopreserved peripheral blood mononuclear cells (PBMCs) and from hypo-responsive individuals. An assay to identify and phenotype alloreactive cells would be particularly valuable, especially for multi-center clinical trials that often store frozen samples for batch analysis. Herein we demonstrate consistent and reproducible alloreactive T cell detection in cryopreserved PBMC following a short-term mixed lymphocyte reaction (MLR). The inherent background expression levels of activation markers on responder T cells were minimized by including a resting period prior to the assay. Stimulator cells were activated before inclusion in the MLR by addition of CD40L and IL-4. The time frame and markers to identify and phenotype alloreactive T cells following stimulation were optimized using short term co-cultures. We defined subsets of CD4+ and CD8+ T cells co-expressing CD69 and either CD154 or CD137 following allostimulation as alloreactive, and further phenotyped these cells with a variety of surface markers such as PD-1, LAG-3, and TIM-3. This assay may allow for the monitoring of donor-specific T cells in transplant recipients with longitudinally collected and cryopreserved PBMCs and provide a useful tool to identify biomarkers associated with tolerance. These biomarkers may add to mechanistic insights in immune recognition of transplanted tissues and/or cells.
Assuntos
Leucócitos Mononucleares/imunologia , Linfócitos T/imunologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos B/imunologia , Ligante de CD40/análise , Criopreservação , Humanos , Lectinas Tipo C/análise , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
T-Cell development is a major branch of lymphoid development and a key output of hematopoiesis, especially in early life, but the molecular requirements for T-cell potential have remained obscure. Considerable advances have now been made toward solving this problem through single-cell transcriptome studies, interfaced with in vitro differentiation assays that monitor potential efficiently at the single-cell level. This review focuses on a series of recent reports studying mouse and human early T-cell precursors, both in the developing fetus and in stringently purified postnatal samples of intrathymic and prethymic T-lineage precursors. Cross-comparison of results reveals a robustly conserved core program in mouse and human, but with some informative and provocative variations between species and between ontogenic states. Repeated findings are the multipotent progenitor regulatory signature of thymus-seeding cells and the proximity of the T-cell program to dendritic cell programs, especially to plasmacytoid dendritic cells in humans.
Assuntos
Hematopoese , Células Precursoras de Linfócitos T/citologia , Análise de Célula Única/métodos , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Linhagem da Célula , Movimento Celular , Separação Celular , Células Cultivadas , Células Dendríticas/citologia , Feto/citologia , Feto/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Humanos , Camundongos , Células-Tronco Multipotentes/citologia , Células Precursoras de Linfócitos T/classificação , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Repressoras/fisiologia , Especificidade da Espécie , Timo/citologia , Timo/embriologia , Timo/crescimento & desenvolvimento , Transcriptoma , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti-programmed cell death protein 1 (PD-1) treatment is predominantly studied in tumor-infiltrating lymphocytes (TILs). Its impact on PD-1 expressing cells in tumor-draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD-1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD-1, CD69, and HLA-DR on CD4+ and CD8+ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD-1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD-1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD-1 negative TILs expressed high levels of human leukocyte antigen - DR isotype and CD69. In contrast, PD-1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3+ PD-1+ cells in tumor-draining lymph nodes had significantly lower disease-free and overall survival rates (log-rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD-1 levels in TDLNs correspond with survival and potentially with response to anti-PD-1 therapy. Such knowledge may ultimately help guide anti-PD-1 treatment.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Neoplasias Bucais/imunologia , Linfonodo Sentinela/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Citometria de Fluxo , Antígenos HLA-DR/análise , Antígenos HLA-DR/metabolismo , Humanos , Lectinas Tipo C/análise , Lectinas Tipo C/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Receptor de Morte Celular Programada 1/análise , Receptor de Morte Celular Programada 1/metabolismo , Linfonodo Sentinela/citologia , Linfonodo Sentinela/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Aromatic antiepileptic drugs (AEDs) are common causes of cutaneous adverse drug reactions, which range from morbilliform drug eruption to life-threatening severe cutaneous adverse reactions, including drug reaction with eosinophilia and systemic symptoms, StevensâJohnson syndrome, and toxic epidermal necrolysis. Different in vitro methods for identifying the culprit drugs have been developed; however, it is particularly challenging for StevensâJohnson syndrome-toxic epidermal necrolysis. In this study, we enrolled 63 patients (39 with StevensâJohnson syndrome-toxic epidermal necrolysis, 13 with drug reaction with eosinophilia and systemic symptoms, and 11 with morbilliform drug eruption) and 30 tolerant controls to examine the performance of lymphocyte activation tests by measuring the expression of granulysin, granzyme B, and IFN-γ. Granulysin-based lymphocyte activation tests displayed the best sensitivity and specificity to identify the causality: 73.9% sensitivity and 96.7% specificity for carbamazepine and 68.2% sensitivity and 96.7% specificity for phenytoin. Oxcarbazepine and lamotrigine show weak antigenicity. Granulysin-based lymphocyte activation tests expanded predominantly memory cytotoxic T lymphocytes with characteristics of drug-specific T-cell receptor, major histocompatibility complex I dependence, and cross reactivity to different aromatic AEDs. Among 29 follow-up patients, 28 alternatively used nonaromatic AEDs, and none developed cutaneous adverse drug reactions. Our data suggest that granulysin-based lymphocyte activation tests represent in vitro cytotoxic T-lymphocyte memory response to offending drugs and are useful to confirm drug causality of AED-induced severe cutaneous adverse reactions. Implementing these tests will improve the AED-induced severe cutaneous adverse reactions prevention and clinical care.
Assuntos
Anticonvulsivantes/efeitos adversos , Antígenos de Diferenciação de Linfócitos T/análise , Síndrome de Hipersensibilidade a Medicamentos/diagnóstico , Ativação Linfocitária/efeitos dos fármacos , Síndrome de Stevens-Johnson/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação de Linfócitos T/metabolismo , Criança , Síndrome de Hipersensibilidade a Medicamentos/sangue , Síndrome de Hipersensibilidade a Medicamentos/imunologia , Feminino , Granzimas/análise , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Síndrome de Stevens-Johnson/sangue , Síndrome de Stevens-Johnson/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Adulto JovemRESUMO
BACKGROUND: Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. METHODS: The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. RESULTS: Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. CONCLUSIONS: This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.
Assuntos
Adenocarcinoma/patologia , Anticorpos Biespecíficos/farmacologia , Glipicanas/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias da Próstata/patologia , Anticorpos de Cadeia Única/farmacologia , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T Citotóxicos/imunologia , Adenocarcinoma/imunologia , Anticorpos Biespecíficos/imunologia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Citotoxicidade Imunológica , Glipicanas/antagonistas & inibidores , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Subunidade alfa de Receptor de Interleucina-2/análise , Lectinas Tipo C/análise , Ativação Linfocitária , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias da Próstata/imunologia , Proteínas Recombinantes/imunologia , Anticorpos de Cadeia Única/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39-CD69-) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39+CD69+) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39+ state. However, ACT responders retained a pool of CD39- stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/transplante , Melanoma/terapia , Neoplasias Cutâneas/terapia , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Apirase/análise , Linfócitos T CD8-Positivos/química , Feminino , Humanos , Lectinas Tipo C/análise , Melanoma/imunologia , Camundongos , Camundongos Mutantes , Neoplasias Cutâneas/imunologiaRESUMO
Arsenic (As) readily crosses the placenta and exposure of the fetus may cause adverse consequences later in life, including immunomodulation. In the current study, the question was asked how the immune repertoire might respond in postnatal life when there is no further As exposure. Here, pregnant mice (Balb/c [H-2d]) were exposed to arsenic trioxide (As2O3) through their drinking water from time of conception until parturition. Their offspring, 4-week-old mice who had not been exposed again to As, were used for functional analyses of innate, humoral and cellular immunity. Compared to cells from non-As-exposed dam offspring, isolated peritoneal macro-phages (MÏ) displayed no differences in T-cell stimulating ability. Levels of circulating IgG2a but not IgG1 were decreased in As-exposed dam offspring as compared to control offspring counterparts. Mixed-leukocyte reactions (MLR) indicated that CD4+ T-cells from the prenatal As-exposed mice were significantly less responsive to allogenic stimulation as evidenced by decreases in interferon (IFN)-γ and IL-2 production and in expression of CD44 and CD69 (but not CD25) activation markers. Interestingly, the MÏ from the prenatal As-exposed mice were capable of stimulating normal allogenic T-cells, indicating that T-cells from these mice were refractory to allogenic signals. There was also a significant decrease in absolute numbers of splenic CD4+ and CD8+ T-cells due to prenatal As exposure (as compared to control). Lastly, the impaired immune function of the prenatal As-exposed mice was correlated with a very strong susceptibility to Escherichia coli infection. Taken together, the data from this study clearly show that in utero As exposure may continue to perpetuate a dampening effect on the immune repertoire of offspring, even into the early stages of postnatal life.
Assuntos
Trióxido de Arsênio/toxicidade , Linfócitos T CD8-Positivos/imunologia , Infecções por Escherichia coli/imunologia , Exposição Materna/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/imunologia , Animais , Animais Recém-Nascidos , Antígenos CD/análise , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/metabolismo , Trióxido de Arsênio/administração & dosagem , Trióxido de Arsênio/farmacocinética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças/sangue , Suscetibilidade a Doenças/induzido quimicamente , Suscetibilidade a Doenças/imunologia , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/induzido quimicamente , Feminino , Humanos , Receptores de Hialuronatos/análise , Receptores de Hialuronatos/metabolismo , Interferon gama/análise , Interferon gama/metabolismo , Interleucina-2/análise , Interleucina-2/metabolismo , Lectinas Tipo C/análise , Lectinas Tipo C/metabolismo , Masculino , Troca Materno-Fetal/imunologia , Camundongos , Circulação Placentária/imunologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamenteRESUMO
DNAM-1 (CD226) is an activating immunoreceptor expressed on T cells and NK cells and involved in the pathogenesis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We previously reported that a soluble form of DNAM-1 (sDNAM-1) is generated by shedding from activated T cells. Moreover, higher serum levels of sDNAM-1 in patients before allo-HSCT is a predictive biomarker for the development of aGVHD based on the retrospective univariate and multivariate analyses in allo-HSCT patients. However, it remains unclear how the serum levels of sDNAM-1 are regulated after allo-HSCT and whether they are associated with the development of aGVHD. Here, we constructed a mathematical model to assess the dynamics of sDNAM-1 after allo-HSCT by assuming that there are three types of sDNAM-1 (the first and the second were from alloreactive and non-alloreactive donor lymphocytes, respectively, and the third from recipient lymphocytes). Our mathematical model fitted well to the data set of sDNAM-1 in patients (n = 67) who had undergone allo-HSCT and suggest that the high proportion of the first type of sDNAM-1 to the total of the first and second types is associated with high risk of the development of severe aGVHD. Thus, sDNAM-1 after allo-HSCT can be a biomarker for the development of aGVHD.
Assuntos
Antígenos de Diferenciação de Linfócitos T/sangue , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Modelos Teóricos , Adulto , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/metabolismo , Biomarcadores/análise , Biomarcadores/sangue , Biomarcadores/metabolismo , Feminino , Doença Enxerto-Hospedeiro/sangue , Meia-Vida , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Células Matadoras Naturais/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Linfócitos T/metabolismo , Transplante Homólogo/efeitos adversosRESUMO
BACKGROUND: Adaptive immune cells, including CD4+CD69+ and CD4+CD25+FoxP3+ regulatory T (Treg) cells, are important for maintaining immunological tolerance. In human systemic lupus erythematosus (SLE), CD4+CD25+FoxP3+ Treg cells are reduced, whereas CD69 expression is increased, resulting in a homeostatic immune imbalance that may intensify autoreactive T cell activity. To analyze the mechanisms implicated in autotolerance failure, we evaluated CD4+CD69+ and CD4+CD25+FoxP3+ T cells and interleukin profiles in a pristane-induced SLE experimental model. METHODS: For lupus induction, 26 female Balb/c mice received a single intraperitoneal 0.5 ml dose of pristane, and 16 mice received the same dose of saline. Blood and spleen samples were collected from euthanized mice 90 and 120 days after pristane or saline inoculation. Mononuclear cells from peripheral blood (PBMC), peritoneal lavage (PL) and splenocytes were obtained by erythrocyte lysis and cryopreserved for further evaluation by flow cytometry using the GuavaEasyCyte TM HT. After thawing, cells were washed and stained with monoclonal antibodies against CD3, CD4, CD8, CD25, CD28, CD69, FoxP3, CD14 and Ly6C (BD Pharmingen TM). Interleukins were quantified using Multiplex® MAP. The Mann-Whitney test and the Pearson coefficient were used for statistical analysis, and p < 0.05 considered significant. RESULTS: Compared with the controls, SLE-induced animals presented increased numbers of CD4+CD69+ T cells in the blood on T90 and T120 (p = 0.022 and p = 0.008) and in the spleen on T120 (p = 0.049), but there were decreased numbers in the PL (p = 0.049) on T120. The percentage of Treg was lower in blood (p < 0.005 and p < 0.012) on T90 and T120, in spleen (p = 0.043) on T120 and in PL (p = 0.001) on T90. Increased numbers of CD4 + CD69+ T cells in the PL were positively associated with high IL-2 (p = 0.486) and IFN-γ (p = 0.017) levels, whereas reduced Treg cells in the blood were negatively correlated with TNFα levels (p = 0.043) and positively correlated with TGFß1 (p = 0.038). CONCLUSION: Increased numbers of CD4+CD69+ T cells and reduced numbers of CD4+CD25+FoxP3+ Treg cells with an altered interleukin profile suggests loss of autotolerance in pristane-induced lupus mice, which is similar to human lupus. Therefore, this model is useful in evaluating mechanisms of cellular activation, peripheral tolerance and homeostatic immune imbalance involved in human SLE.
Assuntos
Linfócitos T CD4-Positivos/citologia , Lúpus Eritematoso Sistêmico/imunologia , Lavagem Peritoneal , Baço/citologia , Linfócitos T Reguladores/citologia , Animais , Antígenos CD/análise , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos Ly/análise , Antígenos Ly/imunologia , Antígenos CD28/análise , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/imunologia , Imunossupressores , Subunidade alfa de Receptor de Interleucina-2/análise , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/análise , Lectinas Tipo C/imunologia , Receptores de Lipopolissacarídeos/análise , Receptores de Lipopolissacarídeos/imunologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/induzido quimicamente , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , TerpenosRESUMO
BACKGROUND: Mucosal-associated invariant T (MAIT) cells represent a specialized lymphocyte population associated with chronic inflammatory disorders. Little is known, however, about MAIT cells in diseases of the kidney, including CKD. METHODS: To evaluate MAIT cells in human native kidneys with tubulointerstitial fibrosis, the hallmark of CKD, we used multicolor flow cytometry to identify, enumerate, and phenotype such cells from human kidney tissue biopsy samples, and immunofluorescence microscopy to localize these cells. We cocultured MAIT cells and human primary proximal tubular epithelial cells (PTECs) under hypoxic (1% oxygen) conditions to enable examination of mechanistic tubulointerstitial interactions. RESULTS: We identified MAIT cells (CD3+ TCR Vα7.2+ CD161hi) in healthy and diseased kidney tissues, detecting expression of tissue-resident markers (CD103/CD69) on MAIT cells in both states. Tissue samples from kidneys with tubulointerstitial fibrosis had significantly elevated numbers of MAIT cells compared with either nonfibrotic samples from diseased kidneys or tissue samples from healthy kidneys. Furthermore, CD69 expression levels, also an established marker of lymphocyte activation, were significantly increased on MAIT cells from fibrotic tissue samples. Immunofluorescent analyses of fibrotic kidney tissue identified MAIT cells accumulating adjacent to PTECs. Notably, MAIT cells activated in the presence of human PTECs under hypoxic conditions (modeling the fibrotic microenvironment) displayed significantly upregulated expression of CD69 and cytotoxic molecules perforin and granzyme B; we also observed a corresponding significant increase in PTEC necrosis in these cocultures. CONCLUSIONS: Our findings indicate that human tissue-resident MAIT cells in the kidney may contribute to the fibrotic process of CKD via complex interactions with PTECs.
Assuntos
Rim/patologia , Células T Invariantes Associadas à Mucosa/fisiologia , Insuficiência Renal Crônica/imunologia , Adulto , Idoso , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Comunicação Celular , Técnicas de Cocultura , Células Epiteliais/fisiologia , Feminino , Fibrose , Humanos , Túbulos Renais Proximais/citologia , Lectinas Tipo C/análise , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/patologiaAssuntos
Medula Óssea/patologia , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Subpopulações de Linfócitos T/transplante , Adulto , Antígenos de Diferenciação de Linfócitos T/análise , Exame de Medula Óssea , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/transplante , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/transplante , Linfócitos T CD8-Positivos/ultraestrutura , Núcleo Celular/ultraestrutura , Células-Tronco Hematopoéticas/patologia , Humanos , Ativação Linfocitária , Masculino , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Indução de Remissão , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Subpopulações de Linfócitos T/ultraestruturaRESUMO
BACKGROUND: Immune thrombocytopenia (ITP) is an immune-mediated bleeding disorder in children. Activated T cells have been shown to play important roles in ITP. The aims of this study were to evaluate whether these T cell activation markers could be used as indicators to differentiate ITP patients from controls, and to assess whether they could be used as predictors of IVIG response in ITP patients. METHODS: A cohort of 92 hospitalized ITP patients, 49 unrelated healthy children, and 48 thrombocytosis patients were enrolled in this retrospective study between February 2013 and September 2018. Expression of CD25, HLA-DR, and CD69 on the surfaces of CD4+ and CD8+ T cells were detected by flow cytometry. All statistical analyses were performed using SPSS 20.0 software. RESULTS: Compared to the healthy controls, ITP patients had higher percentages of CD4â¯+â¯CD25+ T cells, CD4â¯+â¯HLA-DR+ T cells, CD8â¯+â¯HLA-DR+ T cells, and CD8â¯+â¯CD69+ T cells. Compared to the thrombocytosis patients, ITP patients had higher percentages of CD4â¯+â¯HLA-DR+ T cells and CD8â¯+â¯HLA-DR+ T cells, and lower CD4â¯+â¯CD69+ T cells and CD8â¯+â¯CD69+ T cells. Platelet count at admission had a negative correlation with CD4â¯+â¯CD25+ T cells in ITP. CD4â¯+â¯CD69+ T cells were decreased in chronic compared to the newly diagnosed and persistent ITP patients. Activated T cell markers had no predictive value for IVIG response in ITP patients. CONCLUSIONS: T cell activation markers were excessively expressed in pediatric ITP, and those markers had no predictive value for IVIG response in ITP patients.
Assuntos
Ativação Linfocitária , Púrpura Trombocitopênica Idiopática/imunologia , Linfócitos T/imunologia , Adolescente , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos CD4/análise , Criança , Pré-Escolar , Doença Crônica , Feminino , Antígenos HLA-DR/análise , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , Lectinas Tipo C/análise , Masculino , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/terapia , Linfócitos T/patologia , Trombocitose/diagnóstico , Trombocitose/imunologiaRESUMO
Immune responses play an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the characteristics of T lymphocyte subsets in PCOS remain insufficiently understood. In this study, lymphocytes of follicular fluid (FF) were obtained from oocyte retrieval before in-vitro fertilization (IVF) in infertile women with or without PCOS. The levels of cluster of differentiation 25 (CD25), CD69, programmed death 1 (PD-1), interferon-γ (IFN-γ), interleukin 17A (IL-17A) and IL-10 in T lymphocytes were determined by flow cytometry. Our results showed that the percentage of FF CD8+ T cells was significantly decreased in infertile patients with PCOS (P < 0.05). Furthermore, the levels of CD69 and IFN-γ were significantly decreased and the level of PD-1 was increased in both CD4+ and CD8+ T cells from infertile patients with PCOS (P < 0.05). Moreover, the expression of PD-1 on CD4+ or CD8+ T cells was positively correlated with the estradiol (E2) levels in the serum and reversely correlated with the expression of IFN-γ in CD4+ or CD8+ T cells in infertile patients with PCOS. These results suggested that T cell dysfunction may be involved in the pathogenesis of PCOS.
Assuntos
Líquido Folicular/imunologia , Síndrome do Ovário Policístico/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Antígenos CD/análise , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/análise , Citocinas/imunologia , Feminino , Fertilização in vitro , Humanos , Infertilidade Feminina/complicações , Infertilidade Feminina/imunologia , Subunidade alfa de Receptor de Interleucina-2/análise , Subunidade alfa de Receptor de Interleucina-2/imunologia , Lectinas Tipo C/análise , Lectinas Tipo C/imunologia , Síndrome do Ovário Policístico/complicações , Adulto JovemRESUMO
Invasive mould infections (IMI) in immunocompromised patients are difficult to diagnose. Early and targeted treatment is paramount, but minimally invasive tests reliably identifying pathogens are lacking. We previously showed that monitoring pathogen-specific CD4+T cells in peripheral blood using upregulation of induced CD154 positive lymphocytes can be used to diagnose acute IMI. Here, we validate our findings in an independent patient cohort. We stimulated peripheral blood cells from at-risk patients with Aspergillus spp. and Mucorales lysates and quantitated mould-reactive CD4/CD69/CD154 positive lymphocytes via flow cytometry. Mould-reactive lymphocytes were quantitated in 115 at-risk patients. In 38 (33%) patients, the test was not evaluable, mainly due to low T cell counts or non-reactive positive control. Test results were evaluable in 77 (67%) patients. Of these, four patients (5%) had proven IMI and elevated mould-reactive T cell signals. Of 73 (95%) patients without proven IMI, 59 (81%) had mould-reactive T cell signals within normal range. Fourteen (19%) patients without confirmed IMI showed elevated T cell signals and 11 of those received antifungal treatment. The mould-reactive lymphocyte assay identified presence of IMI with a sensitivity of 100% and specificity of 81%. The mould-reactive lymphocyte assay correctly identified all patients with proven IMI. Assay applicability is limited by low T cell counts during bone marrow suppression. The assay has the potential to support diagnosis of invasive mould infection to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative.
Assuntos
Aspergillus/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções Fúngicas Invasivas/diagnóstico , Mucorales/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos T CD4-Positivos/química , Ligante de CD40/análise , Feminino , Citometria de Fluxo , Humanos , Hospedeiro Imunocomprometido , Lectinas Tipo C/análise , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Subpopulações de Linfócitos T/química , Adulto JovemRESUMO
Erythroid differentiation regulator 1 (Erdr1) has been identified as a stromal survival factor released under stressful conditions. Previously, Erdr1 was reported to be expressed highly in thymus, but roles of Erdr1 in thymus were not known. Here, the effects of Erdr1 on T cell development were investigated. The expression of Erdr1 was higher in thymus than bone marrow and Erdr1 was detected in both the cortex and medulla of thymus. Erdr1 treatment significantly induced the expression of activation marker, CD69, from thymocytes in the presence of TCR stimuli in vitro and the induction was dependent on increased Ca2+ influx. In addition, in vivo administration of Erdr1 resulted in significant increase of total and positive selected thymocyte numbers, particularly in the number of CD3TCRhiCD69+ DP thymocytes. Taken together, our results show that Erdr1 enhances the strength of TCR signaling and cellularity of thymocytes by amplifying Ca2+ influx in thymocytes receiving TCR signals.
Assuntos
Cálcio/metabolismo , Proteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Timócitos/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Lectinas Tipo C/análise , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Explants of lymphoid tissue provide a rare opportunity to assess the organization of the immune system in a living, dynamic environment. Traditionally, ex vivo immunostaining is conducted in fixed tissue sections, while live tissues are analyzed using genetically engineered fluorescent reporters or adoptively transferred, pre-labelled cell populations. Here, we validated a protocol for immunostaining and imaging in live, thick slices of lymph node tissue, thus providing a spatial "map" of the lymph node while maintaining the viability and functionality of the slices. Using anti-B220/CD45R (B cell) as a prototype antibody, the procedure for immunostaining was tested for sufficient signal to noise with respect to staining time, temperature, and wash time, and the specificity was verified in comparison to isotype controls. Immunostaining signal in live tissue slices was detectable to atleast 120⯵m deep for both whole antibodies and F(ab')2 fragments using the staining procedure. This procedure revealed the expected changes in B cell organization in lymph nodes from immunized mice. Cell surface staining with most antibodies did not induce cytokine secretion, and cytokine secretion in response to T cell stimulation was unaffected by immunostaining. Staining with known a mitogenic antibody (anti-CD3) simultaneously labelled the cells and activated the tissue, confirming that reagents for live immunostaining must be selected judiciously. As a proof of concept, this method was used to reveal the dynamic distribution of CD69, a T cell activation marker, in lymph node slices before and after ex vivo stimulation.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Complexo CD3/análise , Imunofluorescência , Secções Congeladas , Lectinas Tipo C/análise , Antígenos Comuns de Leucócito/análise , Linfonodos/imunologia , Animais , Biomarcadores/análise , Feminino , Interferon gama/metabolismo , Linfonodos/metabolismo , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Reprodutibilidade dos Testes , Técnicas de Cultura de Tecidos , Sobrevivência de TecidosRESUMO
In Kawasaki disease (KD), the effect of plasma exchange (PE) on immune cells has not been fully elucidated. Therefore, we examined the changes in the number of CD14+ CD16+ activated monocytes, regulatory T (Treg ), and T-helper type 17 (Th17) cells in KD patients treated with PE. The percentage of total monocytes and subclasses of lymphocytes, including CD4+ and CD8+ T cells, and CD19+ B cells, showed no significant difference before and after PE. However, the percentage of CD14+ CD16+ monocytes in total leukocytes decreased significantly after PE (1.1% ± 1.5% vs. 2.1% ± 2.3%, P < 0.05). Furthermore, while the percentage of Th17 cells in CD4+ T cells did not change, the percentage of Treg cells in CD4+ T cells increased significantly after PE (11.1% ± 5.1% vs. 8.0% ± 4.4%, P < 0.05). Therefore, PE downregulates activated monocytes and upregulates Treg cells toward normal levels and thus attenuates inflammation in KD.