RESUMO
Envelhecer compreende um fenômeno complexo, natural e irreversível, que submete o organismo a inúmeras alterações nos processos biológicos, fisiológicos, ambientais, psicológicos, comportamentais e sociais. Esse processo é caracterizado por um declínio gradual dos mecanismos homeostáticos do organismo, intimamente relacionados com o estado senescente. A senescência, quando diz respeito ao sistema imunológico, é denominada de imunossenescência, que pode ser definida como uma parada estável do ciclo celular associada a mudanças, com uma resposta que limita a proliferação de células envelhecidas ou danificadas. A autofagia está diretamente relacionada com a manutenção do fenótipo senescente, em que a atividade autofágica exerce um papel essencial e ativo na influência da biossíntese de proteínas e organelas. Essa via é regulada naturalmente pela proteína mTOR e quimicamente pelo fármaco rapamicina. Assim, pretendemos investigar: (1) as alterações no perfil corporal e hematimêtrico dos animais ao longo do tratamento com rapamicina; (2) avaliar o perfil de citocinas; (3) observar as modificações histológicas em órgãos linfoides primários e secundário; (4) analisar as populações de células linfoides e mieloides; e (5) avaliar a capacidade proliferativa de linfócitos in vitro. Camundongos SAMP-8 e SAMR-1 foram tratados com rapamicina durante dois meses. A mensuração da massa corporal e análises hematológicas foram realizadas antes e durante o tratamento. Amostras de soro, medula óssea, timo e baço foram analisados em ensaios de ELISA, histologia, população e subpopulações de células. Alterações na massa corporal, parâmetros hematológicos e celularidade de células foram nítidas entre os dois modelos utilizados. Diferenças também foram percebidas na detecção de citocinas IL-1ß. IL-6 e TNF-α, com resultados significantes nas amostras de baço, timo e medula óssea. As citocinas IL-7 e IL-15 apresentaram diferenças de secreção entre os grupos, sendo a primeira maior detectada em camundongos com senescência acelerada tratados com rapamicina. Em nossa análise histológica observamos que os camundongos SAM-P8 apresentaram involução tímica. E nas subpopulações de linfócitos T do baço, células TCD4+ e TCD8+ estavam, respectivamente, em maior e menor quantidade nos camundongos SAM-P8 tratados com rapamicina. Dessa forma, o camundongo da linhagem SAM-P8 é um excelente modelo para se estudar as alterações da senescência, em que o mesmo apresenta características fisiológicas distintas dos camundongos utilizados como controle (SAM-R1). Além disso, verificamos que a dose de rapamicina empregada não desencadeou alterações que pudessem comprometer a resposta imunológica desses camundongos, bem como na possibilidade de atuar na resposta contra os efeitos complexos do envelhecimento
Aging comprises a complex, natural, and irreversible phenomenon, which subjects the organism to countless alterations in biological, physiological, environmental, psychological, behavioral, and social processes. This process is characterized by a gradual decline in the organism's homeostatic mechanisms, closely related to senescence effects. Senescence, when it concerns the immune system, is called immunosenescence, which can be defined as a stable cell cycle arrest associated with changes and is a response that limits the proliferation of aged or damaged cells. Autophagy is a genetically regulated, conserved cellular process and a metabolic pathway essential for maintaining cellular homeostasis, which plays a constitutive and active role in controlling the biosynthesis of proteins and organelles. This pathway is regulated naturally by mTOR or chemically by the drug rapamycin, having a direct relationship with cellular homeostasis and maintenance of the senescent phenotype. Thus, we intend to investigate: (1) the changes in the body and hematimetic profile of the animals throughout the rapamycin treatment; (2) evaluate the cytokine profile; (3) observe histological changes in primary and secondary lymphoid organs; (4) analyze lymphoid and myeloid cell populations; and (5) evaluate the proliferative capacity of lymphocytes in vitro. SAMP-8 and SAMR-1 mice were treated with rapamycin for two months. Body mass measurement and hematological analyses were performed before and during treatment. Serum, bone marrow, thymus and spleen samples were analyzed in ELISA assays, histology, cell population and subpopulations. Changes in body mass, hematological parameters, and cellularity were clear between the two models used. Differences were also noticed in the detection of cytokines IL-1ß. IL-6 and TNF-α, with significant results in the spleen, thymus and bone marrow samples. The cytokines IL-7 and IL-15 showed differences in secretion between groups, the former being higher detected in mice with accelerated senescence treated with rapamycin. In our histological analysis we observed that SAM-P8 mice showed thymic involution. And in the spleen T-lymphocyte subpopulations, TCD4+ and TCD8+ cells were, respectively, in higher and lower quantities in SAM-P8 mice treated with rapamycin. Thus, the SAM-P8 mouse is an excellent model to study the changes of senescence, since it presents physiological characteristics different from the control mice (SAM-R1). Furthermore, we verified that the dose of rapamycin used did not trigger changes that could compromise the immune response of these mice, as well as the possibility of acting in the modulatory response against the complex effects of aging
Assuntos
Animais , Masculino , Camundongos , Envelhecimento , Sirolimo/efeitos adversos , Imunossenescência , Autofagia/imunologia , Técnicas In Vitro/métodos , Ensaio de Imunoadsorção Enzimática/instrumentação , Preparações Farmacêuticas/administração & dosagem , Subpopulações de Linfócitos T/classificação , HomeostaseRESUMO
OBJECTIVE: To investigate the quantity of CD4+T, CD4+T, CD8+T, and γδT cells in peripheral blood of HIV-infected/AIDS patients as well as to explore the possible role of CD4/CD8 ratio and γδT cells in the progression of HIV/AIDS, aimed at providing evidence for the diagnosis and treatment of AIDS. METHODS: The quantity levels of CD3+T cells, CD4+T cells, CD8+T cells, and γδT cells in peripheral blood of 46 HIV-infected/AIDS patients and 30 healthy controls were detected by using flow cytometry. RESULTS: The count of CD3+T, CD4+T, CD8+T, and γδT cells ( x ¯ ± s , A/µl) in the peripheral blood was 1183.64 ± 132.58, 278.39 ± 122.38, 863.13 ± 82.38, and 22.53 ± 1.74 in the experimental group as well as 1456.46 ± 124.37, 788.74 ± 189.67, 569.61 ± 46.49, and 10.96 ± 0.28 in the control group, respectively. The p values of the two groups were <0.005 after the t-test, revealing a statistically significant difference. The proportion of CD3+T, CD4+T, CD8+T, and γδT cells in total lymphocytes in the two groups ( x ¯ ± s , %) was 71.83 ± 5.37, 13.39 ± 2.23, 62.93 ± 5.81, and 3.67 ± 0.87 in the experimental group, respectively. In the control group, the values were expressed as 66.72 ± 5.48, 42.77 ± 3.38, 31.41 ± 3.62, and 1.73 ± 0.36, respectively. After performing the t-test, p values in the two groups were <0.005 except CD3+T, with statistically significant differences. Besides, CD4/CD8 was 0.33 ± 0.11 in the experimental group and 1.48 ± 0.29 in the control group, t = 26.528, p < 0.001, exhibiting a significant statistical difference. CONCLUSION: HIV infection induces the activation and proliferation of CD8+T and γδT cells, contributing to the decrease of CD4+T cells, while CD8+T and γδT cells are involved in the immune response and tissue damage after HIV infection.
Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Infecções por HIV/imunologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/imunologia , Síndrome da Imunodeficiência Adquirida/sangue , Adulto , Idoso , Complexo CD3/sangue , Contagem de Linfócito CD4 , Relação CD4-CD8 , Estudos de Casos e Controles , Proliferação de Células , Biologia Computacional , Feminino , Infecções por HIV/sangue , Humanos , Ativação Linfocitária , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T gama-delta/sangue , Adulto JovemRESUMO
The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immune-mediated gastrointestinal diseases. The lymphocytes at these effector sites are principally T cells which present important morphological, phenotypical and functional differences. Flow cytometry (FC) is one of the most commonly used techniques to characterize intestinal lymphocytes in human and animal models. Published studies with a focus on dogs for intraepithelial lymphocytes (IEL) immunophenotyping exist in very limited numbers. Moreover, no lamina propria lymphocytes (LPL) isolation protocols in the canine species have been described for FC evaluation. In addition to immune intestinal dysregulation, imbalances in the peripheral blood immune system have been described in both human and animal gastrointestinal disorders. The aim of this study was to provide a protocol for canine IEL and LPL isolation for FC immunophenotyping of T cells subsets. Specifically, T helper, T cytotoxic, activated Th and Tc lymphocytes, regulatory, double negative, double positive, IFN-γ and IL-4 producing T cells, and to compare their respective populations between these effector sites and with the blood stream compartment in healthy dogs. The potential relationship of these cells distributions with age, sex and breed was also evaluated. This study included sixteen healthy dogs of different sexes and breeds with a mean age of 4.55 ± 2.93 years old. The selected protocols for the three immune compartments showed proper cell yield, purity, viability, and the absence of phenotypic and functional disturbances. Histologically, an adequate separation of the duodenal epithelium from the lamina propria was also observed. All the proposed T cells subsets were identified in the three immune compartments studied, showing some statistically significant differences in their distributions at these locations that highlight the importance of their individual evaluation. This study provides an adequate method for canine small intestine IEL and LPL isolation for FC immunophenotyping and is key for future studies on the gastrointestinal immune system associated with different canine diseases.
Assuntos
Imunofenotipagem , Mucosa Intestinal , Intestino Delgado , Subpopulações de Linfócitos T , Animais , Cães , Citometria de Fluxo/veterinária , Imunofenotipagem/veterinária , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Subpopulações de Linfócitos T/classificaçãoRESUMO
As high-throughput genomics assays become more efficient and cost effective, their utilization has become standard in large-scale biomedical projects. These studies are often explorative, in that relationships between samples are not explicitly defined a priori, but rather emerge from data-driven discovery and annotation of molecular subtypes, thereby informing hypotheses and independent evaluation. Here, we present K2Taxonomer, a novel unsupervised recursive partitioning algorithm and associated R package that utilize ensemble learning to identify robust subgroups in a 'taxonomy-like' structure. K2Taxonomer was devised to accommodate different data paradigms, and is suitable for the analysis of both bulk and single-cell transcriptomics, and other '-omics', data. For each of these data types, we demonstrate the power of K2Taxonomer to discover known relationships in both simulated and human tissue data. We conclude with a practical application on breast cancer tumor infiltrating lymphocyte (TIL) single-cell profiles, in which we identified co-expression of translational machinery genes as a dominant transcriptional program shared by T cells subtypes, associated with better prognosis in breast cancer tissue bulk expression data.
Assuntos
Algoritmos , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Linfócitos do Interstício Tumoral/classificação , Linfócitos do Interstício Tumoral/metabolismo , Prognóstico , Reprodutibilidade dos Testes , Análise de Sobrevida , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/metabolismoRESUMO
BACKGROUND: Previously, we have found that IL-22 could be not only secreted outside of cells, but also highly expressed on the T cells membrane in HIV-1 negative patients with tuberculosis (TB). However, the study on membrane-bound IL-22+ cells of HIV-1 infected patients is rare. Therefore, we investigated antigen-specific membrane-bound IL-22+ T cell subsets in Mycobacterium tuberculosis (M.tb) coinfection of HIV-1 infected individuals. METHODS: A case-control study that enrolled 74 HIV-1 infected participants was carried out, including HIV-1 monoinfection (HIV+TB-, n = 43), HIV-1 infected patients with latent TB (HIV+LTB, n = 18) and HIV-1 coinfected patients with active TB (HIV+TB+, n = 13). We made use of an IFN-γ release assay (IGRA) to screen LTB individuals. Purified protein derivative (PPD) and phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) were used as specific-stimulators to detect the levels of peripheral blood membrane-bound IL-22+ T cell subsets via cell surface staining and flow cytometry among three groups. RESULTS: An approximate rate of 24.3% (n = 18 out of 74) of latent M.tb infection among HIV-1 positive population in Eastern China. Interestingly, HMBPP-specific CD3+Vγ2+ T cells were impaired in HIV+TB+patients compared with HIV+LTB patients (P < 0.05). Furthermore, increases of PPD-specific and HMBPP-specific membrane-bound IL-22+ T cell subsets including CD3+, CD3+CD4+ and CD3+Vγ2+ T cells were observed in HIV+TB+group rather than HIV+LTB groups (all P < 0.05). CONCLUSION: Antigen-specific membrane-bound IL-22+ T cells were highly expressed in M.tb coinfection of HIV-1 infected individuals, and may play an important role in anti-TB immune response during coinfection with HIV-1.
Assuntos
Infecções por HIV/imunologia , Interleucinas/imunologia , Interleucinas/metabolismo , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Tuberculose/imunologia , Adolescente , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Citocinas/análise , Citocinas/imunologia , Feminino , Infecções por HIV/microbiologia , Humanos , Interleucinas/genética , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/classificação , Adulto Jovem , Interleucina 22RESUMO
BACKGROUND AIMS: Adoptive cellular therapy with immune effector cells (IECs) has shown promising efficacy against some neoplastic diseases as well as potential in immune regulation. Both inherent variability in starting material and variations in cell composition produced by the manufacturing process must be thoroughly evaluated with a validated method established to quantify viable lymphocyte subtypes. Currently, commercialized immunophenotyping methods determine cell viability with significant errors in thawed products since they do not include any viability staining. We hereby report on the validation of a flow cytometry-based method for quantifying viable lymphocyte immunophenotypes in fresh and cryopreserved hematopoietic cellular products. METHODS: Using fresh or frozen cellular products and stabilized blood, we report on the validation parameters accuracy, uncertainty, precision, sensitivity, robustness and contamination between samples for quantification of viable CD3+, CD4+ T cells, CD8+ T cells, CD3-CD56+CD16+/- NK cells, CD19+ B cells and CD14+ monocytes of relevance to fresh and cryopreserved hematopoietic cellular products using the Cytomics FC500 cytometer (Beckman Coulter). RESULTS: The acceptance criteria set in the validation plan were all met. The method is able to accommodate the variability in absolute numbers of cells in starting materials collected or cryopreserved from patients or healthy donors (uncertainty of ≤20% at three different concentrations), stability over time (compliance over 3 years during regular inter-laboratory comparisons) and confidence in meaningful changes during cell processing and manufacturing (intra-assay and intermediate precision of 10% coefficient of variation). Furthermore, the method can accurately report on the efficacy of cell depletion since the lower limit of quantification was established (CD3+, CD4+ and CD8+ cells at 9, 8 and 8 cells/µL, respectively). The method complies with Foundation for the Accreditation of Cellular Therapy (FACT) standards for IEC, FACT-Joint Accreditation Committee of ISCT-EBMT (JACIE) hematopoietic cell therapy standards, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2(R1) and International Organization for Standardization 15189 standards. Furthermore, it complies with Ligand Binding Assay Bioanalytical Focus Group/American Association of Pharmaceutical Scientists, International Council for Standardization of Hematology/International Clinical Cytometry Society and European Bioanalysis Forum recommendations for validating such methods. CONCLUSIONS: The implications of this effort include standardization of viable cell immunophenotyping of starting material for cell manufacturing, cell selection and in-process quality controls or dosing of IECs. This method also complies with all relevant standards, particularly FACT-JACIE standards, in terms of enumerating and reporting on the viability of the "clinically relevant cell populations."
Assuntos
Linfócitos B/classificação , Criopreservação , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Subpopulações de Linfócitos T/classificação , Adulto , Linfócitos B/metabolismo , Humanos , Imunofenotipagem , Masculino , Reprodutibilidade dos TestesRESUMO
Transient partial remission, a period of low insulin requirement experienced by most patients soon after diagnosis, has been associated with mechanisms of immune regulation. A better understanding of such natural mechanisms of immune regulation might identify new targets for immunotherapies that reverse type 1 diabetes (T1D). In this study, using Cox model multivariate analysis, we validated our previous findings that patients with the highest frequency of CD4+CD25+CD127hi (127-hi) cells at diagnosis experience the longest partial remission, and we showed that the 127-hi cell population is a mix of Th1- and Th2-type cells, with a significant bias toward antiinflammatory Th2-type cells. In addition, we extended these findings to show that patients with the highest frequency of 127-hi cells at diagnosis were significantly more likely to maintain ß cell function. Moreover, in patients treated with alefacept in the T1DAL clinical trial, the probability of responding favorably to the antiinflammatory drug was significantly higher in those with a higher frequency of 127-hi cells at diagnosis than those with a lower 127-hi cell frequency. These data are consistent with the hypothesis that 127-hi cells maintain an antiinflammatory environment that is permissive for partial remission, ß cell survival, and response to antiinflammatory immunotherapy.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Alefacept/uso terapêutico , Linfócitos T CD4-Positivos/classificação , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/terapia , Progressão da Doença , Feminino , Humanos , Imunoterapia/métodos , Lactente , Subunidade alfa de Receptor de Interleucina-2/sangue , Subunidade alfa de Receptor de Interleucina-7/sangue , Masculino , Análise Multivariada , Modelos de Riscos Proporcionais , Subpopulações de Linfócitos T/classificação , Adulto JovemRESUMO
Immunological memory, defined as the ability to respond in an enhanced manner upon secondary encounter with the same pathogen, can provide substantial protection against infectious disease. The improved protection is mediated in part by different populations of memory CD8 T cells that are retained after primary infection. Memory cells persist in the absence of pathogen-derived antigens and enable secondary CD8 T-cell responses with accelerated kinetics and of larger magnitude after reencounter with the same pathogen. At least three subsets of memory T cells have been defined that are referred to as central memory CD8 T cells (Tcm), effector memory CD8 T cells (Tem), and tissue-resident memory CD8 T cells (Trm). Tcm and Tem are circulating memory T cells that mediate bodywide immune surveillance in search of invading pathogens. In contrast, Trm permanently reside in peripheral barrier tissues, where they form a stationary defensive line of sentinels that alert the immune system upon pathogen reencounter. The characterization of these different subsets has been instrumental in our understanding of the strategies that memory T cells employ to counter invading pathogens. It is clear that memory T cells not only have a numerical advantage over naive T cells resulting in improved protection in secondary responses, but also acquire distinct sets of competencies that assist in pathogen clearance. Nevertheless, inherent challenges are associated with the allocation of memory T cells to a limited number of subsets. The classification of memory T cells into Tcm, Tem, and Trm may not take into account the full extent of the heterogeneity that is observed in the memory population. Therefore, in this review, we will revisit the current classification of memory subsets, elaborate on functional and migratory properties attributed to Tcm, Tem, and Trm, and discuss how potential heterogeneity within these populations arises and persists.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Memória Imunológica/fisiologia , Subpopulações de Linfócitos T/fisiologia , Animais , Linfócitos T CD8-Positivos/classificação , Diferenciação Celular , Humanos , Ativação Linfocitária , Subpopulações de Linfócitos T/classificaçãoRESUMO
t(8;21)(q22;q22) acute myelogenous leukemia (AML) is morphologically characterized by a continuum of heterogeneous leukemia cells from myeloblasts to differentiated myeloid elements. Thus, t(8;21) AML is an excellent model for studying heterogeneous cell populations and cellular evolution during disease progression. Using integrative analyses of immunophenotype, RNA-sequencing (RNA-seq), and single-cell RNA-sequencing (scRNA-seq), we identified three distinct intrapatient leukemic cell populations that were arrested at different stages of myeloid differentiation: CD34+CD117dim blasts, CD34+CD117bri blasts, and abnormal myeloid cells with partial maturation (AM). CD117 is also known as c-KIT protein. CD34+CD117dim cells were blocked in the G0/G1 phase at disease onset, presenting with the regular morphology of myeloblasts showing features of granulocyte-monocyte progenitors (GMP), and were drug-resistant to chemotherapy. Genes associated with cell migration and adhesion (LGALS1, EMP3, and ANXA2) were highly expressed in the CD34+CD117dim population. CD34+CD117bri blasts were blocked a bit later than the CD34+CD117dim population in the hematopoietic differentiation stage and displayed high proliferation ability. AM cells, which bear abnormal myelocyte morphology, especially overexpressed granule genes AZU1, ELANE, and PRTN3 and were sensitive to chemotherapy. scRNA-seq at different time points identified CD34+CD117dim blasts as an important leukemic cluster that expanded at postrelapse refractory stage after several cycles of chemotherapy. Patients with t(8;21) AML with a higher proportion of CD34+CD117dim cells had significantly worse clinical outcomes than those with a lower CD34+CD117dim proportion. Univariate and multivariate analyses identified CD34+CD117dim proportion as an independent factor for poor disease outcome. Our study provides evidence for the multidimensional heterogeneity of t(8;21)AML and may offer new tools for future disease stratification.
Assuntos
Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/metabolismo , Adulto , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/metabolismo , TranscriptomaRESUMO
We investigated the phenotype and molecular signatures of CD8+ T cell subsets in kidney-transplant recipients (KTRs) with biopsy-proven T cell-mediated rejection (TCMR). We included 121 KTRs and divided them into three groups according to the pathologic or clinical diagnosis: Normal biopsy control (NC)(n = 32), TCMR (n = 50), and long-term graft survival (LTGS)(n = 39). We used flowcytometry and microarray to analyze the phenotype and molecular signatures of CD8+ T cell subsets using peripheral blood from those patients and analyzed significant gene expressions according to CD8+ T cell subsets. We investigated whether the analysis of CD8+ T cell subsets is useful for predicting the development of TCMR. CCR7+CD8+ T cells significantly decreased, but CD28nullCD57+CD8+ T cells and CCR7-CD45RA+CD8+ T cells showed an increase in the TCMR group compared to other groups (p<0.05 for each); hence CCR7+CD8+ T cells showed significant negative correlations to both effector CD8+ T cells. We identified genes significantly associated with the change of CCR7+CD8+ T, CCR7-CD45RA+CD8+ T, and CD28nullCD57+CD8+ T cells in an ex vivo study and found that most of them were included in the significant genes on in vitro CCR7+CD8+ T cells. Finally, the decrease of CCR7+CD8+ T cells relative to CD28nullCD57+ T or CCR7-CD45RA+CD8+ T cells can predict TCMR significantly in the whole clinical cohort. In conclusion, phenotype and molecular signature of CD8+ T subsets showed a significant relationship to the development of TCMR; hence monitoring of CD8+ T cell subsets may be a useful for predicting TCMR in KTRs.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Transplante de Rim/efeitos adversos , Subpopulações de Linfócitos T/imunologia , Adulto , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD57/genética , Antígenos CD57/imunologia , Linfócitos T CD8-Positivos/classificação , Estudos Transversais , Feminino , Perfilação da Expressão Gênica , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/genética , Voluntários Saudáveis , Humanos , Imunofenotipagem , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Receptores CCR7/genética , Receptores CCR7/imunologia , Subpopulações de Linfócitos T/classificaçãoRESUMO
T cells are crucial for the success of immune-based cancer therapy. Reinvigorating antitumor T cell activity by blocking checkpoint inhibitory receptors has provided clinical benefits for many cancer patients. However, the efficacy of these treatments varies in cancer patients and the mechanisms underlying these diverse responses remain elusive. The density and status of tumor-infiltrating T cells have been shown to positively correlate with patient response to checkpoint blockades. Therefore, further understanding of the heterogeneity, clonal expansion, migration, and effector functions of tumor-infiltrating T cells will provide fundamental insights into antitumor immune responses. To this end, recent advances in single-cell RNA sequencing technology have enabled profound and extensive characterization of intratumoral immune cells and have improved our understanding of their dynamic relationships. Here, we summarize recent progress in single-cell RNA sequencing technology and current strategies to uncover heterogeneous tumor-infiltrating T cell subsets. In particular, we discuss how the coupling of deep transcriptome information with T cell receptor (TCR)-based lineage tracing has furthered our understanding of intratumoral T cell populations. We also discuss the functional implications of various T cell subsets in tumors and highlight the identification of novel T cell markers with therapeutic or prognostic potential.
Assuntos
Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , RNA Neoplásico/genética , Subpopulações de Linfócitos T/imunologia , Transcriptoma , Comunicação Celular/genética , Comunicação Celular/imunologia , Expressão Gênica , Heterogeneidade Genética , Humanos , Ativação Linfocitária , Linfócitos do Interstício Tumoral/patologia , Neoplasias/genética , Neoplasias/patologia , RNA Neoplásico/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Sequência de RNA , Análise de Célula Única , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/patologia , Microambiente Tumoral/imunologiaRESUMO
Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.
Assuntos
Alérgenos/administração & dosagem , Asma/dietoterapia , Diacilglicerol O-Aciltransferase/imunologia , Dieta Cetogênica/métodos , Interleucina-33/imunologia , Gotículas Lipídicas/metabolismo , Subpopulações de Linfócitos T/imunologia , Alternaria/química , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citocinas/administração & dosagem , Diacilglicerol O-Aciltransferase/genética , Modelos Animais de Doenças , Ácidos Graxos/imunologia , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Glucose/imunologia , Glucose/metabolismo , Imunidade Inata , Interleucina-33/administração & dosagem , Interleucina-33/genética , Interleucinas/administração & dosagem , Gotículas Lipídicas/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR gama/genética , PPAR gama/imunologia , Papaína/administração & dosagem , Fosfolipídeos/imunologia , Fosfolipídeos/metabolismo , Cultura Primária de Células , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Linfopoietina do Estroma do TimoRESUMO
The intestinal microbiota shapes and directs immune development locally and systemically, but little is known about whether commensal microbes in the stomach can impact their immunological microenvironment. Here, we report that group 2 innate lymphoid cells (ILC2s) were the predominant ILC subset in the stomach and show that their homeostasis and effector functions were regulated by local commensal communities. Microbes elicited interleukin-7 (IL-7) and IL-33 production in the stomach, which in turn triggered the propagation and activation of ILC2. Stomach ILC2s were also rapidly induced following infection with Helicobacter pylori. ILC2-derived IL-5 resulted in the production of IgA, which coated stomach bacteria in both specific pathogen-free (SPF) and H. pylori-infected mice. Our study thus identifies ILC2-dependent IgA response that is regulated by the commensal microbiota, which is implicated in stomach protection by eliminating IgA-coated bacteria including pathogenic H. pylori.
Assuntos
Microbioma Gastrointestinal/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/patogenicidade , Imunoglobulina A/biossíntese , Interleucina-5/imunologia , Estômago/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Feminino , Regulação da Expressão Gênica , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/imunologia , Imunidade Humoral , Imunidade Inata , Interleucina-33/genética , Interleucina-33/imunologia , Interleucina-5/genética , Interleucina-7/genética , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Transdução de Sinais , Estômago/microbiologia , Simbiose/imunologia , Subpopulações de Linfócitos T/classificaçãoRESUMO
BACKGROUND: Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project. METHODS: The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration. RESULTS: Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting. CONCLUSION: Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.
Assuntos
Citometria de Fluxo/normas , Imunofenotipagem/normas , Subpopulações de Linfócitos T/classificação , Biomarcadores/sangue , Complexo CD3/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Cor/normas , Citometria de Fluxo/métodos , Humanos , Memória Imunológica , Itália , Antígenos Comuns de Leucócito/sangue , Leucócitos Mononucleares/imunologia , Variações Dependentes do Observador , Receptores CCR7/sangue , Subpopulações de Linfócitos T/imunologiaRESUMO
Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.
Assuntos
Imunidade Celular , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Interleucina-33/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Subpopulações de Linfócitos T/imunologia , Triptofano Hidroxilase/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Suscetibilidade a Doenças , Regulação da Expressão Gênica/imunologia , Imunidade Inata , Imunidade nas Mucosas , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interleucina-33/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/patogenicidade , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nippostrongylus/crescimento & desenvolvimento , Nippostrongylus/patogenicidade , Cultura Primária de Células , Transdução de Sinais , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/parasitologia , Triptofano Hidroxilase/genéticaRESUMO
We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level.
Assuntos
Regiões Determinantes de Complementaridade/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Gêmeos Monozigóticos , Recombinação V(D)J/imunologia , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/classificação , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologiaRESUMO
PURPOSE: To evaluate peripheral blood immunological parameters and the possible correlation with age, gender and adenoid size in children with adenoid hypertrophy with OME. METHODS: A total of 664 children with adenoid hypertrophy were initially enrolled in our study, of which 83 had concomitant OME. To minimize selection bias, we performed one to two propensity score matching (PSM) between children with and without OME. After PSM, 80 children with OME (OME group) and 157 children without OME (adenoid hypertrophy [AH] group) were selected. The patients' peripheral blood samples were prepared prior to surgery and their immunological parameters were compared between groups. RESULTS: Compared to the AH group, the serum level of C3 was significantly higher in the OME group (0.88 ± 0.01 g/L vs. 0.94 ± 0.02 g/L; p = 0.014), which was the only independent risk factor for OME (odds ratio 13.58, 95% confidence interval 1.25-147.99; p = 0.032). However, no such difference was seen for serum immunoglobulin (IgG, IgA, IgM, IgE), T cell subsets (CD3+, CD4+ and CD8+ T cells), or lymphocytes and monocytes. Further subgroup analyses showed that in children ≤ 5 years old, the C3 level was significantly higher in OME patients (p = 0.023). A subgroup analysis based on sex indicated that there was a significantly higher level of serum C3 (p = 0.009) and lower CD3+ and CD4+ T cells (p = 0.010 and p = 0.021, respectively) in girls with OME compared to those without OME. No association between immunological parameters and adenoid size was found. CONCLUSIONS: There were no significant differences in cellular immunology and humoral immune indicators in children with adenoid hypertrophy with or without OME. In children ≤ 5 years old, significantly higher serum C3 levels in patients with OME demonstrate excessively activated C3 in comparison to patients without OME. For girls, a higher serum level of C3 with a lower amount of CD3+ and CD4+ T cells may be associated with OME.
Assuntos
Tonsila Faríngea , Complemento C3/análise , Imunoglobulinas/sangue , Otite Média com Derrame , Subpopulações de Linfócitos T/imunologia , Tonsila Faríngea/imunologia , Tonsila Faríngea/patologia , Pré-Escolar , Feminino , Humanos , Hipertrofia , Imunoglobulinas/classificação , Testes Imunológicos/métodos , Masculino , Tamanho do Órgão , Otite Média com Derrame/sangue , Otite Média com Derrame/diagnóstico , Pontuação de Propensão , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/classificaçãoRESUMO
BACKGROUND: Impaired T cell immune function exists in end-stage renal disease (ESRD) patients. Dialysis treatment may lead to changes in T cell subsets. In the present study, we aimed to identify alterations of T cell phenotypes in ESRD patients, especially in those receiving peritoneal dialysis (PD), and analyze the potential associated factors. METHODS: In the present study, 110 PD patients and 110 age/gender-matched hemodialysis (HD) patients who met the inclusion criteria were studied. Pre-dialysis blood samples were obtained and analyzed by flow cytometry to detect the expression of CD45RO and CCR7. Univariate and multivariate regression analyses were used to determine the factors associated with the alteration of T cell phenotypes. RESULTS: In all dialysis patients, age was associated with the frequencies of both CD4+ and CD8+ naïve T cells, effector memory (EM) T cells and effector memory RA (EMRA) T cells but not central memory (CM) T cells. Dialysis modality was also associated with T cell subsets. Compared with HD patients, PD patients showed an increase in both CD4+ and CD8+ CM T cells and a reduction in both CD4+ and CD8+ EM and EMRA T cells. However, the number of CD4+ naïve T cells was lower and the number of CD8+ naïve T cells was higher in PD patients than those in HD patients. In PD patients, further multivariate analysis revealed that the frequency of CD4+ naïve T cells was positively associated with nPCR, while the frequency of CD8+ naïve T cells was negatively associated with age. CONCLUSION: In dialysis patients, the dialysis modality and age influence T cell subsets. There is a progression from naïve to effector T cells in HD patients compared with PD patients. In PD patients, different factors may influence the frequencies of CD4+ and CD8+ naïve T cells.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Falência Renal Crônica/imunologia , Falência Renal Crônica/terapia , Fenótipo , Diálise Renal/métodos , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal , Receptores CCR7/análise , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/citologia , Adulto JovemRESUMO
The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age-related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR-specific antibodies available to study its immune system with respect to age- and cancer-related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross-reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing- and cancer-related research areas.
Assuntos
Anticorpos/isolamento & purificação , Subpopulações de Linfócitos B/imunologia , Células-Tronco Hematopoéticas/imunologia , Ratos-Toupeira/imunologia , Células Mieloides/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Anticorpos/química , Subpopulações de Linfócitos B/classificação , Subpopulações de Linfócitos B/citologia , Biomarcadores/análise , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Reações Cruzadas , Resistência à Doença/genética , Resistência à Doença/imunologia , Cobaias , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Longevidade/genética , Longevidade/imunologia , Camundongos , Células Mieloides/classificação , Células Mieloides/citologia , Baço/citologia , Baço/imunologia , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/citologiaRESUMO
How the age-associated decline of immune function leads to increased cancer incidence is poorly understood. Here, we have characterised the cellular composition of the γδ T-cell pool in peripheral lymph nodes (pLNs) upon ageing. We find that ageing has minimal cell-intrinsic effects on function and global gene expression of γδ T cells, and γδTCR diversity remains stable. However, ageing alters TCRδ chain usage and clonal structure of γδ T-cell subsets. Importantly, IL-17-producing γδ17 T cells dominate the γδ T-cell pool of aged mice-mainly due to the selective expansion of Vγ6+ γδ17 T cells and augmented γδ17 polarisation of Vγ4+ T cells. Expansion of the γδ17 T-cell compartment is mediated by increased IL-7 expression in the T-cell zone of old mice. In a Lewis lung cancer model, pro-tumourigenic Vγ6+ γδ17 T cells are exclusively activated in the tumour-draining LN and their infiltration into the tumour correlates with increased tumour size in aged mice. Thus, upon ageing, substantial compositional changes in γδ T-cell pool in the pLN lead to an unbalanced γδ T-cell response in the tumour that is associated with accelerated tumour growth.