RESUMEN
CD8+ T cells, a fundamental part of the adaptive immune system, employ cytotoxic responses important for targeting pathogenic bacteria, viruses, and tumor cells. During early activation, CD8+ T cells undergo many changes in metabolism and gene expression. The bridge between epigenetic and metabolic influences on gene expression and cell fate has yet to be fully understood. Here, we investigated the importance of ATP citrate lyase (Acly), an enzyme involved in both metabolism and histone acetylation, for early stages of CD8+ T cell activation. We performed polyclonal activation of murine CD8+ T cells in vitro in the presence or absence of the Acly inhibitor BMS303141. We found that inhibiting Acly during early activation results in decreased expression of early activation markers. Consistent with impaired early activation, we found that inhibition also resulted in increased uptake of fatty acids and decreased glucose uptake without changing mitochondrial ATP levels. On an epigenetic and transcriptional level, early stage Acly inhibition specifically downregulated promoter histone H3 acetylation (H3ac) and expression of the key transcription factor IRF4; however, global levels of H3ac remained similar. Most importantly, the study was able to highlight the importance of Acly in early stages of CD8+ T cell activation and histone regulation.
Asunto(s)
ATP Citrato (pro-S)-Liasa , Antineoplásicos , ATP Citrato (pro-S)-Liasa/genética , ATP Citrato (pro-S)-Liasa/metabolismo , Acetilación , Animales , Linfocitos T CD8-positivos/metabolismo , Histonas/metabolismo , RatonesRESUMEN
With the release and use of the Becton Dickenson FACS Diva Software, the use of Area as the default parameter came into play. As such, the use of area as a calculated parameter, methods were needed to be employed to ensure doublet discrimination and proper display on standard FSC/SSC. Improper setting of forward area scaling can alter the display cell populations. This combined with improper area gating strategy can lead to doublet inclusion which in sorting rare events can compromise sort purity. In extreme cases where area scaling with the individual lasers is ignored, differences can exist between Area and Height where compensation will likely not be optimal, particularly if one parameter - usually height is saturated. In addition, area scaling can impact population grouping. As FSC and individual laser area scaling is a function of event size, the most common error is to accept the setting determined by CS&T, which are 3.2⯵m particles and proceed with the sample(s) without regard to the sample's actual size. With cellular events smaller or more likely larger than the CS&T beads, this will make the area scaling settings less than optimal. Analysis and sorting rare events with populations larger than the CS&T beads can be compromised if adjustments in FSC area scaling are not addressed. Proper FSC and laser area scaling must be determined empirically for each sample. Examples of the effects of sample size on area scaling will be presented in addition to gating and templates for determining area scaling.
Asunto(s)
Citometría de Flujo/métodos , Programas Informáticos , Células Hep G2 , Humanos , Rayos Láser , LuzAsunto(s)
COVID-19/epidemiología , Guías como Asunto/normas , Exposición Profesional/normas , Pandemias , Equipo de Protección Personal/normas , Distanciamiento Físico , COVID-19/prevención & control , Humanos , Exposición Profesional/prevención & control , Pandemias/prevención & control , Teletrabajo/normasRESUMEN
Granulocytes play a key role in innate immunity and the most common functional assays are phagocytosis and oxidative burst. The purpose of this technical note is to use image-based flow cytometry to divide activated granulocytes into unique subsets based on their degree of phagocytosis and oxidative burst in response to different experimental incubations. Prior to the experiments, all reagents were titered to determine the lowest dose that resulted in an acceptable signal to noise ratio. Heparinized, whole blood (100 µl) was mixed with one of two bioparticles (E. coli and S. aureus) and DHE (10 µg/ml) and incubated for 5, 10, 20, 40, 60, 80, 100, 120, and 140 min in a 37°C water bath. An additional tube kept on ice was used as a negative control. All subsequent processing steps were completed on ice in the dark to minimize additional activation of cells. After the 37°C incubation, N-ethylmaleimide (15 mM) was added to halt phagocytosis, preventing the uptake of additional microparticles. Suspensions were labeled with CD66b-APC and CD45-APCeFluor780 for 60 min and a fix/lyse solution was added. Prior to acquisition, 7AAD was added to stain nuclear DNA. A minimum of 5,000 granulocyte (CD66b+) events were acquired using a Millipore-Amnis FlowSight equipped with blue (488 nm, 60 mW), red (642 nm, 100 mW), and side scatter (785 nm, 12 mW) lasers. Samples were compensated and analyzed using Amnis IDEAS software (v.5.0.983.0). Image-based analysis allowed us to divide activated granulocytes into three distinct subsets, whose relative abundance changed as a function of both bioparticle type and incubation length. The method described in this technical note represents a potential novel adaptation to common methods of assessing granulocyte function. More research is needed to test and validate our image-based method in clinical conditions that impair granulocyte function.
Asunto(s)
Citometría de Flujo/métodos , Granulocitos/fisiología , Fagocitosis , Estallido Respiratorio , Escherichia coli/inmunología , Granulocitos/inmunología , Granulocitos/microbiología , Humanos , Relación Señal-Ruido , Staphylococcus aureus/inmunologíaRESUMEN
A major rate-limiting step in developing more effective immunotherapies for GBM is our inadequate understanding of the cellular complexity and the molecular heterogeneity of immune infiltrates in gliomas. Here, we report an integrated analysis of 201,986 human glioma, immune, and other stromal cells at the single cell level. In doing so, we discover extensive spatial and molecular heterogeneity in immune infiltrates. We identify molecular signatures for nine distinct myeloid cell subtypes, of which five are independent prognostic indicators of glioma patient survival. Furthermore, we identify S100A4 as a regulator of immune suppressive T and myeloid cells in GBM and demonstrate that deleting S100a4 in non-cancer cells is sufficient to reprogram the immune landscape and significantly improve survival. This study provides insights into spatial, molecular, and functional heterogeneity of glioma and glioma-associated immune cells and demonstrates the utility of this dataset for discovering therapeutic targets for this poorly immunogenic cancer.
Asunto(s)
Inmunoterapia , Proteína de Unión al Calcio S100A4/aislamiento & purificación , Análisis de la Célula Individual/métodos , Animales , Neoplasias Encefálicas/inmunología , Femenino , Glioma/inmunología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Mieloides , Pronóstico , Proteína de Unión al Calcio S100A4/genética , Microambiente Tumoral/inmunologíaRESUMEN
During acquired immunity to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production. As IL-12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL-12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5-deficient (C5(-/-)) and -sufficient (C5(+/+)) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5(-/-) DCs secreted less IL-12p70 (600 pg/mL vs. 100 pg/mL) than C5(+/+) DCs, and they secreted more IL-10. Using immunophenotyping, reduced CD40 expression was found on C5(-/-) DCs after BCG infection. BCG-primed DCs were then cocultured with naive or BCG-immune T cells to differentiate them into IFN-gamma-secreting Th1 T cells. Coincident with increased IL-12p70 levels, BCG-primed C5(+/+) DCs cocultured with naive or immune C5(+/+) T cells showed a larger increase in CD4+ IFN-gamma/CD8+ IFN-gamma+ T cells compared with cocultured DCs and T cells from C5(-/-) mice. Thus, BCG-primed C5(+/+) DCs were better able to drive a Th1 response. Furthermore, BCG aerosol-infected C5(-/-) mice showed reduced CD4 and CD8 IFN-gamma-secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL-12p70 secretion from DCs.
Asunto(s)
Complemento C5a/inmunología , Células Dendríticas/inmunología , Interleucina-12/inmunología , Mycobacterium bovis/inmunología , Células TH1/inmunología , Tuberculosis/inmunología , Animales , Antígenos CD40/biosíntesis , Antígenos CD40/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Células Cultivadas , Técnicas de Cocultivo , Complemento C5a/biosíntesis , Complemento C5a/genética , Células Dendríticas/metabolismo , Células Dendríticas/patología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-12/biosíntesis , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Noqueados , Células TH1/metabolismo , Células TH1/patología , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis/patología , Tuberculosis/veterinariaRESUMEN
Chronic vascular disease is partially characterized by the presence of lesions along the vascular endothelial wall. Current FDA-approved clinical techniques lack the ability to measure very early changes in endothelial cell health. When endothelial cells are damaged, they release endothelial microparticles (EMPs) into circulation. Thus, blood EMP concentration may represent a useful cardiovascular disease biomarker. Despite the potential value of EMPs, current flow cytometry techniques may not consistently distinguish EMPs from other small cell particles. The purpose of this study was to use imaging flow cytometry to modify existing methods of identifying EMPs based on cell-surface receptor expression and visual morphology. Platelet poor plasma (PPP) was isolated using four different techniques, each utilizing a two-step serial centrifugation process. The cell-surface markers used in this study were selected based on those that are commonly reported in the literature. PPP (100µL) was labeled with CD31, CD42a, CD45, CD51, CD66b, and CD144 for 30-min in dark on ice. Based on replicated experiments, EMPs were best identified by cell-surface CD144 expression relative to other commonly reported EMP markers (CD31 & CD51). It is important to note that contaminating LMPs, GMPs, and PMPs were thought to be removed in the preparation of PPP. However, upon analysis of prepared samples staining CD31 against CD51 revealed a double-positive population that was less than 1% EMPs. In contrast, when using CD144 to identify EMPs, ~87% of observed particles were free of contaminating microparticles. Using a counterstain of CD42a, this purity can be improved to over 99%. More research is needed to understand how our improved EMP measurement method can be used in experimental models measuring acute vascular responses or chronic vascular diseases.
Asunto(s)
Aterosclerosis/diagnóstico , Micropartículas Derivadas de Células/inmunología , Células Endoteliales/inmunología , Receptores de Superficie Celular/inmunología , Antígenos CD/inmunología , Antígenos de Superficie/inmunología , Aterosclerosis/inmunología , Biomarcadores/sangre , Plaquetas/citología , Cadherinas/inmunología , Centrifugación/métodos , Células Endoteliales/citología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Citometría de Flujo , Humanos , Integrina alfaV/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Coloración y EtiquetadoRESUMEN
Prior to imaging agent use in preclinical studies and clinical diagnostics, biological activity must be validated. The Lindmo assay has been used conventionally to quantify radiolabeled antibody (Ab) immunoreactivity, although published findings suggest it does not provide consistently accurate results. We developed and tested a near-infrared (NIR) flow cytometry (FC) method for quantifying biological activity of a dual-labeled Ab for use as a multimodal contrast agent in small animal and human positron emission tomography and NIR fluorescence imaging. Antibody specific for epithelial cell adhesion molecule was conjugated to DOTA-NHS-ester, labeled with IRDye 800CW and further labeled with (64)Cu or nonradioactive Cu prior to reacting with human prostate cancer cells for testing by the Lindmo or FC method, respectively. Immunoreactivity of the dual-labeled agent was found to be 76.4 ± 15.7% by the Lindmo assay. When tested with and without Cu labeling using NIR FC, the biological activity was found to be 73.1 ± 7.7 and 79.4 ± 8.1%, respectively. No significant differences were found between these activity levels (p > 0.05), supporting NIR FC as an alternative method for measuring immunoreactivity and demonstrating that Cu labeling does not significantly affect the agent's ability to bind to its target. Biological activity was significantly reduced when the NIR dye-to-protein ratio was increased 3- to 4-fold in agent preparations when tested by FC and the Lindmo assay. In summary, NIR FC is an alternative with similar specificity and sensitivity, and greater reproducibility relative to the Lindmo assay for quantifying biological activity of NIR fluorophore-labeled, multimodal imaging agents.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Medios de Contraste , Citometría de Flujo , Colorantes Fluorescentes , Imagen Molecular/métodos , Radioinmunoensayo/métodos , Espectroscopía Infrarroja Corta , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/inmunología , Molécula de Adhesión Celular Epitelial , Humanos , Indoles , Masculino , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Sensibilidad y Especificidad , Succinimidas/química , Células Tumorales CultivadasRESUMEN
Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Alveolos Pulmonares/citología , Diferenciación Celular , Línea Celular , Complemento C3/metabolismo , Complemento C5/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Pulmón/metabolismo , Microscopía Electrónica de Transmisión , Modelos Genéticos , Regiones Promotoras Genéticas , Tensoactivos/química , alfa 1-Antitripsina/metabolismoRESUMEN
The C3a anaphylatoxin is a 77-amino acid peptide that is generated by enzymatic cleavage of C3 during activation of the complement system. C3a mediates numerous biological functions on binding its receptor (C3aR), which is present on both myeloid and nonmyeloid cells. To investigate the biological impact of C3a-mediated effects during acute pneumonia caused by Pseudomonas aeruginosa, we subjected C3aR-deficient mice and matched wild-type (WT) mice to P. aeruginosa pulmonary infection. C3aR-deficient mice exhibited increased killing of P. aeruginosa in the lungs, less dissemination of bacteria into the bloodstream, and a decreased inflammatory response to P. aeruginosa pulmonary infection compared with WT mice. To examine whether the absence of C3aR would impact the humoral immune response to P. aeruginosa, we immunized WT and C3aR-deficient mice via intraperitoneal injection with live P. aeruginosa. Both groups of mice developed similar levels of antibody specific to P. aeruginosa. Immunized C3aR-deficient and WT mice were subjected to P. aeruginosa pulmonary infection, and C3aR-deficient mice again displayed increased killing of P. aeruginosa in the lungs, less dissemination of bacteria into the bloodstream, and a decreased inflammatory response in the lungs. Collectively, these data demonstrate that independently of antibody production, absence of C3aR causes enhanced killing of P. aeruginosa despite a diminished inflammatory response in a mouse model of pneumonia.
Asunto(s)
Proteínas de la Membrana/metabolismo , Neumonía , Pseudomonas aeruginosa/inmunología , Receptores de Complemento/metabolismo , Animales , Anticuerpos/sangre , Líquido del Lavado Bronquioalveolar/citología , Quimiocinas/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Pulmón/citología , Pulmón/metabolismo , Pulmón/patología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Receptores de Complemento/genéticaRESUMEN
To identify additional members of the murine N-formyl-Met-Leu-Phe peptide receptor family (fMLF-R), a mouse macrophage cDNA library was screened using the open reading frame of murine N-formyl peptide receptor. Four individual hybridizing cDNA clones were maintained through tertiary screening. One cDNA clone was a truncated, polyadenylated version of the previously described murine-fMLF-R. The other three cDNA clones varied in length, but contained identical open reading frame sequences. One clone, 8C10, was selected for further study and shared 70% sequence identity with murine-fMLF-R and 89% sequence identity with murine lipoxin A4 receptor cDNA. When placed into the pcDNA-3 expression vector and cotransfected with Galpha16 cDNA into COS-1 cells, 8C10 cDNA induced the production of inositol-1,4,5-triphosphate when concentrations of 1-1600 nM lipoxin A4 (LXA4) were tested as ligands. Northern blot analysis of murine organs indicated that the 8C10 message is present in lung, spleen, and adipose tissue. Moreover, mice treated with LPS demonstrated increased expression of 8C10 message in spleen and adipose tissue, while showing a slight reduction in lung. We have also characterized the 8C10 structural gene from a 129Sv/J genomic library and have determined its size to be >6.1 kb in length and comprised of two exons separated by a 4.8-kb intron. Collectively, these data indicate that this homologue receptor is closely related to the murine LXA4 receptor and functionally responds to LXA4 as a ligand.