Journal: PLoS Pathogens

Article Title: LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma

doi: 10.1371/journal.ppat.1006503

Figure Lengend Snippet: ( A ) Representative IHC staining for LMP1 and GLUT1 in NPC specimens. ( B-D ) Statistical analyses of the correlations between LMP1 expression and CD33 + MDSC number ( B ), GLUT1 expression and CD33 + MDSC number ( C ), and LMP1 and GLUT1 expression level ( D ), Pearson’s chi-square test was used to assess statistical significance. ( E-F ) Kaplan-Meier estimates of the progression-free interval were determined based on the median levels of LMP1 ( E ) and GLUT1 ( F ) in the patients with NPC. P < 0.05 was considered statistically significant.

Article Snippet: Tumor-associated MDSCs were generated from CD33 + cells isolated by anti-CD33 beads (Miltenyi Biotec Company) from peripheral blood mononuclear cells (PBMCs) of healthy donors in a co-culture Transwell system (0.4-μm pore, Corning) with the NPC cell lines TW03, CNE2, TW03-LMP1 or CNE2-LMP1 as previously described.

Techniques: Immunohistochemistry, Expressing

Journal: PLoS Pathogens

Article Title: LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma

doi: 10.1371/journal.ppat.1006503

Figure Lengend Snippet: CD33 + cells were isolated from healthy PBMCs using human CD33 MicroBeads and were co-cultured with NPC or NPC-LMP1 cells in a Transwell system for 48 h. The percentage of CD33 + CD11b + HLA-DR - MDSCs was measured by FACS staining. ( A ) Expression of the NLRP3, ASC, caspase-1, IL-1β, COX-2, Arg-1 and iNOS mRNAs was determined via real-time qRT-PCR, and the results were normalized against GAPDH expression. ( B ) NPC cells, including CNE2-vector, CNE2-LMP1, TW03-vector, and TW03-LMP1 cells, were cultured overnight following stimulation with LPS (0.1 μg/mL) and ATP (5 mM, 30 min) or no stimulation. Then, the culture supernatants were collected, and the concentrations of IL-1β, IL-6 and GM-CSF were measured by ELISA. ( C ) Representative density plots are shown as the CD33 + CD11b + cells in the HLA-DR - gate induced by NPC or NPC-LMP1 cells in different combinations. Representative data from 1 of 5 experiments are presented. ( D ) Statistical analysis of the percentage of MDSCs mediated by NPC cells in different combinations. CFSE-labeled PBMCs were co-cultured with M-MDSCs, CNE2-vector-MDSCs, CNE2-LMP1-MDSCs, TW03-vector-MDSCs or TW03-LMP1-MDSCs at a ratio of 1:1 in OKT3-coated 96-well plates. After 3 days, the cells were collected, stained with anti-human mAbs against CD4 and CD8 and quantified by flow cytometry. Representative FACS density plots ( E ) and the statistical data ( F ) showed that the proliferation of PBMCs and CD4 + and CD8 + T cells was markedly suppressed by CNE2-LMP1-MDSCs or TW03-LMP1-MDSCs compared with the effects of CNE2-vector-MDSCs or TW03-vector-MDSCs. Data are presented as the means ± SEM of representative experiments performed in triplicate. *P < 0.05, **P < 0.01 compared with the control treatment.

Article Snippet: Tumor-associated MDSCs were generated from CD33 + cells isolated by anti-CD33 beads (Miltenyi Biotec Company) from peripheral blood mononuclear cells (PBMCs) of healthy donors in a co-culture Transwell system (0.4-μm pore, Corning) with the NPC cell lines TW03, CNE2, TW03-LMP1 or CNE2-LMP1 as previously described.

Techniques: Isolation, Cell Culture, Staining, Expressing, Quantitative RT-PCR, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry

Journal: PLoS Pathogens

Article Title: LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma

doi: 10.1371/journal.ppat.1006503

Figure Lengend Snippet: ( A ) WB showed the level of GLUT1 in CNE2-LMP1-siNC, CNE2-LMP1-siGLUT1, TW03-LMP1-siNC and TW03-LMP1-siGLUT1 cells, and GAPD was included as the control. ( B and C ) Representative ECAR assays for CNE2-LMP1-siNC, CNE2-LMP1-siGLUT1, TW03-LMP1-siControl and TW03-LMP1-siGLUT1 cells. The time course and calculations for ( B ) glycolytic capacity and ( C ) statistical analysis of glycolytic capacity and glycolytic reserve are shown. ( D ) Expression of the NLRP3, ASC, caspase-1, IL-1β, COX-2, Arg-1 and iNOS mRNAs was determined via qRT-PCR in CNE2-LMP1-siControl, CNE2-LMP1-siGLUT1, TW03-LMP1-siControl and TW03-LMP1-siGLUT1 cells. ( E ) Representative immunoblotting of NPC-vector, NPC-LMP1, NPC-LMP1-siNC and NPC-LMP1-siGLUT1 cells stained with the indicated antibodies (n = 5). ( F ) Secretion of the cytokines IL-1β, IL-6 and GM-CSF from NPC-LMP1 cells following treatment with siGLUT1, siControl (siNC) or 2-DG was determined via ELISA. ( G ) Statistical analysis of the percentage of CD33 + CD11b + HLA-DR - MDSCs mediated by CNE2-LMP1 or TW03-LMP1 cells following treatment with siGULT1, siNC or 2-DG. Data are presented as the means ± SEM of representative experiments performed in triplicate. *P < 0.05, **P < 0.01 compared with the control treatment.

Article Snippet: Tumor-associated MDSCs were generated from CD33 + cells isolated by anti-CD33 beads (Miltenyi Biotec Company) from peripheral blood mononuclear cells (PBMCs) of healthy donors in a co-culture Transwell system (0.4-μm pore, Corning) with the NPC cell lines TW03, CNE2, TW03-LMP1 or CNE2-LMP1 as previously described.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Staining, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma

doi: 10.1371/journal.ppat.1006503

Figure Lengend Snippet: ( A ) Representative immunoblots of CNE2-LMP1 and TW03-LMP1 cells treated with siNC or siNLRP3 and stained with the indicated antibodies (n = 3). ( B ) Secretion of the cytokines IL-1β, IL-6 and GM-CSF from NPC-LMP1 cells following treatment with si-NLRP3, siControl (siNC) or VX-765 was determined via ELISA. ( C ) Blockade of NLRP3 decreased the differentiation of HLA-DR - CD11b + CD33 + MDSCs mediated by NPC-LMP1. Representative data from 5 independent experiments are shown. ( D ) Statistical analysis of the percentage of CD33 + CD11b + HLA-DR - MDSCs mediated by CNE2-LMP1 or TW03-LMP1 cells following treatment with si-NLRP3, siNC or VX-765. Representative data from 3 independent experiments are shown.

Article Snippet: Tumor-associated MDSCs were generated from CD33 + cells isolated by anti-CD33 beads (Miltenyi Biotec Company) from peripheral blood mononuclear cells (PBMCs) of healthy donors in a co-culture Transwell system (0.4-μm pore, Corning) with the NPC cell lines TW03, CNE2, TW03-LMP1 or CNE2-LMP1 as previously described.

Techniques: Western Blot, Staining, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: The lifespan and kinetics of human dendritic cell subsets and their precursors in health and inflammation

doi: 10.1084/jem.20220867

Figure Lengend Snippet: Characterization of human DC subsets. (a) Polychromatic flow cytometry gating strategy for blood DC subsets. Peripheral blood DC cells were identified as Lin − HLA-DR + cells. The DC subsets consist of pDC (CD123 + AXL − : orange gated population) and ASDC (CD123 + CD5 + AXL + Siglec6 + ; purple gated population). cDC could be identified as CD123 − CD11c + cells with cDC1 being CD141 + (red gated population), while cDC2 expressed CD1c + FceR1a + and was further divided into CD5 + DC2 and CD5 − DC3 ( ; ). (b) Polychromatic flow cytometry gating strategy for blood DC subsets sometimes identified a distinct HLA-DR + Lin − CD45RA − CD123 + population (red square). Histogram below shows this population stained positive for the basophil marker CD203c (black) or FMO (grey). (c) Flow cytometry analysis of human DC subsets in the bone marrow. (d) Pie chart depicting the percentage of each DC population in the bone marrow and blood. The table shows the mean total DC in the blood and bone marrow, cells/person, from eight individuals. (e) The approximate daily proliferation rate was estimated for each population. The proliferation rate was defined as p = (fraction_SG2M/SG2M_length) * 24 h. Combining 10,000 bootstrap samples of the S/G2/M measurments (only including non-zero measurements) with samples drawn from a Uniform distribution (U[5, 15 h]), the approximate proliferation rate of all subsets was calculated. The prior distribution of a subset’s proliferation rate was approximated by fitting a log-normal distribution to the bootstrap samples. The fitted parameters µ and σ, which are the mean and standard deviation of the samples’ natural logarithm, respectively, are shown.

Article Snippet: Siglec6 , Human , PE , REA852 , Miltenyi Biotec , 130 112 710.

Techniques: Flow Cytometry, Staining, Marker, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: The lifespan and kinetics of human dendritic cell subsets and their precursors in health and inflammation

doi: 10.1084/jem.20220867

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: Siglec6 , Human , PE , REA852 , Miltenyi Biotec , 130 112 710.

Techniques:

Journal: Frontiers in Molecular Biosciences

Article Title: ARPC1B is a novel prognostic biomarker for kidney renal clear cell carcinoma and correlates with immune infiltration

doi: 10.3389/fmolb.2023.1202524

Figure Lengend Snippet: Relationship between CD8 + T cell and MDSCs and the expression of ARPC1B. (A–B) CD8 + T-cell expression was observed in KIRC and normal renal tissues (arrow indicates the clustering of CD8 + T cells). (C) Correlation between the number of CD8 + T cells and the expression of ARPC1B in KIRC. (D) Differences in the number of CD8 + T cells between KIRC and normal tissues. (E–F) CD33 + MDSCs expression observed in KIRC and normal renal tissue (arrow indicates the clustering of CD33 + MDSCs). (G) Correlation between the number of CD33 + MDSCs and the expression of ARPC1B in KIRC. (H) Differences in the number of CD33 + MDSCs between KIRC and normal tissues. (I) Correlation between the number of CD8 + T cells and number of CD33 + MDSCs in KIRC. NS (not significant with a p -value greater than 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Subsequently, endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min. Then, the FFPE sections were incubated with ARPC1B rabbit polyclonal antibody (1:500, Bioss Biotechnology, China), CD8 monoclonal antibody (working solution, Agilent Dako, Denmark), and CD33 rabbit polyclonal antibody (1:400, Bioss Biotechnology, China), followed by incubation with HRP secondary antibody, and finally stained with peroxidase/DAB immunohistochemistry.

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: STAT3 promotes differentiation of monocytes to MDSCs via CD39/CD73-adenosine signal pathway in oral squamous cell carcinoma

doi: 10.1007/s00262-022-03336-9

Figure Lengend Snippet: Correlation of phosphorylated STAT3 with markers of monocytes, MDSCs and CD39/CD73 in OSCC. a Representative HE-stained images of tumor tissue and representative immunohistochemical staining of p-STAT3, CD39, CD73, CD14 and CD33 in OSCC tissue (Scale bar, 100 μm (left), 50 μm (right)). b and c The expression of p-STAT3 was significantly correlated with CD39 (p < 0.05, r = 0.29), CD73 (p < 0.05, r = 0.26), CD14 (p < 0.01, r = 0.35) and CD33 (p < 0.05, r = 0.31) in human OSCC by analyzing the tissue microarray immunohistochemical staining (n = 63). d Hierarchical clustering reveals close relation of p-STAT3, CD39, CD73, CD14 and CD33 in human OSCC (n = 63)

Article Snippet: The primary antibodies for our experiment included the following: p-STAT3 Tyr705 (CST, 9145), CD39 (Proteintech, 19229-1-AP), CD73 (CST, 13160), CD14 (Affinity, DF13278), CD33 (Affinity, DF6789), CD4 (CST, 92599), CD8 (CST, 98941), STAT3 (Proteintech, 60199-1-Ig).

Techniques: Staining, Immunohistochemical staining, Expressing, Microarray