Journal: Scientific Reports

Article Title: Indoxyl sulfate (IS)-mediated immune dysfunction provokes endothelial damage in patients with end-stage renal disease (ESRD)

doi: 10.1038/s41598-017-03130-z

Figure Lengend Snippet: CD4 + CD28 − T cells expressing CX3CR1, a receptor for CX3CL1, are expanded under the TNF-α rich environment in ESRD patients. ( A ) Frequencies (%) of CD28 − cells in CD4 + and CD8 + T cells in ESRD patients (n = 50) and age-matched HCs (n = 28). ( B ) Purified CD4 + CD28 + cells from ESRD patients were stimulated with α-CD3/CD28 Ab-coated beads and IL-2 in the absence or presence of TNF-α. At 4 days, beads were removed using a magnet and the cytokines were re-supplemented every 3–4 days. CD28 expression was analyzed every 7 days by flow cytometry. Representative FACS plot of change in CD28 expression on cultured CD4 + CD28 + T cells in ESRD patients under indicated culture conditions (Left). On the indicated day, cultured cells were harvested and the frequency of CD28 − cells was determined by flow cytometry (n = 4) (Right). ( C ) Purified naive CD4 + T cells were stimulated with α-CD3/CD28 Ab-coated beads and IL-2. At 4 days, beads were removed using a magnet and the cell were co-cultured with monocytes, which were stimulated with IS (1,000 μM) for 24 hr. IS-stimulated CD14 + monocytes were re-supplemented every 3–4 days. CD28 expression was analyzed every 7 days by flow cytometry. Representative FACS plot of change in CD28 expression on cultured naive CD4 + T cells under indicated culture conditions (Left). On the indicated day, cultured cells were harvested and the frequency of CD28 − cells was determined by flow cytometry (n = 3) (Right). ( D ) Representative contour plot of CX3CR1 expression on CD4 + CD28 + and CD4 + CD28 − T cells from ESRD patients and HCs ( E ) Expanded CX3CR1 + CD4 + T cells in patients with ESRD compared with HCs. ( F ) Frequency (%) of CX3CR1 + cells positively correlates with the frequency of CD28 − cells in CD4 + T cells of ESRD patients (n = 46). Each data point represents an individual subject. ( G ) Freshly-purified CD4 + memory T cells from ESRD patients were stained with APC-conjugated anti-CD28 mAb and a chemotaxis assay was performed at various concentrations of CX3CL1 (0 to 10 ng/ml) for 2 hours using a transwell system. The frequency (%) of CD28 − T cells in migrated cells at various concentrations of CX3CL1 was analyzed by flow cytometry. Bar graphs show the mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.005 by two-tailed unpaired t -test ( A and E ) or 2 way ANOVA test ( B and C ). P value in ( F ) was obtained using the Pearson correlation analysis. Box plots displaying medians, 25th and 75th percentiles as boxes, and minimum and maximum values as whiskers (n = 6). * p < 0.05 by two-tailed paired non-parametric test ( G ).

Article Snippet: The following antibodies were used for flow cytometric analysis: Anti-Perforin-Alexa fluor 488, anti-CD28-allophycocyanin (APC), anti-CD19-APC, anti-CD3-APC, anti-CD56-APC, anti-CD3-APC-cyanin7 (Cy7), anti-CD8-APC-Cy7, anti-CD14-APC-Cy7, anti-CD14-fluorescein isothiocyanate (FITC), anti-HLA-DR-FITC, anti-CD16-R-phycoerythrin (PE), anti-GATA3-PE, anti-CD45RA-PE-cyanin 5 (Cy5), anti-CD4-PE-Cy5, anti-CD16-PE-Cy5, anti-CCR7-PE-Cy7, anti-CD4-PE-Cy7, anti-CD4-V450, anti-GranzymeB-V450, anti-CD8-V500, anti-CD3-V500 (all from BD Bioscience, Franklin Lakes, NJ), anti-CD57-FITC, anti-CD4-FITC, anti-CD85j-PE, anti-CX3CR1-PE, anti-T-bet-PE-Cy7, anti-Eomes-Peridinin chlorophyll (PerCP)-efluor710 (six from eBioscience, San Diego, CA), anti-HLA-DR-PE-Cy5, anti-IL-7Rα-V450, anti-CD57-V450 (three from BioLegend, San Diego, CA), anti-CX3CR1-FITC (MBL International Corporation, Woburn, MA).

Techniques: Expressing, Purification, Flow Cytometry, Cell Culture, Staining, Chemotaxis Assay, Two Tailed Test

Journal: Oncoimmunology

Article Title: Dasatinib promotes Th1-type responses in granzyme B expressing T-cells

doi: 10.4161/onci.28925

Figure Lengend Snippet: Figure 1. The proportion of granzyme B positive (GrB+) T-cells is increased in CML patients at diagnosis and further expands during dasatinib therapy. Fresh or frozen PBMNCs were first stained for surface markers (α-CD45, α-CD3, α-CD4 and α-CD8), and after fixation and permeabilization intracellular GrB was stained, and cells were analyzed with flow cytometry. ( A ) The relative proportions of GrB+CD4+ T-cells and ( B ) GrB+CD8+ T-cells in samples obtained from healthy controls (n = 5) and CML patients at diagnosis (dg) (n = 18). ( C ) The proportion of GrB+CD4+ T-cells and ( D ) GrB+CD8+ T-cells 6 mo after start on dasatinib (DA, n = 7), imatinib (IM, n = 6), or nilotinib (NI, n = 6) therapy. The absolute number of GrB+CD4+ ( E ) and GrB+CD8+ T-cells ( F ) was measured in CML patients 6 mo after the start of DA (n = 8), IM (n = 6), or NI (n = 5) therapy. Panels A and B were analyzed by nonparametric Mann Whitney t test and panels C, D and E by 1way ANOVA.

Article Snippet: For the analysis of GrB+ T-cells, the cells were first stained for cell surface markers; α-CD45 APC-H7 (BD 641417), α-CD3 APC (BD 345767), α-CD4 PerCP (BD 345770), and α-CD8 PE-Cy7 (BD 335822), and then fixed and permeabilized with Fix/Perm (BD 554714), and intracellular GrB was stained (Alexa Fluor 700, BD 561016)).

Techniques: Staining, Flow Cytometry, MANN-WHITNEY