Journal: bioRxiv

Article Title: IL-21 and IFN-alpha have both opposite and redundant role on human innate precursors and memory B-cell differentiation

doi: 10.1101/2021.03.31.437810

Figure Lengend Snippet: (A) Representative flow cytometry plots of CD38 and CD27 expression on CD19 + B cells. Three populations, observed after stimulation (priming), are gated: CD27 high CD38 high B cells (PBs, plasmablasts in black), CD27 + CD38 int (Int, intermediate B cells in dark grey), CD27 - CD38 + (Act, activated B cells in light grey). Mean ± SEM percentages (below) of Act, Int and PBs in each B-cell subset. (N= 5 to 14) (B) Assessment by ELISPOT of the Ig secretory function in primed B cells. The right plot depicted the percentages of IgM-(blue), IgA-(yellow), IgG-(red) secreting B cells (C) Representative flow cytometry plots of IRF4 and IRF8 intracellular staining. Black gates depicted IRF4 + IRF8 low differentiated B cells and grey gates the IRF4 low IRF8 + activated B cells. Mean ± SEM percentages (right) of IRF4 + IRF8 low B cells. Data are representative of N= 14 (NA); N= 5 (DN); N= 7 (USM), and N= 11 (SM) independent experiments. Below are the histogram overlays of the expression of BLIMP1 in IRF4 + IRF8 low (black) and IRF4 low IRF8 + (grey) populations. Statistical significance in (A, C) was determined by a one-way paired ANOVA test using Tukey’s correction for multiple comparisons. Error bars indicate mean ± SEM. ** p<0.01, *** p<0.001. NA: Naive B cells, USM: Unswitched memory B cells, SM: Switched memory B cells, DN: Double-negative B cells.

Article Snippet: CD19 + B cells were purified from human PBMCs using the REAlease® CD19 Microbead Kit (Miltenyi Biotec) according to the manufacturer’s recommendations with purity greater than 98 %.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunospot, Staining

Journal: Communications Biology

Article Title: CISH, a key intracellular checkpoint, in comparison and combination to existing and emerging cancer immune checkpoints

doi: 10.1038/s42003-026-09579-x

Figure Lengend Snippet: a NALM6 (GFP + FF-Luc + ) target cell killing by CD8 + CD19-CAR + T cells in the presence (Control) or absence of CISH (CISH) at effector: target ratio 1:2. Cytolysis was measured by remnant luciferase activity assay after 18 h of co-culture. b Left, CD19 expression (colored) on engineered NALM6 target cells compared with unstained cells (gray). Right, CD19-CAR + T cell killing of NALM6 cells expressing varying levels of CD19. c Effector cytokine (IFNγ) levels in CD19 stimulated CD19-CAR + CD8 + cells, as measured by ELIZA. a–c : Statistical significance was determined by Two-way ANOVA vs. Control: Not shown = not significant, P values shown in graphs or *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. All data is representative of at least three independent experiments. N = 10 donors. Error bars represent mean ± SEM. d–f Cytokine profile of CISH depleted CD19-CAR + T cells after overnight co-culture with CD19 expressing NALM6 cells (as measured by nELISA). d Pathway analysis by Reactome.org. Graph shows significantly regulated pathways; p ≤ 0.05 (x-axis: Pathway Hierarchy/ Go Biological Process; y-axis; -Log 10 (p-value)). e Volcano plot (Log 2 FC vs. -Log 10 (p-value); Left panel) and bar graph (Log 2 FC ≥ 1.2); Right panel) of differentially regulated secreted factors in CISH KO vs. Control CD19-CAR + CD8 + cells after overnight co-culture with CD19 WT NALM6 cells. f Significantly downregulated factors in CISH depleted CD19 stimulated CD19-CAR + CD8 + cells. d–f Statistical significance was determined by either multiple t-test or Two-way ANOVA vs. Control in each treatment. Not shown = not significant, P values shown in graphs or * P ≤ 0.05; ** P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. N = 3 donors. Data are mean ± SD.

Article Snippet: If needed, CD19-CAR + CD8 + cells were enriched using the MACSprepTM CD19 CAR MicroBead Kit following manufacturer’s instructions (Miltenyi Biotec, 130-127-866).

Techniques: Control, Luciferase, Activity Assay, Co-Culture Assay, Expressing