Journal: bioRxiv

Article Title: Multiparametric optimization of human primary B-cell cultures using Design of Experiments

doi: 10.1101/2025.01.16.633474

Figure Lengend Snippet: Stepwise gating of human PBMCs, exemplifying the lymphocyte/singlet-FSC/singlet-SSC/live/CD19 + hierarchy used in subsequent analyses.

Article Snippet: Human CD19 + B cells were purified from buffy coats using the StraightFrom Buffy Coat CD19 MicroBead purification Kit (Miltenyi Biotec, cat. #130-114-974) following the manufactures instructions.

Techniques:

Journal: bioRxiv

Article Title: Multiparametric optimization of human primary B-cell cultures using Design of Experiments

doi: 10.1101/2025.01.16.633474

Figure Lengend Snippet: ( A-D ) Pareto plot of parameter effect sizes on IgE ( A ), IgA ( B ), IgG1 ( C ), and IgG3 ( D ) detection in the medium of DOE 6 cultures measured by TRIFMA. ( E-G ) Measured IgG1 and IgG3 in culture medium of each sample from DOE 6 where color and mean indicated by dotted lines are specific for time ( E ), level of IL-4 ( F ), N.40 expression ( G ). ( H ) As ( E ) but for measuring IgG1 and IgA. ( I ) Flow plotting IgD vs. CD27 of CD19 + live singlet lymphocytes after 10 days of naïve B-cell iGB culturing to illustrate the difference of their activation and differentiation depending on the stimuli provided, with the feeder-cell expression level of CD40L being a very apparent contributor. ( J ) as ( I ) but plotting CD38 vs. CD95.

Article Snippet: Human CD19 + B cells were purified from buffy coats using the StraightFrom Buffy Coat CD19 MicroBead purification Kit (Miltenyi Biotec, cat. #130-114-974) following the manufactures instructions.

Techniques: Expressing, Activation Assay

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