Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 (BCR method) in the absence (control; CTRL) or presence of TAC (6ng/ml) or SRL (2ng/ml or 6ng/ml) and flow cytometric analyses were performed after 6 days in culture. (A) A representative experiment: cells were gated on viable lymphocytes and analyzed for CFSE diluting proliferating cells. This scheme of analysis was used in all subsequent experiment, unless indicated otherwise. (B) The percentage of proliferating CD19 + B cells as obtained in A from 7 different experiments. (C) Absolute number of proliferating CD19 + B cells was calculated in each experiment by multiplying the recovered cell counts with the percentage of proliferating cells as in A (n = 7). Statistically significant (*p < 0.05) inhibition of B cell proliferation was observed with SRL at both subtherapeutic (2ng/ml) and therapeutic (6ng/ml) doses.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Control, Inhibition

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were stimulated with anti-IgM, anti-CD40 mAb and IL-21 and multi-color flow cytometric analyses were performed on day 6 as described in . The figures A , B and C show the expression of various surface markers on stimulated B cells (Percentage of positive cells/total proliferating CD19 + cells) p < 0.05, **p < 0.01. CTRL, control; TAC6, 6 ng/ml TAC; SRL2, 2 ng/ml SRL; SRL6, 6 ng/ml SRL.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Expressing, Control

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: B cells were purified by depleting non-B cells resulting in >95% CD19 + cells which subsequently sorted into CD27 − (naïve) and CD27 + (memory) B-cell fractions. These subsets were labeled with CFSE, stimulated with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of TAC or SRL at 6ng/ml and were analyzed by multicolor flow cytometry after 6 days in culture. (A) A representative experiment: histogram plots show dilution of CFSE in the proliferating CD19 + CD27 − (upper panel) or CD19 + CD27 + (lower panel) cells. (B) Data are from four different independent experiments are shown as mean ± SD percent proliferating naïve CD19 + CD27 − and memory CD19 + CD27 + B cells. (C) B cell subsets showing indicated surface markers were analyzed and plotted as mean ± SD (n = 4) percent of proliferating cells in the cultures of naïve CD19 + CD27 − (upper panel) and memory CD19 + CD27 + B cells (lower panel). The residual cells that proliferated in presence of SRL demonstrated an activated phenotype. *p < 0.05, **p < 0.01.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were cultured as in Figs and . (A) A representative experiment showing flow cytometric profile indicative of putative plasma cells (CD19 low ) in proliferated B cells (gated on viable lymphocytes; see ). (B) The mean ± SD percentage of such CD19 low B cells from 4 different independent experiments. (C) Mean ± SD (n = 4) percentage of CD19 low CD38 ++ plasmablasts, CD19 low CD138 + plasma cells, Blimp1 + PAX5 - cells and CD138 + Blimp1 + cells in the proliferating CD19 low cells. *p < 0.05, **p < 0.01. CTRL, control; TAC, 6 ng/ml TAC; SRL, 6 ng/ml SRL.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Cell Culture, Clinical Proteomics, Control

Journal: PLoS ONE

Article Title: Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells

doi: 10.1371/journal.pone.0129658

Figure Lengend Snippet: Purified CD19 + B cells were pre-stimulated for 6 days with anti-IgM, anti-CD40 mAb and IL-21 in the absence (CTRL) or presence of 6ng/ml TAC or SRL. These cultured B cells were used as stimulators in 6-day MLRs of allogeneic CFSE-labelled CD4 + CD25 − T cell responders. ( A ) Level of proliferation differentially induced by pre-cultured B cells as detected by CFSE dilution in the allogeneic CD4 responder cells (representative experiment on the left, and compiled data from 8 independent experiments on the right). ( B ) Percentage of responding T cells positive for memory marker (CD45RO) and activation markers (CD62L, CD25, CD69, CD95) after co-culture with pre-stimulated B cells (n = 8); ( C ) Mean ± SD (n = 4) percentage of responding proliferating T cells expressing intracellular cytokines (top row) and transcription factors (bottom row). Taken together the data indicated that B cells that proliferated in presence of SRL on a per cell basis were capable of inducing alloreactive T cell proliferation towards a Th1 phenotype. *p < 0.05. ** p < 0.01.

Article Snippet: Total B cells were isolated from PBMC by positive selection using human CD19 MicroBeads kit (MACS, Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Cell Culture, Marker, Activation Assay, Co-Culture Assay, Expressing

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: Malaria Journal

Article Title: High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories

doi: 10.1186/s12936-018-2547-3

Figure Lengend Snippet: List of fluorophore-conjugated antibodies

Article Snippet: Still, other positive B cell isolation kits from whole blood such as the DynabeadsTM CD19 Pan B kit [ ] or the Miltenyi Biotec MACSprep Chimerism CD19 MicroBeads human kit [ ] reside within the same price range with 36–60 USD per isolation and a magnet cost between 1076 USD and 13,200 USD.

Techniques:

Journal: Malaria Journal

Article Title: High purity high yield tandem B and T helper cell isolation for qRT-PCR analysis suitable for basically equipped laboratories

doi: 10.1186/s12936-018-2547-3

Figure Lengend Snippet: Flow cytometry analysis of purity and efficiency of B and Th cell isolation. Shown is a representative analysis of three independent isolations. a Flow cytometry analysis of whole blood after erythrocyte lysis (= before B cell isolation). Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper right: total T cells (CD3+) versus B cells (CD19+). Lower left: total T cells (CD3+) versus Th cells (CD3+CD4+). b Flow cytometry analysis of isolated CD19+ B cells (= positive fraction after B cell isolation). Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper middle: total T cells (CD3+) versus B cells (CD19+). Lower middle: total T cells (CD3+) versus Th cells (CD3+CD4+). Upper right: histogram of CD19 positive cells. Lower right: histogram of CD4 positive cells. c Flow cytometry analysis of isolated CD4+ Th cells (= positive fraction after Th cell isolation). Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper middle: total T cells (CD3+) versus B cells (CD19+). Lower middle: total T cells (CD3+) versus Th cells (CD3+CD4+). Upper right: histogram of CD19 positive cells. Lower right: histogram of CD4 positive cells. d Flow cytometry analysis of the negative fraction after Th cell isolation. Upper left: gating of live lymphocytes. Lower left: gating of single cells. Upper middle: total T cells (CD3+) versus B cells (CD19+). Lower middle: total T cells (CD3+) versus Th cells (CD3+CD4+). Upper right: histogram of CD19 positive cells. Lower right: histogram of CD4 positive cells

Article Snippet: Still, other positive B cell isolation kits from whole blood such as the DynabeadsTM CD19 Pan B kit [ ] or the Miltenyi Biotec MACSprep Chimerism CD19 MicroBeads human kit [ ] reside within the same price range with 36–60 USD per isolation and a magnet cost between 1076 USD and 13,200 USD.

Techniques: Flow Cytometry, Cell Isolation, Lysis, Isolation

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