Journal: Nature Communications

Article Title: Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

doi: 10.1038/s41467-024-48570-0

Figure Lengend Snippet: a Number of clonal families of BCRs found within the different BMPC clusters (diversity) and probability of finding different clonal families by random selection of cells (Simpson diversity index). A clonal family was defined by V and J gene composition and a CDR3 region with <20% Hamming distance in both the heavy and light chains originating from one donor. b Mutation rates in the framework regions FR1-3 (left) and in the CDR1-3 regions (right) of the heavy and light chain rearrangements across BMPC clusters. c Bubble plot of the mutation rates in the framework regions FR1-3 (left) and in the CDR1-3 regions (right) of BCRs per isotype and cluster. Colour scale indicates median mutation rates. Values below or above the scale limits are shown in blue or red, respectively. Bubble sizes correspond to the percentage of cells expressing a defined isotype within the indicated cluster. d Violin plot depicting the mutation rates in the V gene of the BCRs of BMPC per cluster. Statistical significance between clusters is shown in Supplementary Data (two-tailed Mann–Whitney U test). Horizontal lines represent the median mutation rate. Violins are coloured by the z score of CD19 gene expression in each cluster. e Identification of SARS-CoV-2 spike-specific and tetanus toxoid-specific public clones (in black) among analysed BMPC. Public clones were defined by exhibiting over 80% CDR3 sequence identity in both heavy and light chains when compared to the BCR of sequenced peripheral blood and bone marrow spike- and tetanus-specific cells from vaccinated individuals (see Supplementary Fig. and Supplementary Data ). f Relative distribution of spike-specific (red) and tetanus-specific (blue) BMPC (depicted in e ) per cluster. g Comparison of mutation rates within the framework regions FR1-3 (left) and the CDR1-3 regions (right) of spike-specific (red) and tetanus-specific (blue) BMPC per isotype. Horizontal lines represent the median mutation rate. Statistics were performed using a two-tailed Mann–Whitney U test. Source data are provided as a Source Data file.

Article Snippet: Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Selection, Mutagenesis, Expressing, Two Tailed Test, MANN-WHITNEY, Gene Expression, Clone Assay, Sequencing, Comparison

Journal: Nature Communications

Article Title: Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

doi: 10.1038/s41467-024-48570-0

Figure Lengend Snippet: a , b Gene Set Enrichment Analysis (GSEA) based on gene signatures from ex vivo-differentiated plasmablasts in presence of different cytokine combinations (data sets from Stephenson et al. ). Briefly, naive B cells were cultured in differentiating conditions for 6 days after which different cytokines were added to the culture. After 12 or 24 h, cells were harvested and sequenced. Gene signatures were identified by comparing the different conditions and time points to 0 h. a Density plots of ASC (time points and vaccine protocols combined) with significant enrichment identified by GSEA of gene signatures from ex vivo-differentiated plasmablasts. b Density plots of BMPC with significant enrichment identified by GSEA of gene signatures from ex vivo-differentiated plasmablasts. Violin plots of the normalised enrichment score (NES) per BMPC cluster are depicted in Supplementary Fig. . Statistical significance between NES scores is shown in Supplementary Data (two-tailed Mann–Whitney U test). c , d T follicular helper cells (Tfh, CD19 − CD4 + CD45RA − CXCR5 ++ ) from tonsils or T peripheral helper cells (Tph, CD19 − CD4 + CD45RA − CXCR5 − ) from BAL of sarcoidosis patients were co-cultured 1:1 with tonsillar memory B cells (CD19 + CD4 − IgD − CD38 − ) for 7 days in presence of staphylococcal enterotoxin B (SEB). Where indicated, T-cell help was blocked with an anti-CD40L antibody and/or recombinant soluble IL-21R. c Histograms of CD19 expression by differentiated plasmablasts (see Supplementary Fig. 7). Two Tfh and two Tph donors were analysed. d Frequencies of CD19 hi plasmablasts. Statistics were performed using an RM parametric one-way ANOVA followed by multiple testing according to Fischer’s LSD test. Source data are provided as a Source Data file.

Article Snippet: Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Ex Vivo, Cell Culture, Two Tailed Test, MANN-WHITNEY, Recombinant, Expressing

Journal: Nature Communications

Article Title: Recruitment of plasma cells from IL-21-dependent and IL-21-independent immune reactions to the bone marrow

doi: 10.1038/s41467-024-48570-0

Figure Lengend Snippet: a Representative pseudocolour plots of intracellular double-positive SARS-CoV-2 RBD (left) or tetanus toxoid (TT, right) staining in CD38 high CD138 + CD14 − CD3 − live singlet BMPC (gating strategy in Supplementary Fig. ). b – e Each symbol represents one donor/sample (see Supplementary Table ). Filled symbols represent BMPC samples which were also analysed by single-cell sequencing (Fig. ). b Frequencies of RBD-specific and TT-specific BMPC within total BMPC. Horizontal lines indicate the median. Statistics were performed using the one-tailed Mann–Whitney U test. n = 20 BM samples. c Frequencies of CD19 low cells within RBD-specific BMPC (red), TT-specific BMPC (blue) and total BMPC (black). Horizontal lines indicate the median. Statistics were performed using the Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. n = 20 BM samples. d Frequencies of IgG+ (left) and IgA+ (right) cells within RBD-specific BMPC (red), TT-specific BMPC (blue) and total BMPC (black). Horizontal lines indicate the median. Statistics were performed using the Kruskal–Wallis tests with Dunn’s correction for multiple comparisons. n = 20 BM samples. e Correlation between the frequency of CD19 low RBD-specific BMPC and days after 3rd vaccination against SARS-CoV-2. Statistics were performed using one-tailed Spearman’s correlations. n = 11 BM samples. Source data are provided as a Source Data file.

Article Snippet: Plasma cells were enriched from bone marrow using StraightFrom Whole Blood and Bone Marrow CD138 MicroBeads and StraightFrom Whole Blood CD19 MicroBeads (Miltenyi Biotec) according to manufacturer’s instructions.

Techniques: Staining, Sequencing, One-tailed Test, MANN-WHITNEY

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