Journal: PLoS ONE

Article Title: Evaluation of Peripheral Blood Mononuclear Cell Processing and Analysis for Survival Motor Neuron Protein

doi: 10.1371/journal.pone.0050763

Figure Lengend Snippet: SMN levels were analyzed by CD+ cell subtype at the 7d timepoint from Study 4. A : Analysis of SMN by PBMC cell subpopulation revealed that the CD14+ population had statistically significant reductions in SMN levels. B : Fractionation of PBMCs with normalization by cell count showed no differences in SMN signal in group analysis. C : Evaluation of total soluble protein levels by CD+ population revealed that CD14+ cells had double the protein concentrations of CD4+, CD8+, CD19+, and CD56+ cells. This differential is sufficient to drive variability in situations that cause CD14+ populations to fluctuate. D : SMN levels (normalized by protein) show consistently lower levels in CD14+ fractions compared to all other fractions, with differences up to 7x within individual PBMC subpopulations. E : SMN in individuals as measured by cell counts were also variable ranging up to 3.5x between individuals’ subcellular populations, but was overall less variable than protein normalized SMN measures. In the bodies of the boxplots indicate the first and third quartiles, while the horizontal bar indicates the median. In error bars depict standard deviation.

Article Snippet: The order of addition of magnetic beads with antibodies for positive selection of PBMC subsets were CD14, CD56, CD19, CD8, and CD4 (Miltenyi OctoMACs #130-050-201, #130-050-401, #130-050-301, #130-045-201, #130-045-101 respectively).

Techniques: Fractionation, Cell Counting, Standard Deviation

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