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: European Journal of Immunology

Article Title: Granulysin Antimicrobial Activity Promotes Dormancy in Mycobacterium tuberculosis

doi: 10.1002/eji.70004

Figure Lengend Snippet: Cytotoxic, activated NK and (unconventional) T cells are significantly enriched in patient immune cells exposed to Mtb . (A) Volcano plot (x: Hedge's g effect size, y: false discovery rate (FDR) adjusted p values of pairwise Wilcoxon rank sum tests) of functional marker expression (patients vs healthy controls). (B) Frequency of host cell phenotype hits identified in (A). (C) T cell subset proportions among T cells, and (D) frequency of T cell 9 subset within all cells (irrespective of functional marker positivity), split by disease group (inset p ‐values from Wilcoxon tests (unadjusted)). Each dot (panels B and D) and stacked bar (panel C) represents an independent study participant (HC: n = 12, TB: n = 11). (E) UMAP visualizations of host cell phenotype hits identified in (A) (left), and cells’ median expression of perforin (second left) and CD38 (second right) and CD56 positivity (right).

Article Snippet: The CD56 + fraction of PBMCs was depleted using Miltenyi's human REAlease CD56 MicroBead kit (Miltenyi, 130‐117‐033), following the manufacturer's instructions.

Techniques: Functional Assay, Marker, Expressing

Journal: European Journal of Immunology

Article Title: Granulysin Antimicrobial Activity Promotes Dormancy in Mycobacterium tuberculosis

doi: 10.1002/eji.70004

Figure Lengend Snippet: Exposure to submycobactericidal concentrations of granulysin drives Mtb to adopt a dormant‐like state. (A) Spearman's correlation analysis between the frequency of dormant‐like Mtb retrieved from ex vivo infection responses and the prevalence of indicated host cell phenotypes in the respective sample (asterisks for significant p ‐values after Benjamini–Hochberg correction). (B) Percentage of dormant‐like (Nile red + , auramine‐O − ) bacilli observed one day postinfection (p.i.) within ex vivo infection responses from TB patients, depleted or not of CD56 + cells ( n = 10 independent patients). Inset p ‐value of a paired t ‐test. (C) Composition of the CD56 + fraction ( n = 10 independent patients) depleted in b) as percentage of total live singlet cells: NK (CD3 − CD56 + ), NKT (CD3 + CD56 + ), γδ T (CD3 + TCRγδ + CD56 + ), MAIT (CD3 + TCRVa7.2 + CD56 + ); gating strategy provided as Figure . (D) Frequency of dormant‐like Mtb upon 24 h incubation with strontium‐degranulated (+Sr 2+ ) or not (‐Sr 2+ ) cell‐free supernatants of CD56 + cells, in the presence or absence of anti‐granulysin (GNLY) or isotype control (ISO) antibodies (AB) ( n = 4 independent patients). The red dotted line indicates the Mtb dormancy level measured following incubation in cell‐free strontium‐containing medium. Inset p‐ values from an ANOVA with Holm–Sidak's multiple comparisons test. (E) Dose‐response effect of native 9 kDa granulysin on Mtb dormancy induction (based on auramine‐O/Nile red staining). Error bars indicate the range, and open circles the mean of two experimental replicates. The shaded area depicts the mycobactericidal concentrations based on the study by Stenger et al. .

Article Snippet: The CD56 + fraction of PBMCs was depleted using Miltenyi's human REAlease CD56 MicroBead kit (Miltenyi, 130‐117‐033), following the manufacturer's instructions.

Techniques: Ex Vivo, Infection, Incubation, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Large expansion of phenotypically defined sx-MDSCs comprised mainly of M-MDSC and PMN-MDSCs immediately after surgery (POD1). ( A ) Representative flow plots showing CD33 versus Lineage (CD3/CD56/CD19) for baseline (left) and POD1 (right) PBMCs. ( B ) Representative flow plots gated down on CD33 + Lin − cells showing CD15 versus CD14 expression. ( C ) Healthy controls (n=15), Baseline, and POD1 patients (n=44) proportion of CD33 + Lin − cells (left) and MDSC subsets (right). ( D ) The MFI of HLA-DR gated on M-MDSCs (left) and the proportion of M-MDSCs that are HLA-DR lo (right) in healthy controls, baseline and POD1 patients with cancer. ( E ) Representative histograms of common M-MDSC and PMN-MDSC markers, before and after surgery. Statistical analysis was performed using Wilcoxon matched pairs signed-rank test or Kruskal-Wallis with Dunn’s post test. **** p≤0.0001. FMO, fluorescence minus one control; HLA-DR, human leukocyte antigen - DR isotype; E-MDSC, early stage-MDSC; M-MDSC, monocytic-MDSC; MDSC, myeloid derived suppressor cell; sx-MDSC, surgery-induced MDSC, MFI; mean fluorescence instensity; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: NK cells were isolated from whole blood using the autoMACS Pro Separator and StraightFrom Whole Blood CD56 MicroBeads (Miltenyi Biotec, #130-090-875), following the manufacturer’s protocol.

Techniques: Expressing, Fluorescence, Control, Derivative Assay