Journal: Nature Communications

Article Title: Outcomes of controlled human malaria infection after BCG vaccination

doi: 10.1038/s41467-019-08659-3

Figure Lengend Snippet: In vivo activation of lymphocytes, monocytes and neutrophils after CHMI. In vivo leucocyte activation was determined by direct staining of fresh whole blood with fluorescent antibodies every 2 days post-challenge. Lymphocytes were defined based on forward scatter and sideward scatter characteristics, and duplet events were excluded. a NK cell activation was defined as the percentage of CD3 - CD56 dim CD16 + live cells expressing CD69. b γδT cell activation was defined as the percentage of CD3 + γδTCR + live cells expressing CD69. c NKT cell activation was defined as the percentage of CD3 + γδTCR - CD56 + live cells expressing CD69. d αβT cell activation was defined the as percentage of CD3 + γδTCR - CD56 − live cells expressing CD69. e Monocytes were defined based on forward and side scatter characteristics, and the as HLA-DR + CD14 + . Within the monocyte population, cells were then divided into CD16 - and CD16 + monocytes. f – g Within the CD16 - monocyte population, the relative change in mean fluorescent intensity of HLA-DR and CD86 compared to pre-malaria challenge values was determined. h Neutrophils were defined based on forward and side scatter characteristics, and then defined as HLA-DR - CD14 - CD16 + CD11b + . Activated neutrophils were defined as CD62L dim CD11b high . i – j IFN-γ and granzyme B were measured by Luminex assay in citrate plasma taken ever 2 days. Circulating cytokine levels are corrected for baseline levels (pre-BCG vaccination time point) at each time point. In all graphs the grey dots represent non-BCG vaccinated control group volunteers ( n = 10), and each coloured dot shows an individual BCG vaccinated volunteer ( n = 9). Statistical analysis are between BCG vaccinated and control volunteers at a single time point, and p -values are the results of Mann–Whitney U -test. * p < 0.05. k Circulating CRP levels were measured in citrate plasma are shown for each BCG vaccinated volunteer (colours consistently represent the same volunteers across each graph)

Article Snippet: For neutrophil analysis samples were stained with CD14-PerCP (Biolegend; clone HCD14; catalogue number 325632; final dilution 1:30), HLA-DR-APC (Biolegend; clone L243; catalogue number 307610; final dilution 1:80), CD16 − APC-eFluor780 (eBiosciences; clone CB16; catalogue number 47–0168–42; final dilution 1:1280), CD62L-PE-Cy7 (eBioscience; clone DREG-56; catalogue number 25–0629–42; final dilution 1:1280) and CD11b-BV510 (Biolegend; clone ICRF44; catalogue number 301334; final dilution 1:180).

Techniques: In Vivo, Activation Assay, Staining, Expressing, Luminex, MANN-WHITNEY

Journal: PLoS Neglected Tropical Diseases

Article Title: Following successful anti-leishmanial treatment, neutrophil counts, CD10 expression and phagocytic capacity remain reduced in visceral leishmaniasis patients co-infected with HIV

doi: 10.1371/journal.pntd.0010681

Figure Lengend Snippet: Neutrophils were isolated from whole blood by dextran sedimentation as described ibn Materials and Methods: A. The expression levels (MFI = Median Fluorescence Intensity) of CD63 expression levels were measured on neutrophils from controls (n = 12), VL (ToD: n = 26, EoT: n = 14 3m: n = 22, 6-12m: n = 18) and VL/HIV (ToD: n = 18, EoT: n = 14, 3m: n = 15, 6-12m: n = 11) patients by flow cytometry as described in Materials and Methods. B. The expression levels (MFI) of CD62L expression levels were measured on neutrophils from controls (n = 18), VL (ToD: n = 25, EoT: n = 19 3m: n = 24, 6-12m: n = 34) and VL/HIV (ToD: n = 27, EoT: n = 25, 3m: n = 18, 6-12m: n = 23) patients by flow cytometry as described in Materials and Methods; C. The intracellular expression levels (MFI) of Arginase 1 levels were measured in neutrophils from controls (n = 11), VL (ToD: n = 23, EoT: n = 11, 3m: n = 14, 6-12m: n = 17) and VL/HIV (ToD: n = 15, EoT: n = 9, 3m: n = 9, 6-12m: n = 11) patients by flow cytometry as described in Materials and Methods. Each symbol represents the value for one individual. Results are presented as median with interquartile range. Statistical differences between VL and VL/HIV patients at each time point were determined using a Mann-Whitney test and statistical differences between the 4 different time points for each cohort of patients were determined by Kruskal-Wallis test. ToD = Time of Diagnosis; EoT = End of Treatment; 3m = 3 months post EoT; 6-12m = 6–12 months post EoT. ns = not significant.

Article Snippet: The following antibodies were used: anti-human CD62L PE/Cy7 (DREG-56), anti-human CD15 APC (H198), anti-human CD10 FITC (eBioSN5c), anti-human CD63 PE/Cy7 (H5C6) (eBiosciences) and anti-human arginase 1 PE (14D2C43) (BioLegend).

Techniques: Isolation, Sedimentation, Expressing, Fluorescence, Flow Cytometry, MANN-WHITNEY, Biomarker Discovery

Journal: PLoS Neglected Tropical Diseases

Article Title: Following successful anti-leishmanial treatment, neutrophil counts, CD10 expression and phagocytic capacity remain reduced in visceral leishmaniasis patients co-infected with HIV

doi: 10.1371/journal.pntd.0010681

Figure Lengend Snippet: Comparison of neutrophil CD62L expression levels between VL and VL/HIV patients and controls over time.

Article Snippet: The following antibodies were used: anti-human CD62L PE/Cy7 (DREG-56), anti-human CD15 APC (H198), anti-human CD10 FITC (eBioSN5c), anti-human CD63 PE/Cy7 (H5C6) (eBiosciences) and anti-human arginase 1 PE (14D2C43) (BioLegend).

Techniques: Comparison, Expressing

Journal: Oncoimmunology

Article Title: Functional T cells targeting tumor-associated antigens are predictive for recurrence-free survival of patients with radically operated non-small cell lung cancer

doi: 10.1080/2162402X.2017.1360458

Figure Lengend Snippet: Gating strategy for T cell subsets. (A) Representative data from one NSCLC patient showing CD4+ (upper line) and CD8+ (lower line) T cell subset populations in blood and tumor. Tumor-infiltrating lymphocytes (TILs), immediately isolated from freshly resected NSCLC tumor tissue and peripheral blood-derived T cells (PBTCs) were used for flow cytometry to define T cell subsets (for cell preparation, see Patients and Methods section). The same gating strategy was used for TILs and PBTCs. First, using forward (FSC-A) vs. side scatter (SSC-A), the cells were gated for lymphocytes (plot marked with asterisk) and singlets (FSC-H vs. FSC-A). The viability of the gated cells was further analyzed using a live/dead stain, taking only the pacific orange-low population. CD4+ and CD8+ surface expression was used to determine the CD4+ and CD8+ T cell populations. By using CD62L and CD45RA, the different T cell subsets were identified from CD4+ and CD8+ populations as follows: CD62L+/CD45RA− cells (central memory T cell; TCM), CD62L+/CD45RA+ cells (naïve/stem-like memory T cell; TN/SCM), CD62L−/CD45RA+ cells (effector T cell; Teff), and CD62L−/CD45RA− cells (effector memory T cell; TEM). The numbers in the dot plots indicate the proportions (%) of gated populations among gated cells (B) Relative proportions of CD4+ and CD8+ cell subsets enumerated in freshly prepared PBTCs (PB, n = 4) and NSCLC tissue (TILs, n = 6) immediately processed after resection by flow cytometry. (C) The CD4/CD8 T cell ratio is shifted toward CD8+ predominance in TILs compared with PBTCs as determined by flow cytometry. (D) Effector to naïve/TSCM ratios for CD4+ and CD8+ PBTCs and TILs. (C–D) Each symbol represents data from an individual patient. Black lines indicate mean values. Error bars show mean ± SEM. p-values were determined by an unpaired 2-tailed t-test.

Article Snippet: 20 Dead cells were excluded by staining with a fixable yellow dead-cell stain (Life Technologies GmbH; {"type":"entrez-nucleotide","attrs":{"text":"L34959","term_id":"522202","term_text":"L34959"}} L34959 ; 1:1,000), followed by T cell subset staining: anti-human CD4-PerCp-Cy5.5 (clone RPA T4, BD), CD8-V450 (RPA T8, BD), CD45RA-APC-H7 (clone H100, BD), and CD62L-PeCy7 (clone DREG56, eBioscience).

Techniques: Isolation, Derivative Assay, Flow Cytometry, Staining, Expressing

Journal: Thoracic Cancer

Article Title: Effectiveness of EGFR‐TKI rechallenge immediately after PD ‐1 blockade failure

doi: 10.1111/1759-7714.13864

Figure Lengend Snippet: Transition of lymphocytes using PBMC analysis (CD4, CD8, Foxp3 and CD62L low ) in patients with (a) PR and (b) non‐PR after EGFR‐TKI rechallenge. Analysis of PBMC was performed at two points before ICI and EGFR‐TKI treatment. No statistically significant difference in the percentage of these lymphocytes between before ICI and EGFR‐TKI was observed in (a) PR and (b) non‐PR groups

Article Snippet: The following mAbs were used in the study: fluorescein isothiocyanate (FITC)‐conjugated anti‐CD3 (HIT3a) and anti‐CD4 (RPA‐T4), phycoerythrin (PE)‐conjugated anti‐CD8 (RPAT8) and anti‐CD25 (M‐A251), PE‐Cy7‐conjugated anti‐CD25 (MA251), PE‐Cy5‐conjugated anti‐CD62L (Dreg 56; all from BD Pharmingen), and FITC‐conjugated anti‐CD62L (Dreg 56; eBioscience).

Techniques: