Journal: Journal of Virology

Article Title: Upregulation of Interleukin 7 Receptor Alpha and Programmed Death 1 Marks an Epitope-Specific CD8 + T-Cell Response That Disappears following Primary Epstein-Barr Virus Infection

doi: 10.1128/jvi.00141-09

Figure Lengend Snippet: FIG. 2. IL-7R statuses of YVL-, GLC-, and TLD-specific re- sponses over time post-IM. (A) FACS profiles of IL-7R and either YVL- or TLD-tetramer staining from the blood samples of an HLA- A*0201-positive IM patient (IM179) taken during acute infection (IM179.1), and after a further 4 (IM179.2), 10 (IM179.3), and 27 (IM179.4) months. Data from a healthy EBV carrier are shown for comparison. Profiles are gated on CD8 T cells. Numbers in the upper-right quadrants refer to the percentages of tetramer-positive,

Article Snippet: Goat anti-human IL-7R antibody (R&D Systems, Minneapolis, MN) was detected by fluorescein isothiocyanate (FITC)-conjugated swine anti-goat immunoglobulin G antibody (Caltag Laboratories, Burlingame, CA) as previously described (33, 34); cells were dually stained with Tricolorlabeled anti-human CD8 monoclonal antibody (MAb) (Caltag).

Techniques: Staining, Infection, Comparison

Journal: bioRxiv

Article Title: Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

doi: 10.1101/262410

Figure Lengend Snippet: 1 a) Representative flow cytometry plots from live gated positively selected CD14 monocytes from one individual where cells were incubated for 24h alone (top panel) or in presence of LPS (bottom panels) from same individual 1 b) Violin plot demonstrating significant induction of IL7R + monocytes in across paired cultures of positively selected monocytes (Monocyte IL7R + post LPS-median:23.8, min:6.5, max:55.8, IQR:15.6-34.9%; n=84, paired T-test) 1 c) Comparative effects of LPS on IL7R+ counts across CD14 + monocytes, CD4+ and CD8 + T-cells and CD56 + NK cells from the same PBMC cultures, untreated or with LPS for 24h (PBMC IL7R + post LPS-median:29.9%, min:5.9, max:66.40, IQR:22.3-40.0; n=103, paired T-test) 1 d) Pearson correlation analyses were performed between indicated cells and treatments on number of IL7R + cells. *** P <0.001, ** P<0.01. 1 e) Comparative induction of IL7R + monocytes from 6 individuals with monocytes treated for 24h with either the TLR7 agonist Imiquimod, TLR1/2 agonist Pam3CysK4 or TLR4 agonist LPS (one way ANOVA). 1 f) Expression of TNF at 2h LPS assayed versus expression of IL7R from monocytes from same individuals at 24h LPS. 1 g) Comparative incubation of monocytes alone (n=69), with LPS or with LPS + anti-TNF monoclonal antibody (Infliximab). Incubation with TNF antagonist significantly reduces LPS induced IL7R + monocyte counts (paired t-test). 1 h) Violin plots of responses across cell types of PBMCs treated with TNF alone leads to significant induction of IL7R + monocytes and significantly reduces CD4 and CD8 T-cell IL7R + positivity (n=78, paired T-test).

Article Snippet: Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Flow Cytometry, Incubation, Expressing

Journal: bioRxiv

Article Title: Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

doi: 10.1101/262410

Figure Lengend Snippet: 2 a) Microarray data demonstrating eQTL to IL7R noted at rs931555 after both 2 and 24 hour treatment with LPS (2h LPS: n=261, 24h LPS n=322) 2 b) Local association plot for monocyte surface IL7R after exposure to LPS from positively selected monocytes demonstrates peak association to rs6897932 (n=84) 2 c) Batch corrected log values for surface IL7R from positively selected monocytes demonstrating significant effect of rs6897932 carriage after stimulation in comparison to baseline monocytes (n=84, ANOVA for linear fit model) 2 d) Surface IL7R in PBMCs across cell subsets after exposure to LPS. (n= 87, Note a significant effect of rs6897932 on surface IL7R is only observed in CD14 + monocytes, ANOVA for linear fit model) 2 e) As per 2 d) but PBMCs treated with TNF for 24h (n=62)

Article Snippet: Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Microarray, Comparison

Journal: bioRxiv

Article Title: Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

doi: 10.1101/262410

Figure Lengend Snippet: 3 a) Elisa of sIL7R performed on supernatants of monocytes treated with LPS for 24h. Samples were randomly chosen from original eQTL dataset and genotypes revealed post-hoc – a significant association was observed at rs6897932 (n=161, ANOVA for linear fit model). 3 b) Elisa data from 3 a) showing additional effect of rs931555 genotype on sIL7R (linear model). 3 c) Correlation analysis performed between 15421 microarray probes and soluble IL7R (y-axis) identified DDX39A expression (x-axis) to be significantly anti-correlated with soluble IL7R levels, supporting evidence for its regulation of soluble IL7R. Trend lines indicate correlation per by allele, with rho and P-value for all data (n=161) 3 d) Elisa of sIL7R from isolated primary CD14 and CD4 cells cultured for 24 hours either alone or in the presence of LPS, LPS + IL-7 and CD3/CD28 activating beads. (n=6).

Article Snippet: Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Enzyme-linked Immunosorbent Assay, Microarray, Expressing, Isolation, Cell Culture

Journal: bioRxiv

Article Title: Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

doi: 10.1101/262410

Figure Lengend Snippet: 4 a) Violin plot demonstrating monocytes from PBMC cultures in the untreated state, after exposure to LPS and after exposure to LPS with recombinant IL-7. IL-7 leads to marked downregulation of IL7R on LPS stimulated monocytes, indicative sensitivity to exogenous cytokine (paired T-test). 4 b) Volcano plot of mRNA from RNAseq experiments of 8 paired monocyte samples either treated with LPS alone for 24h or with LPS with additional IL-7 added for the last 2h of culture. Treatment leads to widespread differential transcript expression with the most significant 20 transcripts labelled (FDR<0.05). 4 c) Example boxplots of genes differentially regulated by recombinant IL-7 in monocytes (linear model) (n=8 subjects). 4 d) Results from gene ontology biological pathway analysis was performed on IL-7 induced genes with all significant pathways labelled. 4 e) Single-cell real-time quantitative PCR (RT-qPCR) data from 3 individuals (192 cells) either untreated or in the presence of IL-7 alone, LPS alone, or LPS with IL-7 added in the last 2h of culture showing expression of CSF1R, CCL5, CD14, FCGR3B , membrane-bound IL7R (IL7Rmb) and sIL7R. 4 f) t-SNE plot of single-cell RT-qPCR data showing expression of IL7Rmb, sIL7R, LTA, CD14, MYB, CCL5, LYZ, FCGR3B, STAB1 and TCF7 overlaid on the unbiased clustering.

Article Snippet: Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Recombinant, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Membrane

Journal: bioRxiv

Article Title: Context-specific regulation of monocyte surface IL7R expression and soluble receptor secretion by a common autoimmune risk allele

doi: 10.1101/262410

Figure Lengend Snippet: 5 a) Representative flow cytometry of IL7R staining of matched PBMC (left) and SFMC (middle isotype control, right) from a spondyloarthritis patient. Gated on CD3 − CD14 + . 5 b) Results from 4 patients (2 As and 2 PsA) showing monocyte IL7R staining in matched PBMC and SFMC using flow cytometry. 5 c) t-SNE clustering of monocytes from single-cell RNA sequencing of spondyloarthritis patient (n=3) SFMC showing 5 clusters (n=3). 5 d) Single-cell monocyte expression of IL7R, LTB, CD14, CCL5, IL32 and TCF7 overlaid on t-SNE clustering in (c). 5 e) Volcano plot of genes significantly upregulated in the SFMC monocyte cluster 4 compared to all other SFMC monocyte clusters with top 20 genes annotated. 5 f) Heatmap of top 20 genes from the each of the five SFMC monocyte clusters identified in (c).

Article Snippet: Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Flow Cytometry, Staining, RNA Sequencing Assay, Expressing

Journal: Immunity

Article Title: The Proteomic Landscape of Human Ex Vivo Regulatory and Conventional T Cells Reveals Specific Metabolic Requirements

doi: 10.1016/j.immuni.2016.01.028

Figure Lengend Snippet: Effector Functions of Human Tconv Cells Are Mainly Sustained by Glycolysis (A) Representative flow cytometry plots showing expression of activation markers (CD25, CD127, CD71, ICAM-1, VLA-4, and CTLA-4) in Tconv cells upon 36 hr activation with anti-CD3 and anti-CD28 in the presence or absence of 2-DG or etomoxir. Percentage and MFI of positive cells are indicated. One representative out of two independent experiments is shown. (B) MFI of IL-2 and IFN-γ expression in Tconv cells stimulated for 12 hr with anti-CD3 and anti-CD28 in the presence or absence of 2-DG or etomoxir, analyzed by flow cytometry. Data are expressed as mean ± SEM (n = 3). (C) Immunoblot for P-ERK 1/2, P-STAT5, and P-S6 on Tconv cells upon 12 hr anti-CD3 and anti-CD28 stimulation in the presence or absence of 2-DG or etomoxir. Total ERK 1/2, total STAT5, and total S6 served as a loading control. One representative out of two independent experiments is shown. The graphs show the relative densitometric quantitation of each phosphorylated protein normalized on its total form, in Tconv cells treated for 12 hr with anti-CD3 and anti-CD28 in the presence or absence of 2-DG or etomoxir (n = 6, data are shown as mean ± SEM of two independent experiments, in triplicates). All statistical analysis by paired two-tailed Student’s t test ( ∗ p < 0.05, ∗∗ p < 0.005).

Article Snippet: We isolated human Treg (CD4 + CD25 high CD127 − ) and Tconv (CD4 + CD25 − ) cells from PBMCs from buffy coats of human healthy donors by high-performance cell sorting (MoFlo, Dako Beckman-Coulter or BD FACS-Jazz, BD Bioscience) in the IEOS-CNR Sorting Facility in Napoli, after staining with the following antibodies at 1:100 dilution: FITC anti-human CD4 (BD PharMingen, clone RPA-T4, Cat.# 555346), PE anti-human CD25 (BD PharMingen, clone M-A251 Cat.# 555432) and APC anti-human CD127 (R&D Systems, clone 40131, Cat.# FAB306A), or by magnetic cell separation with Dynabeads Regulatory T Cell Kit (Invitrogen).

Techniques: Flow Cytometry, Expressing, Activation Assay, Western Blot, Control, Quantitation Assay, Two Tailed Test