Journal: Mucosal immunology

Article Title: Maturation of mast cell progenitors to mucosal mast cells during allergic pulmonary inflammation in mice

doi: 10.1038/mi.2014.91

Figure Lengend Snippet: Expression of β7 and β1 integrins and forward and side scatter characteristics of inducible lung MC populations at D7 after challenges. ( a ) Representative histograms (top panel) and mean (± SEM) net MFI (bottom panel) of β7 integrin expression by the 3 populations of lung MCs: MCp -green, eMMC -red and MMC -blue; black dotted line -isotype control. Net MFI was determined by subtracting the value of the isotype control. ( b ) Representative histograms (top panel) and mean (± SEM) net MFI (bottom panel) of β1 (CD29) integrin expression by the 3 populations of lung MCs (top panel) colored as in a. ( c ) Representative histograms show the side scatter profile of the 3 lung MC populations colored as in a . Grey shaded area indicates immature spleen MCp. ( d ) Representative histograms show the forward scatter profile of the 3 lung MC populations compared to spleen MCp as in c . Histograms in c and d show the mean value from 2-3 mice/group from one of 5 experiments. Bar graphs present means ± SEMs from 3-5 separate experiments with 5-13 mice per group.

Article Snippet: Fluorescently labeled anti–β7 integrin (M293) and anti-αE integrin mAb (M290), as well as blocking anti-αE integrin mAb (M290) were obtained from BD Biosciences (San Diego, CA).

Techniques: Expressing

Journal: Mucosal immunology

Article Title: Maturation of mast cell progenitors to mucosal mast cells during allergic pulmonary inflammation in mice

doi: 10.1038/mi.2014.91

Figure Lengend Snippet: Expression of β7 and β1 integrins and forward and side scatter characteristics of tracheal constitutive and inducible MC populations at D7 after challenges. ( a ) Representative histograms (top panel) and mean (± SEM) net MFI (bottom panel) of the β7 integrin expression on the 3 populations of tracheal MCs, CMCs (orange shaded), eMMCs (red) and MMCs (blue); black dotted line -isotype control. ( b ) Representative histograms (top panel) and mean (± SEM) net MFI (bottom panel) of the β1 (CD29) integrin expression on the 3 populations of tracheal MCs colored as in a. ( c ) Representative histograms of the side scatter characteristics of the 3 MC populations in trachea (colored as in a and compared with splenic MCp (grey shaded) and intraperitoneal MCs (back dotted line). ( d ) Forward scatter characteristics of tracheal, splenic and intraperitoneal MC populations as in c . Histograms in c and d show the mean value from 2-3 mice/group from one of 5 experiments. Bar graphs represent mean (± SEM) from 4 experiments with 5-8 mice per group * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Fluorescently labeled anti–β7 integrin (M293) and anti-αE integrin mAb (M290), as well as blocking anti-αE integrin mAb (M290) were obtained from BD Biosciences (San Diego, CA).

Techniques: Expressing

Journal: bioRxiv

Article Title: Salivary gland macrophages assist tissue-resident CD8 + T cell immune surveillance

doi: 10.1101/723791

Figure Lengend Snippet: A. Experimental layout. B. OT-I T PLN-M and T RM speeds after combined anti-αL, α4 and αE integrin mAb (αItg) inhibition. Arrows indicate median values (µm/min). C. 2PM image of T RM – tissue macrophage colocalization in αItg-treated SMG. Arrows indicate T cell – tissue macrophage contacts. Scale bar, 20 µm. D. OT-I T RM speeds in SMG after WD administration of RAD or RGD peptide . E. WT and CXCR3 -/- OT-I T RM speeds in SMG in memory phase (≥ day 30 p.i.). Arrows indicate median values (µm/min). F. OT-I T PLN-M and T RM speeds after systemic treatment with active PTx or inactive (mutant) PTx (PTx mut ). Arrows indicate median values (µm/min). G. 2PM image of T RM – tissue macrophage colocalization in PTx-treated SMG. Arrows indicate T cell – tissue macrophage contacts. Scale bar, 20 µm. H. Flow cytometry plot of mixed T RM and macrophages. I. Quantification of cluster formation as shown in H. J. Example image sequences showing T RM in transient contact with macrophages under agarose on fibronectin-coated plates. T RM displacement is shown by segmented line. Scale bar, 50 μm. Time in min:s. K. T RM – macrophage contact duration for individual tracks. Data in B, D, E and F are pooled from 2-5 independent experiments with a total of 2-7 mice with at least 111 tracks per condition and analyzed with unpaired Student’s t-test. Data in I are pooled from 2 independent experiments and analyzed using unpaired Student’s t-test. ***, p < 0.001.

Article Snippet: Anti-α1-integrin Ha31/8 was from BD Bioscience, anti-α4 integrin PS/2 and anti-αE integrin M290 were from BioXCell, anti-αV integrin RMV-7 was from BioLegend, and anti-Mac1 mAb M1/70 was purified from hybridoma supernatant.

Techniques: Inhibition, Mutagenesis, Flow Cytometry

Journal: bioRxiv

Article Title: Salivary gland macrophages assist tissue-resident CD8 + T cell immune surveillance

doi: 10.1101/723791

Figure Lengend Snippet: A. 2PM time-lapse image sequence of T RM in DTx-treated CD11c-YFP or CD11c-DTR -> Ubi-GFP chimeras. Magenta lines indicate outlines of acini, white segmented lines indicate cell tracks. Scale bar, 50 µm (overview) and 20 µm (insert). Time in min:s. B. Example T RM tracks in presence or absence of macrophage. Scale bar, 10 µm. C. Frequency distribution of T RM speeds in DTx-treated CD11c-YFP or CD11c-DTR bone marrow chimera. Arrows indicate median (µm/min). D. Track analysis outline. Top panel. U-turns (red) describe tracks reversing direction while excluding continuous turns. Bottom panel. Synthetic tracks were generated to assess dwell time in an 80 µm-diameter sphere (black). One example track is shown for control (light blue) and macrophage-depleted (dark blue) condition. E. Percent of tracks making U-turn. Bars indicate 95% confidence intervals. F. In silic o dwell times for T RM tracks in 80 µm-spheres based on measured track parameters. G . 2PM time-lapse image sequences of T RM crawling along a macrophage to enter acini. Epithelial signal was manually masked to show an isolated acinus in zoomed panels. Dashed white line indicates area displayed in xz-view, and arrow indicates T RM -macrophage contact. Top: Scale bar, 50 μm (overview) and 20 μm (insert); bottom: Scale bar, 20 μm (overview) and 10 μm (insert). Time in min:s. H. Percentage of T RM transitions into or out of acini and ducts in CD11c-YFP -> Ubi-GFP chimeras (n = 42) with and without contact to macrophages. I, J. 2PM time-lapse image sequence of CD11cYFP -> Ubi-GFP and DTx-treated CD11cDTR->Ubi-GFP chimeras were analyzed for T RM crossing events (leaving or entering acini). I shows average transitions per hour track duration, and J depicts transitions per 1000 μm total distance migrated. Data points represent individual image sequences. Line indicates mean. K. Experimental layout for analysis of T RM response to local chemokine. CXCL10 was injected with a fluorescent tracer for 4 h to allow T RM accumulation. Integrin blocking mAbs prevent recruitment of circulating T cells. L. T RM per cm 2 at sites of CXCL10 injection in presence or absence of macrophages. Numbers indicate mean ± SD. Data in C, I, J and L are pooled from 2-4 independent experiments with 4-6 mice total. Data in C, I and J were analyzed with Mann-Whitney and data in L were analyzed with Wilcoxon rank test. *, p < 0.05; ***, p < 0.001.

Article Snippet: Anti-α1-integrin Ha31/8 was from BD Bioscience, anti-α4 integrin PS/2 and anti-αE integrin M290 were from BioXCell, anti-αV integrin RMV-7 was from BioLegend, and anti-Mac1 mAb M1/70 was purified from hybridoma supernatant.

Techniques: Sequencing, Generated, Control, Isolation, Injection, Blocking Assay, MANN-WHITNEY