Journal: Annals of Laboratory Medicine

Article Title: Autoantibodies with Mimicking Specificity Detected by the Dilution Technique in Patients with Warm Autoantibodies

doi: 10.3343/alm.2013.33.5.343

Figure Lengend Snippet: Red cell transfusion workflow in 71 patients having warm autoantibodies evaluated with the combination of dilution technique and red cell phenotyping.

Article Snippet: We determined the RBC phenotype of the patients at least three months after the last transfusion by using the commercially available phenotyping gel card RhD+Phenotype (DiaMed GmBH, Cressier, Switzerland).

Techniques:

Journal: Immunity

Article Title: Programming of distinct chemokine-dependent and -independent search strategies for T helper-1 and Th2 cells optimizes function at inflamed sites

doi: 10.1016/j.immuni.2019.06.026

Figure Lengend Snippet: (a) Maximal z-projection images of Th1 and Th2 cells (green) and SHG (blue) (left panel, scale bar 50µm.) and circularity of T cells (right panel) in the CFA + OVA peptide inflamed dermis (pOVA/CFA) in mice treated with PBS or PTx (1µg) 5h prior to imaging. (b) Average velocity, meandering index and displacement rate of T cells in the inflamed dermis, as in (a). Symbols represent individual cells, 1 of 3 independent experiments: 10 mice for Th1 cells and 9 mice for Th2 cells, 15–17 imaging volumes each. (c) Co-transferred Th1 and Th2 cells into WT mice prior to pOVA/CFA immunization and PTx treatement. Average velocity of cells in the same tissue volume, 4–6 independent experiments, 18–24 imaging volumes. (d) Average velocity and meandering index of T cells in the inflamed dermis of mice treated with Gallein 2h prior to imaging. (e) Average velocity of T cells in mice immunized with pOVA/CFA (cognate Ag, left ear) or pLACK/CFA (non-cognate Ag, right ear) and treated with PBS or Gallein. (d) and (e) 1 of 3 independent experiments: 10 mice for Th1 cells and 8 mice for Th2 cells, 12–13 imaging volumes each. (f) Average velocity of T cells in the inflamed dermis of mice with or without 500µg anti-CXCL9 and anti-CXCL10 Ab i.v. 2h prior to imaging. (g) Th1 and Th2 cells co-transferred into mice prior to immunization and anti-CXCL9 and anti-CXCL10 Ab treatment. Same tissue volume, 2 independent experiments >12 imaging volumes. (h) Average velocity of Th2 cells in the pOVA/CFA inflamed ear dermis of PTx-treated mice before and immediately after 100µg i.v. anti-β1 and β3 Abs. (d)-(f), (h) Symbols represent individual cells, 1 of 3 independent experiments, 6 imaging volumes per experiment. Statistics by Mann-Whitney test, ** p ≤ 0.01, *** p ≤0.001, **** p<0.0001. Also see Figure S2 and S3 and Videos 2–4.

Article Snippet: For skewing to a Th2 phenotype, IL-4 (20ng/ml; Peprotech) and anti-IFNγ (40 mg/ml; XMG-1.2) were added to the culture.

Techniques: Imaging, MANN-WHITNEY

Journal: Immunity

Article Title: Programming of distinct chemokine-dependent and -independent search strategies for T helper-1 and Th2 cells optimizes function at inflamed sites

doi: 10.1016/j.immuni.2019.06.026

Figure Lengend Snippet: OVA-specific Th2 cells were adoptively transferred to WT mice and recipients immunized in the ear dermis with CFA or Alum + OVA peptide (pOVA/CFA and pOVA/Alum respectively). (a) Relative proportions and (b) ratios of Type-2 (CCL17, CCL22, CCL11) and Type-1 (CXCL10, CXCL9, CCL5) chemokine proteins in homogenates of pOVA/CFA or pOVA/Alum inflamed ears d3 post-immunization. (c) Average velocity of Th2 cells in the pOVA/CFA or pOVA/Alum inflamed dermis. 1 of 3 independent experiments: 15 mice, 10–15 imaging volumes. (d) Average velocity of Th2 cells in the pOVA/Alum inflamed dermis before and after i.v. anti-β1 and β3 Abs. (e) Average velocity and meandering index of Th2 cells in the pOVA/Alum inflamed dermis of mice treated with PBS or PTx. Three independent experiments. Statistics by Mann-Whitney test, *** p ≤0.001. Also see Figure S3 and Video 6.

Article Snippet: For skewing to a Th2 phenotype, IL-4 (20ng/ml; Peprotech) and anti-IFNγ (40 mg/ml; XMG-1.2) were added to the culture.

Techniques: Imaging, MANN-WHITNEY

Journal: Immunity

Article Title: Programming of distinct chemokine-dependent and -independent search strategies for T helper-1 and Th2 cells optimizes function at inflamed sites

doi: 10.1016/j.immuni.2019.06.026

Figure Lengend Snippet: OVA-specific DO11.10 TCR Tg+ Th1 and Th2 cells were adoptively transferred to WT mice and recipents immunized in the ear dermis with CFA + OVA peptide (pOVA/CFA). Motility within the dermal interstitium was analyzed by IV-MPM, d3 post-immunization. (a) Maximal z projection image of co-transferred Th1 (red) and Th2 (green) cells and SHG (blue) in the pOVA/CFA inflamed dermis. Representative image from 10 mice, 20 imaging volumes. Scale bar: 100µm. (b) Average velocity of T cells in the pOVA/CFA inflamed dermis before and immediately after i.v. 100µg anti-β1 and β3 integrin blocking Abs. Dots represent individual cells, 1 of 4 independent experiments, 6 mice, 6 imaging volumes (Th1) and 5 mice, 5 imaging volumes (Th2). (c) Mean velocity and (d) displacement rate of T cells from 8–10 independent experiments. (e) Mean squared displacement of T cells in the pOVA/CFA inflamed dermis, one of 5–9 independent experiments. (f) Motility coefficient of T cells in the pOVA/CFA inflamed dermis. Dots represent individual cells. Statistics by Mann Whitney. Kolmogorov-Smirnov test of Th1 and Th2 cell squared displacement in xy plane, p < 0.05. (e) and (f) representative data from 8–10 independent experiments, 20 mice, >50 imaging volumes. (g) Migratory paths of Th1 and Th2 cells (75–140 cells per plot) over 60 mins (presented as x-y projections), p=0.03 for frequency of tracks >100 mm displacement between Th1 and Th2 cells. (h,i) Arrest coefficient for T cells in the CFA + OVA (OVA protein, cognate-ligand) or CFA + KLH (KLH protein, non-cognate ligand) inflamed dermis, 3 of >6 independent experiments, 12–18 imaging volumes, statistics by ANOVA. (j-l) T cell contacts with CD11c+ cells in the OVA or KLH inflamed dermis. (j) Frequency of T cells that made contact with a CD11c+ cell over 60 minutes, 3 independent experiments, statistics by student t test. (k) representative time-lapse images of T cells (red) and CD11c+ cells (green) and the 3D surface overlap (white). (l) Duration of 3D surface overlap between T cells and CD11c+ cells. 3 independent experiments, statistics by 2 way ANOVA with Sidak’s multiple comparisons: Th1 vs. Th2 OVA/CFA, p < 0.0001; Th1 vs. Th2 KLH/CFA, not significant. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤0.001, **** p <0.0001. Also see Figure S1 and S2 and Videos 1 and 2.

Article Snippet: For skewing to a Th2 phenotype, IL-4 (20ng/ml; Peprotech) and anti-IFNγ (40 mg/ml; XMG-1.2) were added to the culture.

Techniques: Imaging, Blocking Assay, MANN-WHITNEY

Journal: Immunity

Article Title: Programming of distinct chemokine-dependent and -independent search strategies for T helper-1 and Th2 cells optimizes function at inflamed sites

doi: 10.1016/j.immuni.2019.06.026

Figure Lengend Snippet: (a)(b) Representative histograms of integrins αV and β3 on non-transduced Th1 (blue), Th2 (red) cells and (a) Th1 cells transduced with β3 integrin-containing retrovirus (blue filled) or the same for each plot(b) Th2 cells transduced with αV-shRNA-containing retrovirus (green filled). (a) x and y axis the same for each plot. (b) x and y axis the same for each plot. (c) Motility coefficient of Emerald+ αVβ3hi Th1 and GFP+ αVβ3lo Th2 cells compared to GFP+ control virus transduced Th1 and Th2 cells in the CFA + OVA peptide (pOVA/CFA) inflamed dermis. 3–4 independent experiments, >10 imaging volumes per group. (d) Migratory paths of T cells (>30 cells per plot) in the CFA-inflamed dermis over 60 mins (presented as x-y projections). Representative of >10 imaging volumes. (e) Mean squared displacement of cells in (c). (f) Average velocity of αVβ3hi Th1 and αVβ3lo Th2 cells in immunized mice treated with PBS or PTx. Pooled data from 3–4 independent experiments. (g) Control-GFP+ (Ctrl) and β3-Emerald+ (αVβ3hi) transduced Th1 cells were co-transferred to WT mice and immunized with pOVA/CFA. Cytokine producing cells in the inflamed skin d3 post-transfer by ex vivo intracellular cytokine staining. (h) Control-GFP+ (Ctrl) and αV-shRNA-GFP+ (αVβ3lo) transduced Th2 cells co-transferred to WT mice as in (g). Ex vivo cytokine producing cells in the inflamed skin d3 post-transfer. (g, h) Paired symbols represent data from a single recipient mouse of co-transferred cells. Three independent experiments, 4–5 mice per group per experiment. (i, j) WT C57BL/6 and BALB/c mice were infected with L. major and 3 wks post-infection mice were treated with Gallein (+) or PBS (−) (i.p. daily for 7 days). (i) Frequency of CD4+ T cell IFNγ producers in C57BL/6 mice (left panel) and IL-4 producers in BALB/c mice (right panel) in the infected skin by ex vivo intracellular cytokine staining. Mean of each of three independent experiments, 4–5 mice per group per experiment. (j) Fold change in frequency of IFNγ producers (left) and IL-4 producers (right) for individual mice relative to the mean of the PBS group in each experiment in (i), 10–13 mice per group. Statistics by Mann-Whitney Test, or paired t Test, and, for (j), Anova with Tukey’s multiple comparisons. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p <0.0001. Also see Figure S5.

Article Snippet: For skewing to a Th2 phenotype, IL-4 (20ng/ml; Peprotech) and anti-IFNγ (40 mg/ml; XMG-1.2) were added to the culture.

Techniques: Transduction, shRNA, Control, Virus, Imaging, Ex Vivo, Staining, Infection, MANN-WHITNEY

Journal: Immunity

Article Title: Programming of distinct chemokine-dependent and -independent search strategies for T helper-1 and Th2 cells optimizes function at inflamed sites

doi: 10.1016/j.immuni.2019.06.026

Figure Lengend Snippet: (a) Schematic of data analysis. (b) Principal Component (PC) analysis of selected gene expression (Table S1) in naïve Th1, Th2, Th17, nTreg and iTreg cell subsets. PC1 p = 2.699879e-07; PC2 p = 5.735851e-07 by ANOVA. (c) Heat map of candidate gene expression in Th1 and Th2 cells, Th1-a and Th1-b cells represent separate biological samples. (d) Itgb3 expression of CD4+ T cell subsets relative to naïve CD4+ T cells. Based on RNAseq data from Stubbington et. al. Biology Direct 2015. (e) Integrin β3 cell surface expression on in vitro generated Th1, Th2 and Th17 cells by flow cytometry. Representative data from 2–4 independent experiments.

Article Snippet: For skewing to a Th2 phenotype, IL-4 (20ng/ml; Peprotech) and anti-IFNγ (40 mg/ml; XMG-1.2) were added to the culture.

Techniques: Gene Expression, Expressing, In Vitro, Generated, Flow Cytometry

Journal: Immunity

Article Title: Programming of distinct chemokine-dependent and -independent search strategies for T helper-1 and Th2 cells optimizes function at inflamed sites

doi: 10.1016/j.immuni.2019.06.026

Figure Lengend Snippet: (a) Integrin αV and β3 transcripts in in vitro-generated DO11.10 CD4+ Th1 and Th2 cells measured by qPCR. (b) Cell surface expression of integrins αV, β3 and β1 on Th1 and Th2 cells by flow cytometry. Gray histogram, isotype control. (c) Integrin αV and β3 expression and (d) MFI, of adoptively-transferred Th1 (blue) and Th2 (red) cells in the cervical lymph node (dLN) and inflamed ear dermis (Ear) on d3 following immunization with CFA + OVA peptide (pOVA/CFA). Gray histogram, isotype control; x and y axis the same for all plots. (e) Integrin αV and β3 expression on naïve DO11.10 CD4+ T cells (black line) and APC + pOVA activated CD4+ T cells under Th1 (blue) or Th2 (red) skewing conditions analyzed by flow cytometry. Gray histogram, isotype control; x and y axis the same for all plots. Numbers represent the percent of naïve (black), Th1 (blue) or Th2 (red) cells that fall within the shown gate. (f) Th2:Th1 ratio of integrin staining as in (e). (g) Th2:Th1 cell ratio of integrin staining of WT and Stat6−/− CD4+ T cells following activation with plate-bound anti-TCR and anti-CD28 Abs under Th1 and Th2 cell differentiation conditions. (h) Th2:Th1 cell ratio of integrin staining of WT and Stat1−/− CD4+ T cells activated as in (g). (i-k) In vivo generated Th1 and Th2 cells. Naïve cytokine-reporter (Yeti or 4get) WT15 TCR Tg+ cells (LACK-specific) adoptively transferred to WT recipients and immunized with pLACK in CFA + 11B11 Ab (anti-IL-4) (for Th1 cell polarization) or pLACK in Alum + XMG1.2 Ab (anti-IFNγ) (for Th2 polarization): cells gated on Yeti IFNγ-YFP+ cells (Th1) or 4get IL-4-GFP+ cells (Th2) in the inflamed skin, (gating, Fig. S4c). (i) Representative dot plots of αVβ3 on in vivo-generated Th1 and Th2 populations. (j) MFI, (k) frequency of cells from (i) in quadrant I (Q I) αVhiβ3hi and in quadrant III (Q III) αVloβ3lo. Symbols represent individual mice, 1 of 3 independent experiments, 4–5 mice per group per experiment. Statistics by Mann Whitney, ** p ≤0.01. Also see Figure S4.

Article Snippet: For skewing to a Th2 phenotype, IL-4 (20ng/ml; Peprotech) and anti-IFNγ (40 mg/ml; XMG-1.2) were added to the culture.

Techniques: In Vitro, Generated, Expressing, Flow Cytometry, Control, Staining, Activation Assay, Cell Differentiation, In Vivo, MANN-WHITNEY