Journal: Journal of Immunology Research

Article Title: Enhanced Cysteinyl-Leukotriene Type 1 Receptor Expression in T Cells from House Dust Mite-Allergic Individuals following Stimulation with Der p

doi: 10.1155/2015/384780

Figure Lengend Snippet: Flow-cytometric analysis of T cell proliferation and Th cell polarization. CFSE-labeled CD4 + T cells from HDM-allergic or HDM-nonallergic individuals were cultured with Der p or glycerin as described in . T cell division was analyzed by flow cytometry and illustrated as a CFSE division profile (a). Dead cells were excluded based on their light scattering properties. Nondividing CD4 + T cells in the absence of allergen are shown as dark grey histograms. CD4 + T cells that have divided in response to Der p are shown in light grey histograms, based on CFSE dilution peaks. One representative experiment of three is illustrated. In vitro polarization of human Th1 and Th2 precursors following 11 days of culture with glycerin or Der p was determined by flow cytometry using Alexa-conjugated Abs for human CXCR3 (b) or CRTH2 (c), respectively. Data are expressed as geometric mean (±SEM) fluorescence intensity (MFI). P values are indicated. HDMA = house dust mite-allergic donors. Th2/Th1 ratio of CD4 + T cells from HDM-allergic donors following 11 days of culture with glycerin or Der p (d), n = 6; Th2/Th1 ratio of Der p-stimulated CD4 + T cells from nonallergic and HDM-allergic individuals (e), n = 6 for controls; n = 10 for HDMA.

Article Snippet: On day 11, cells were harvested and incubated for 30 minutes at 4°C with fluorochrome-conjugated monoclonal antibodies: CD184 PE (CXCR4 Ab), CD183 Alexa 488 (CXCR3 Ab), and CD294 Alexa 647 (CRTH2 Ab) (BD Pharmingen, San Diego, CA, USA) antibodies known to recognize Th cell surface markers associated with Th0, Th1, and Th2, respectively [ , ].

Techniques: Labeling, Cell Culture, Flow Cytometry, In Vitro, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Antigen specificity and cross-reactivity drive functionally diverse anti– Aspergillus fumigatus T cell responses in cystic fibrosis

doi: 10.1172/JCI161593

Figure Lengend Snippet: ( A ) PBMCs from pwCF ( n = 8–14) were stimulated with whole A . fumigatus lysate, and reactive CD154 + Tmem, Th1 (IFN-γ + ), Th17 (IL-17A + ), or Th2 (CRTH2 + ) cells were FACS sorted, expanded, and restimulated with a panel of single A . fumigatus proteins. Reactivity is indicated as the percentage of CD154 + TNF-α + within CD4 + T cells. ( B ) Th2 target proteins (Aspf2, Aspf3, CpcB, CatB, Fg-gap, GliT) and non-Th2 target proteins (Scw4, Aspf22, Pst1, Shm2, CcpA, TpiA, Crf1, Sod3) were pooled and used for ex vivo stimulation of PBMCs from A . fumigatus –sensitized pwCF. Relative cytokine production within reactive CD154 + Tmem cells (upper plots) and absolute frequencies of cytokine producers within CD4 + T cells (lower plots) are shown (IL-4, IFN-γ, IL-17A, n = 20; IL-5, IL-13, n = 13). ( C ) A . fumigatus– reactive T cell lines were generated as in A and restimulated with different A . fumigatus antigen extracts in the presence of autologous FastDCs derived from blood monocytes. Reactivity in relation to restimulation with the initially used total A . fumigatus lysate is shown. ( D ) Reactivity of expanded CD154 + Tmem cells from Th2-high ( n = 7) or Th2-low ( n = 6) patients to the different A . fumigatus protein fractions. Each symbol in A – D represents 1 donor; horizontal lines indicate the mean in upper plots and the geometric mean in lower plots of B ; truncated violin plots with the quartiles and range are shown in C and D . Statistical differences were determined by 2-tailed, paired Wilcoxon rank test in B . and by Kruskal-Wallis test with Dunn’s post hoc test in C and D .

Article Snippet: Cells were stained using different combinations of fluorochrome-conjugated antibodies: CD4-APC-Vio770 (M-T466), CD8-VioGreen (REA734), CD14-VioGreen (REA599), CD20-VioGreen (LT20), CD154-FITC (REA238), Ki-67-Vio667 (REA183), IL-17A–PE–Vio770 (CZ8-23G1), CRTH2-PE-Vio770 (REA598), CCR4-APC (REA279), IL-5–VioR667 (JES1-39D10), IL-13–PE (JES10-5A2.2), CD137-PE (4B4-1), CD4-FITC (REA623), CD154-APC (5C8), TNF-α–PE–Vio770 (cA2), CD127-FITC (REA614), CD25-APC (4E3), LAP-PE (REA1214), GARP–VioBright B515 (REA166) (all from Miltenyi Biotec); CD45RA-PE-Cy5 (HI100), IFN-γ–BV785 (4S.B3), IL-4–BV605 (MP4-25D2), IL-10–PE–Dazzle (JES3-9D7), CCR6-BV421 (G034E3), IL-4–PerCP–Cy5.5 (MP4-25D2), CD25-BV421 (BC96), CD127-BV605 (A019D5), Foxp3-AF647 (206D), Helios PE–Cy7 (22F6), and CTLA4-BV605 (BNI3) (all from BioLegend); and IL-2–BV711 (5344.111; BD Biosciences).

Techniques: Ex Vivo, Generated, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Antigen specificity and cross-reactivity drive functionally diverse anti– Aspergillus fumigatus T cell responses in cystic fibrosis

doi: 10.1172/JCI161593

Figure Lengend Snippet: ( A and B ) A . fumigatus –reactive ( n = 8–14) or S . apiospermum –reactive ( n = 6–9) CD154 + Tmem, Th1 (IFN-γ + ), Th17 (IL-17A + ), or Th2 (CRTH2 + ) cells from pwCF were FACS sorted, expanded, and restimulated with both fungal extracts. ( A ) Representative restimulation of expanded T cell lines. Percentages of CD154 + TNF-α + within CD4 + T cells are indicated in the plots. ( B ) Percentage of cross-reactivity of initially A . fumigatus –stimulated cells to S . apiospermum (left plot) and vice versa (right plot). Each symbol represents 1 donor, and data indicate the mean ± SEM. ( C and D ) PBMCs from Th2-high pwCF were ex vivo stimulated with A . fumigatus Aspf2. Reactive CD154 + Tmem cells were FACS purified, expanded, and restimulated in the presence of autologous FastDCs with Aspf2 or orthologous proteins from S . apiospermum (UniProtKD A0A084G096) or C . albicans (pH-regulated antigen Pra1 UniProtKB P87020). ( C ) Dot plot examples of restimulation. Cells were gated on CD4 + T cells, and the percentages of CD154 + TNF-α + T cells are indicated. ( D ) Percentage of cross-reactivity of Aspf2-reactive cells to the orthologous proteins of S . apiospermum and C . albicans . ( E and F ) Expanded A . fumigatus –reactive ( n = 4) or S . apiospermum– reactive ( n = 2) Th2 cells were restimulated with various fungal species. ( E ) Dot plot examples showing the restimulation of S . apiospermum– reactive Th2 cells with various fungal lysates. The percentages of CD154 + Th cells within CD4 + T cells are indicated in the plots. ( F ) The percentage of cross-reactivity in relation to total reactivity after restimulation with the specific fungal lysate is shown. Each row of the heatmap indicates 1 patient. Each symbol in B and D represents 1 donor; data indicate the mean ± SEM in B . Box-and-whisker plots display the quartiles and range in D .

Article Snippet: Cells were stained using different combinations of fluorochrome-conjugated antibodies: CD4-APC-Vio770 (M-T466), CD8-VioGreen (REA734), CD14-VioGreen (REA599), CD20-VioGreen (LT20), CD154-FITC (REA238), Ki-67-Vio667 (REA183), IL-17A–PE–Vio770 (CZ8-23G1), CRTH2-PE-Vio770 (REA598), CCR4-APC (REA279), IL-5–VioR667 (JES1-39D10), IL-13–PE (JES10-5A2.2), CD137-PE (4B4-1), CD4-FITC (REA623), CD154-APC (5C8), TNF-α–PE–Vio770 (cA2), CD127-FITC (REA614), CD25-APC (4E3), LAP-PE (REA1214), GARP–VioBright B515 (REA166) (all from Miltenyi Biotec); CD45RA-PE-Cy5 (HI100), IFN-γ–BV785 (4S.B3), IL-4–BV605 (MP4-25D2), IL-10–PE–Dazzle (JES3-9D7), CCR6-BV421 (G034E3), IL-4–PerCP–Cy5.5 (MP4-25D2), CD25-BV421 (BC96), CD127-BV605 (A019D5), Foxp3-AF647 (206D), Helios PE–Cy7 (22F6), and CTLA4-BV605 (BNI3) (all from BioLegend); and IL-2–BV711 (5344.111; BD Biosciences).

Techniques: Ex Vivo, Purification, Whisker Assay

Journal: Cell Reports Medicine

Article Title: The single-cell transcriptional landscape of innate and adaptive lymphocytes in pediatric-onset colitis

doi: 10.1016/j.xcrm.2023.101038

Figure Lengend Snippet:

Article Snippet: anti-human CRTH2 PE-CF594 (Clone BM16) , BD , Cat#563501.

Techniques: Software, Staining

Journal: eLife

Article Title: Single-cell glycomics analysis by CyTOF-Lec reveals glycan features defining cells differentially susceptible to HIV

doi: 10.7554/eLife.78870

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Human CD294/CRTH2 (BM16) (Rat, Monoclonal) , Fluidigm , Cat# 3163003B , CyTOF (1:50).

Techniques: Recombinant, Plasmid Preparation, Software