Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: Serum CD95L in SLE Patients Induces Transmigration of T Lymphocytes (A) Soluble CD95L levels were measured in serum from newly diagnosed SLE patients (n = 34) and healthy donors (n = 8) via ELISA (Student’s t test). (B) CD95L in SLE serum was fractionated by size-exclusion column chromatography and measured via ELISA. Inset : CD95L was immunoprecipitated from gel-filtration fractions 40–46 and 76–78 and subjected to immunoblotting. The image is representative of gel-filtration analysis of four different patients. (C) CD95L and IL-17 staining in inflamed skin samples from lupus patients or healthy mastectomy subjects. Numbers represent different patients. The scale bar represents 100 μm. (D) CD95L, CD31, CD4, and IL-17 staining in inflamed skin samples from an SLE patient. “V” represents an endothelial vessel, and arrowheads identify marker-expressing cells. (E) Densitometric analysis of CD95L and IL-17 staining in different patients. (F) Transmigration of human T cell subpopulations in the presence of serum from SLE patients or healthy donors. (G) Th17 T cell transmigration in the presence of SLE serum containing the indicated concentrations of CD95-Fc. Undifferentiated Th 0 T cells served as controls. Data represent means ± SD of five individual serum donors. (H) CD4 + T cell transmigration with or without cl-CD95L (100 ng/mL). Data represent means ± SD of three independent experiments. (I) Treg and Th17 cell transmigration with or without cl-CD95L (100 ng/mL) Data represent means ± SD of three independent experiments (two-way ANOVA).

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Transmigration Assay, Enzyme-linked Immunosorbent Assay, Column Chromatography, Immunoprecipitation, Filtration, Western Blot, Staining, Marker, Expressing

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: Transcriptomic Signature in Human Th17 and Treg Cells Stimulated with cl-CD95L (A) Venn diagram comparing the genes differentially expressed between untreated and cl-CD95L-treated T cell subsets. (B) Heat map depicting the relative n-fold change in the amount of transcripts significantly (p ≤ 0.05) and differentially expressed between Th17 and Treg cells stimulated with cl-CD95L (100 ng/mL). Data for each experimental group (n = 2 per condition) are shown. The color gradient indicates n-fold change, as shown. (C) Pathway enrichment analysis of genes whose expression is significantly modulated by cl-CD95L in Th17 and Treg cells and associated p values. (D) Left panel : endothelial transmigration of human Th17 and Treg cells was evaluated in the presence or absence of cl-CD95L (100 ng/mL) in the Boyden chamber. Right panel : Th17 cells were pre-treated with FTY720 (1 μM), TY-52156 (10 μM), VPC-23019 (10 μM), W146 (1 μM), or CAY-1044 (1 μM) and then exposed to cl-CD95L (100 ng/mL), and endothelial transmigration was evaluated via the Boyden chamber. Data represent means ± SD of three independent experiments.

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Expressing, Transmigration Assay

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: Cl-CD95L Is a Chemoattractant for Th17 Cells In Vivo Mice received a single injection of cl-CD95L (200 ng/animal) or vehicle and were sequentially sampled. (A) T cell populations were obtained from tissues or peritoneal cavity washes. Cells were then restimulated in the presence of PMA and ionomycin for 4 hr and then analyzed by flow cytometry. Cells were identified as follows; Th1 (CD4 + IFN-γ + ), Th17 (CD4 + IL-17 + ), and Treg (CD4 + Foxp3 + ). Numbers of infiltrating cells were calculated. (B) Ratios of Th17/Th1 cells per organ were determined 24 hr after injection. Data represent two independent experiments with six mice/group; means ± SEM are displayed.

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: In Vivo, Injection, Flow Cytometry

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: CD95 Induces a DD-Independent Ca 2+ Response (A) CEM T cells were stimulated with cl-CD95L (100 ng/mL). Cells were lysed, and CD95 was immunoprecipitated. The protein complex was resolved by SDS-PAGE and subjected to immunoblotting. Total lysates served as controls. The column marked “B” indicates treatment with beads alone. Data are representative of three independent experiments. (B) Left panel : Th17 cells from peripheral blood were stimulated with cl-CD95L (100 ng/mL) for indicated times. PLA was performed with anti-CD95 and anti-PLCγ1 mAbs. Nuclei were stained in blue (DAPI). Red dots were observed when the distance between anti-CD95 and anti-PLCγ1 mAbs was close (≈16 nm). Right panels : Red dots were counted in 200 cells taken from different fields. Data represent means ± SD of three independent experiments. (C) Schematic diagram of CD95 constructs. (D) CEM-IRC cells expressing GFP alone or the GFP-fused CD95 constructs shown in (C) were loaded with the Ca 2+ probe, Fluo2-AM (1 μM). Cells were stimulated with cl-CD95L (100 ng/mL; arrow), and the intracellular calcium concentration ([Ca 2+ ] i ) was monitored. Data are given as means ± SD of three experiments performed independently on n = 20 cells. (E) HEK cells transfected with the indicated constructs were stimulated with CD95L (100 ng/mL) for indicated times. The CD95 protein complex was immunoprecipitated from cell lysates and subjected to immunoblotting, as indicated. Total lysates served as controls. The column marked “B” indicates treatment with beads alone. Data are representative of three independent experiments. (F) Activated PBLs were pre-incubated for 1 hr with TAT-control or TAT-CID (10 μM) and stimulated with cl-CD95L (100 ng/mL) for the indicated times. The CD95 protein complex was immunoprecipitated from cell lysates and subjected to immunoblotting, as indicated. Total lysates served as controls. The column marked “B” indicates a treatment with beads alone. Data are representative of three independent experiments. (G) Activated PBLs from healthy donors were loaded with FuraPE3-AM (1 μM) and pre-treated for 1 hr with TAT-control or TAT-CID (10 μM). Cells were stimulated with cl-CD95L (100 ng/mL; arrow). Data represent means ± SD.

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Staining, Construct, Expressing, Concentration Assay, Transfection, Incubation, Control

Journal: Immunity

Article Title: CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice

doi: 10.1016/j.immuni.2016.06.028

Figure Lengend Snippet: TAT-CID Alters Immunological Parameters in Lupus-Prone Mice (A) Transmigration of mouse Th17 cells was monitored with the indicated concentrations of TAT-CID. (B–D) C57BL/6 mice were injected with TAT-control or TAT-CID (40 mg/kg) 2 hr prior to intraperitoneal injection of cl-CD95L (200 ng) or vehicle. Animals were examined 24 hr after cl-CD95L injection. (B) Total cell counts in the peritoneal cavity are shown. (C) PECs were subjected to magnetic bead separation to identify the percentage of infiltrating CD4 + CD62L − (activated) T cells. (D) IL-17A concentrations in the peritoneal cavity were measured via ELISA (two-way ANOVA). Data in (B)–(D) represent two independent experiments performed with six mice/group. Data are means ± SEM. (E–I) MRL. Fas lpr/+ mice received either TAT-CID or TAT-control for 5 weeks. (E) Upon completion of the experimental protocol, ratios of spleen weight to body weight of individual animals were measured and compared to those of age-matched MRL and homozygous MRL. Fas lpr/lpr mice. (F) Total cell number in the spleen is shown. (G) Cellular composition of the spleen was determined in regard to the number of CD4 + CD62L − T cells. (H) mRNA expression levels of il-23r, ccr6 , and ror-γt in cells from (C). (I) Isolated T cells from (G) were re-stimulated with anti-CD3 mAb for 72 hr. IL-17A was then quantified by ELISA (unpaired Student’s t test).

Article Snippet: PBMCs were then subjected to selection with a cocktail of antibody-coated magnetic beads: CCR6 + CXCR3 − CD4 + cells (Th17 cells) were sorted with Human Th17 Enrichment kit (STEMCELL Technologies), and CD4 + CD25 + CD127 − Treg cells were isolated with MACS column (Miltenyi Biotec).

Techniques: Transmigration Assay, Injection, Control, Enzyme-linked Immunosorbent Assay, Expressing, Isolation