Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: Allergic phenotype in WT and Saa–/– C57BL/6 mice sensitized and challenged with HDM was analyzed seventy-two hours after the last challenge. Control mice received PBS at both sensitization and challenge. (a) Airway responses to cholinergic stimulation shown as airway pressure over time (APTI, *P = 0.0378, **P = 0.0019), (b) total serum IgE concentrations (**P = 0.0019), (c) bronchoalveolar lavage (BAL) eosinophilia and (d and e) histological examination of airway inflammation. Sections were stained for mucus production with periodic acid Schiff (PAS; Scale bars 100 μm) and bar graphs represent percentage of mucus positive cells in the airways (*P = 0.0241). Numbers of (f) CD11b+ DCs, (g) IL-13+ CD3+CD4+ and (h) IL-13+ILC2 cells (***P = 0.0003) in the lungs and (i and j) TH2 cytokine production from HDM-restimulated lung cells (**P = 0.0018). Data represent means ± SEM of pooled data from 2 independent experiments containing n=11 WT PBS, n=12 WT HDM, n=11 Saa–/– PBS and n=13 Saa–/– HDM animals per group (a, c, and f-j), n=2 WT PBS, n=3 WT HDM, n=2 Saa–/– PBS and n=4 Saa–/– HDM animals per group (d, e), or are representative of 2 experiments (b) with n=8 WT PBS, n=7 WT HDM, n=8 Saa–/– PBS and n=8 Saa–/– HDM animals per group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc (b, c, e-j) or Holm-Sidak’s (a) analysis that compares WT HDM to counterparts. ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: For ILC2 flow, cells were plated at 2 × 10 6 cells/ml, restimulated with PMA/ionomycin overnight as described above before staining with PerCP-Cy5.5 or APC-Cy7-conjugated lineage marker monoclonal antibodies (FcεRI, MAR-1, BioLegend; CD3ε, 145–2C11, BD Pharmingen; TCRβ, H57–597, BD Pharmingen; B220, RA3–682, BD Pharmingen; CD49b, DX5, eBioscience; CD11b, M1/70, BD Pharmingen; CD11c, N418; BioLegend; Gr1, RB6–85C, BD Pharmingen; NK1.1, PK136, BioLegend), Alexa Fluor700-conjugated CD45 (30-F11; BD Pharmingen), BV605-conjugated CD90.2 (53–2.1, BD), APC-e780-conjugated CD25 (PC615, eBioscience), PE-Cy7-conjugated ICOS (C398.4A; BioLegend), BV421-conjugated IL-33Rα (DIH9, BioLegend) and FITC-conjugated T1/ST2 (DJ8, MD Bioproducts).

Techniques: Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Serum Amyloid A is a Soluble Pattern Recognition Receptor that Drives Type 2 Immunity

doi: 10.1038/s41590-020-0698-1

Figure Lengend Snippet: The parasitic worm Schistosoma mansoni (a Puerto Rican isolate) routinely maintained in the laboratory by cycling between the intermediate snail host, Biomphalaria glabrata, and outbred ICR mice as definitive hosts. (a) Numbers of CD11b+ DCs in the lungs of PBS or Schistosoma mansoni worm extract-treated WT and Saa–/– C57BL/6 mice (***P = 0.0002). (b) Frequency and (c) total numbers of CD3+CD4+ T cells (*P = 0.014). (d) TH2 (***P = 0.0003) and (e) TH17 (**P = 0.0014) cells in the lungs of these mice. (f-i) Cytokine production from worm extract-restimulated lung cells (IL-5: *P = 0.019, IL-10: *P = 0.011, IL-13: *P = 0.017, IL-17: *P = 0.017). Data represents means ± SEM of pooled data from 2 independent experiments containing a total of n= 8 animals in each group. P values were calculated with a two-tailed test using one-way analysis of variance (ANOVA) with Dunett’s post hoc that compares WT worm to counterparts. ****P ≤ 0.0001.

Article Snippet: For ILC2 flow, cells were plated at 2 × 10 6 cells/ml, restimulated with PMA/ionomycin overnight as described above before staining with PerCP-Cy5.5 or APC-Cy7-conjugated lineage marker monoclonal antibodies (FcεRI, MAR-1, BioLegend; CD3ε, 145–2C11, BD Pharmingen; TCRβ, H57–597, BD Pharmingen; B220, RA3–682, BD Pharmingen; CD49b, DX5, eBioscience; CD11b, M1/70, BD Pharmingen; CD11c, N418; BioLegend; Gr1, RB6–85C, BD Pharmingen; NK1.1, PK136, BioLegend), Alexa Fluor700-conjugated CD45 (30-F11; BD Pharmingen), BV605-conjugated CD90.2 (53–2.1, BD), APC-e780-conjugated CD25 (PC615, eBioscience), PE-Cy7-conjugated ICOS (C398.4A; BioLegend), BV421-conjugated IL-33Rα (DIH9, BioLegend) and FITC-conjugated T1/ST2 (DJ8, MD Bioproducts).

Techniques: Two Tailed Test

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: i NKT cells require autophagy to coordinate proliferation and survival signals during differentiation

doi: 10.4049/jimmunol.1402154

Figure Lengend Snippet: Loss of Atg5 expression led to cell cycle arrest. (A) Atg5 deficient iNKT cells exhibited enhanced incorporation of BrdU. 5 h after injection, thymic iNKT cells were collected and analyzed for BrdU incorporation and Ki-67 expression. (B) Thymic iNKT cells from Atg5f/f CD4-Cre mice accumulated in S-phase. 2 h post injection of BrdU, thymic iNKT cells were stained with 7-AAD and anti-BrdU antibody to evaluate cell cycle progression. (C) Increased expression of p21cip1 in Atg5 deficient iNKT cells. Shown are the representative flow cytometry analysis (left) and geometric MFI (right) of data pooled from at least three independent experiments gating on either NK1.1 or NK1.1+, CD1d tetramer+ thymocytes. Filled histogram: isotype control; solid line: wild type mice; dotted line: Atg5f/f CD4-Cre mice. * p<0.01, n≥4. Error bars are SD.

Article Snippet: Antibodies and reagents The following antibodies, with clone designation in parentheses, were from BD PharMingen: CD1d–PE (1B1), CD4-APC (RM4–5), CD8-PerCP-Cy5.5 (53–6.7), CD24-FITC (M1/69), CD45.1-FITC (A20), Fas-FITC (Jo2), anti-BrdU-Alexa Fluor 488 (3D4), IFNγ-PE-Cy7 (XMG1.2), IL-4-Alexa Fluor 647 (11B11), Ki-67-PE (B56), NK1.1-PE-Cy7 (PK136), GATA3-PE-Cy7 (L50-823), phospho-Akt(pS473)-PE (M89-61), phospho-Akt(pT308)-PE (J1–223.371) and purified antibody anti-active caspase 3 (C92-605).

Techniques: Expressing, Injection, BrdU Incorporation Assay, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: i NKT cells require autophagy to coordinate proliferation and survival signals during differentiation

doi: 10.4049/jimmunol.1402154

Figure Lengend Snippet: Atg5 deletion resulted in increased mitochondria, super oxide and cell death. Augmented mitochondrial mass (A) and mitochondrial superoxide production (B) in Atg5 deleted, thymic, NK1.1 and NK1.1+iNKT cells. (C) Higher expression of Fas on Atg5 deficient iNKT cells compared to wild type controls. At least four independent experiments were performed with consistent results, both representative flow cytometry plots (left) and geometric MFI values (right) are shown. Filled histogram: isotype control; solid line: wild type mice; dotted line: Atg5f/f CD4-Cre mice. (D) Dramatically increased apoptosis leading to cell death for NK1.1+ thymic iNKT cells devoid of Atg5. Cells were analyzed after overnight culture and compared with wild type controls. Representative flow cytometry analyses from three separate experiments (left) and geometric MFI (right) of dead cells (Annexin V+, Live/Dead Yellow+) are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n≥4. Error bars are SD.

Article Snippet: Antibodies and reagents The following antibodies, with clone designation in parentheses, were from BD PharMingen: CD1d–PE (1B1), CD4-APC (RM4–5), CD8-PerCP-Cy5.5 (53–6.7), CD24-FITC (M1/69), CD45.1-FITC (A20), Fas-FITC (Jo2), anti-BrdU-Alexa Fluor 488 (3D4), IFNγ-PE-Cy7 (XMG1.2), IL-4-Alexa Fluor 647 (11B11), Ki-67-PE (B56), NK1.1-PE-Cy7 (PK136), GATA3-PE-Cy7 (L50-823), phospho-Akt(pS473)-PE (M89-61), phospho-Akt(pT308)-PE (J1–223.371) and purified antibody anti-active caspase 3 (C92-605).

Techniques: Expressing, Flow Cytometry