Product Protocol: TNFSF10 antibody used to evaluate type i interferon negatively controls plasmacytoid dendritic cell numbers in vivo (OASA07564)

Specificity: CD253
Product Page: TNFSF10 antibody (OASA07564)
Antibody Pair Available: OASA07564 Capture OASA07567 Detection
Experiment Type: Mice and treatments, Antibodies, flow cytometry, and cell sorting

Protocol:
1. Mice and treatments:

All animal studies were approved by the Washington University Animal Studies Committee. IFNAR−/− mice (C57BL/6 background) were provided by A. French (Washington University, St. Louis, MO). Bim−/− and Bid−/− mice (Bouillet et al., 1999; Kaufmann et al., 2007) and were provided by A. Strasser (Walter and Eliza Hall Institute, Victoria, Australia). C57BL/6, BDCA2-DTR Tg and 129/SvJ were bred in house. Heterozygous SiglecH-eGFP gene-targeted mice on a 129/SvJ background have been performed and were bred in house (Swiecki et al., 2010). MyD88−/− mice (C57BL/6 background) were bred in house. All mice were used at 6–12 wk of age. Diphtheria toxin (DT; Sigma-Aldrich) was injected i.p. into BDCA2-DTR Tg mice (100–120 ng/mouse) 24 h before HSV-1, CpGA, or poly(I:C) administration. Approximately 200 ug PK136 mAb was injected i.p. 48 h before HSV-1 infection. Purified anti-FasL mAb (CD178, clone MFL3; BioLegend) or purified anti-TRAIL mAb (CD253, clone N2B2; BioLegend) were injected i.v. at 100 ug per mouse 20 h before HSV-1 infection. Anti-IFNAR and isotype control mAbs were injected i.v. at 625 ug per mouse 4 h before HSV-1. Recombinant mouse IFN-β and IFN-α5 were gifts from D. Fremont (Washington University, St. Louis, MO). Mice were injected i.v. with 15,000 U of both IFN-β and IFN-α5.

2. Antibodies, flow cytometry, and cell sorting:

The following reagents were obtained from BD, eBioscience, or BioLegend. Fluorochrome labeled anti-SiglecH (551), anti-B220 (RA3-6B2), anti-CD11c (HL3), anti-Ly6C (AL-21), anti-CD3 (145-2C11), anti-NK1.1 (PK136), anti–Granzyme B (GB11), anti-CD262 (DR5, MD5-1), anti-CD178 (FasL, MFL3), anti-CD95 (Fas, clone 15A7), anti-CD253 (TRAIL, clone N2B2), and Streptavidin. Anti–PDC-TREM (clone 162.7, rat IgG2a) was purified from ascites and biotinylated using the FluoReporter Minibiotin kit (Invitrogen). Fc receptors were blocked before surface staining with supernatant from HB-197 cells (American Type Culture Collection). Propidium iodide was used to determine pDC frequencies and numbers during virus infections. Propidium iodide was not used in caspase activation or Annexin V experiments. All flow cytometry was conducted on a dual laser FACSCalibur flow cytometer (BD) and analyzed with FlowJo software (Tree Star). For pDC sorting from HSV-1–infected mice, CD11c+ cells were enriched from spleens with anti–mouse CD11c microbeads as recommended by the manufacturer (Miltenyi Biotec). Cells were stained with anti–Siglec-H and anti-CD11c and then sorted on a FACSAria II high speed cell sorter (BD). Purities were always >98%.

Summary:
1. Although Melissa et al. could not detect TRAIL on NK cells or pDCs, they attempted to block any potential TRAIL/DR5 interactions in vivo with an anti-TRAIL mAb before infecting mice with HSV-1. Blocking TRAIL had no impact on IFN-α production or caspase activation in pDCs. 2. Collectively, these data suggest that pDC death is independent of Fas/FasL or TRAIL/DR5 interactions and NK or NKT cell cytolytic activity during HSV-1 infection.

References:
1: Swiecki M, Wang Y, Vermi W, Gilfillan S, Schreiber RD, Colonna M. Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo. J Exp Med. 2011 Nov 21;208(12):2367-74. Epub 2011 Nov 14. PubMed PMID: 22084408; PubMed Central PMCID: PMC3256963.

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