For CD4 T-cell enrichment, single cell suspensions from peripheral LN of TCR-transgenic TS-1 and control BALB/c mice were stained with biotinylated Ab to CD8, CD11b, CD19, GR-1 (all in-house generated), and CD49b (BD bioscience), followed by anti-biotin Ab coupled to MACS beads (Miltenyi Biotec) and isolated by autoMACS (Miltenyi Biotec). CD4
T-cell purities were >96% as determined by staining with anti-CD4 and anti-CD3 Ab. Recipient BALB/c mice received 2.3×106 CD4 T cells from either BALB/c or TS-1 donors 12 h prior to infection. Cell suspensions in medium (RPMI 1640, 292 μg/mL L-glutamine, 100 μg/mL penicillin/streptomycin, 10% heat-inactivated fetal calf serum, 0.03 M 2-ME) were placed in duplicates at 106 cells/well into ELISPOT plates (MultiScreen HA Filtration; Millipore) coated with sucrose-density gradient-purified influenza A/PR8. Twofold www.selleckchem.com/products/dorsomorphin-2hcl.html serial dilutions in medium were
performed. Virus-specific ELISPOT assay were done as described previously 32 revealing with either Ig (H+L)-biotin (Southern Biotech) or with anti-C12Id-biotin (23-1 Id). Mean spot counts±SD/106 input cells were calculated from all wells with countable spots. Virus-specific ELISA was done as previously described 32. For C12Id virus-specific ELISA 3% phosphate buffered PFA solution (pH 7.2) was used following serum incubation to crosslink Ag–Ab complexes and enhance sensitivity of the assay 24. Relative virus-specific Ig units were calculated by Vasopressin Receptor comparison to a standard hyperimmune serum 47. Relative virus-specific Ab concentrations were calculated from PD0325901 in vitro a standard virus-specific IgG C4Id+ mAb (clone H37-41-7) or a virus-specific IgG C12Id+ mAb (clone H35-C12.6.2) both purified from tissue-culture supernatant by protein G affinity chromatography. One relative unit was arbitrarily defined as equivalent to binding of 1 μg/mL of the relevant mAb. ELISA plates were measured on a SpectraMax
M5 (Molecular Devices) ELISA reader, and data were analyzed using Soft MaxPro software (Molecular Devices). Statistical analysis was done using a two-tailed un-paired Student’s t-test with the help of Prism 4 software (GraphPad Software, San Diego, CA, USA). Data were regarded as statistically significant at p<0.05. The authors thank Abby Spinner for help with the FACS Aria, Stefan Tunev (UC Davis) for extensive help with immunohistochemistry and immunofluorescence, Dr. Michael McChesney (UC Davis) for critical reading of the manuscript, and Walter Gerhard (The Wistar Institute) for critical reagents, advice, insight, and inspiration throughout these studies. This work was supported by a grant from the NIH/NIAID AI051354 (to N. B.) and NIH training grant support to K. R. (T32-A160555 and T32 HL07013-31A1). Conflict of interest: The authors declare no financial or commercial conflict of interest. "
“B-cell-derived interleukin-10 (IL-10) is known to act in a paracrine fashion to suppress inflammation.