Cells were permeabilized and stained for 30 min with the surface markers CD3 (clone 17A2), CD4 (clone RM4-5), CD25 (clone PC61.5), for the transcription factor Foxp3 (FJK-16s), and for the cytokines IL-6 (clone MP5-20F3), IFNγ (clone XMG1.2), IL-17 (clone TC11-18H10.1), IL-10 (clone JES5-16E3), and Ly-6G (Gr-1, clone 1A8). All antibodies were purchased from BD Bioscience
(San Jose, CA) or eBioscience (San Diego, CA). For each sample, at least 50 000 cells were analyzed. The data were collected and analyzed using CELLQuest or FlowJo software and a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). To determine the levels of secretion of cytokines, dermal Vadimezan datasheet cells pooled from three mice (six ears), or individual lymph node cells, were resuspended at a concentration of 2×106 cells/well in medium RPMI 1640 (supplemented with FCS and antibiotics), seeded into 24-well plates and incubated for 48 h with 50 μg/mL soluble Leishmania antigen alone or combination with 50 μg/mL CpG DNA. Cytokine levels were measured in the supernatants by either using the BD™ CBA Mouse Inflammation Kit following the manufacturer’s instructions (BD Bioscience) or by ELISA (eBioscience). To neutralize IL-6, C57BL/6 mice were injected
Crenolanib with 5 μg anti IL-6 receptor (R&D Systems, Minneapolis, MN) by intraperitoneal injection on days -1, 1, and 3 relative to vaccination as in 11. To old neutralize IL-17 and IFN-γ, C57BL/6 mice were injected with 10 μg anti IL-17 and/or 10 μg anti IFN-γ (R&D Systems) by intraperitoneal injection on days 6, 9, and 12 days relative to vaccination. Analyses of dermal lymphocytes were performed at different time points post infection. Control mice were inoculated with the same dose of GL113, a rat monoclonal antibody (IgG1) purchased from R&D systems. All comparisons of non-normally distributed continuous data were analyzed with the Mann–Whitney U test or ANOVA using GraphPad Prism (San Diego, CA). The specific test
employed is indicated in each figure. The authors would like to thank Dr. Jay Kolls for providing the IL-17R / mice, and Meleana Hinchman for her technical assistance. This work was supported by the NIH grant no. R21 AI61379. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“Several assays to measure pre-existing allospecific T cell immunity in renal transplant candidates have been developed in the past years.