C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Lymphoid cells from IL-6−/− and IL-23 p19−/− mice were provided by Drs. Robert Fairchild (Cleveland Clinic Foundation) and Steve Stohlman (Cleveland Clinic Foundation). IL-21−/− mice were purchased from the MMRRC (Mutant Mouse Regional Resource Centers). Rag2p-GFP and Tgfβ1−/− mice were previously described (8
). All experimental procedures were conducted according to the guidelines of the Institutional Animal Care and Use Committee of the Cleveland Clinic Foundation, Case Western Reserve University, and the University of Washington.
Ex vivo stimulation
Spleen, pLN (axillary and cervical LN) and mesenteric LN (mLN) cells were separately harvested and ex vivo stimulated with PMA (10ng/ml) and Ionomycin (1 µM) for 4 hrs in the presence of 2 µM monensin (Calbiochem, San Diego, CA) during the last 2 hrs. Cells were immediately fixed with 4% paraformaldehyde, permeabilized, and stained with fluorescence conjugated antibodies (see below).
The following antibodies were used: biotinylated anti-γδ TCR (GL3), PE-anti-γδ TCR (GL3), PE-anti-CD25 (PC61), PE-anti-CD44 (IM7), PE-anti-CD62L (MEL-14), PE-anti-IFNγ (XMG1.2), streptavidin-PE, PE-Cy5-anti-CD44 (IM7), APC-anti-CD4 (RM4-5), APC-anti-CD8 (53-6.7), APC-anti-IL-17 (ebio17B7), FITC-anti-CD4 (RM4-5), FITC-anti-CD8(53-6.7), FITC-anti-NK1.1(PK136), FITC-anti-B220(RA3-6B2), and FITC-anti-IFNγ (XMG1.2) Abs. All antibodies were purchased from eBioscience (San Diego, CA) or PharMingen. Cells were acquired using a FACSCalibur (Becton Dickinson, San Diego, CA), and the data were analyzed using FlowJo software (Treestar, Ashland, OR).
Real time PCR
DN γδ TCR-, DN γδ TCR+, DP, and CD4 SP thymocytes were isolated by FACS sorting using a FACSAria cell sorter (Becton Dickinson). RNA was extracted using RNeasy reagent (Qiagen, Valencia, CA), and cDNA was subsequently generated by Superscript III RTase (Invitrogen). Taqman primers/probes specific for Tgfbr1 (Mm03024015_m1) and Tgfbr2 (Mm03024091_m1) were purchased from ABI (Applied Biosystem Inc., Foster city, CA) and their expression was determined using an ABI 7500 PCR system. Expression level was normalized based on the 18S rRNA (VIC-TAMRA, purchased from ABI) expression.
Statistical significance was determined by the Student’s t-test using the SigmaPlot 9.0 (SPSS Inc., Chicago, IL). p<0.05 was considered to indicate a significant difference.