4, bioXcell, West Lebanon, selleckchem NH) on days 0, 1, 3, 5, 7 and 10 after adoptive transfer of CD4+ T cells and LCMV infection. Efficiency of depletion was verified by flow cytometry analysis. Antibodies and flow cytometry ��CD4-PE, ��CD4-PE-Cy5, ��CD19-FITC, ��CD19-APC-Cy7, ��IgD-PE, ��IgM-PE-Cy7, ��CD93/AA4-APC, ��CD23-Biotin, ��CD21/35-FITC, ��IFN-��-FITC, ��TNF��-PE, were purchased from eBioscience (San Diego, CA). For intracellular staining, lymphocytes (106/wells) were stimulated with GP61 peptide (at 10?6 M per well) in the presence of recombinant IL-2 (25 U/ml, ProSpec, Rehovot, Israel) and 5 mg/ml Brefeldin A (Sigma-Aldrich, Switzerland) for 5 hours. Cells were stained for surface molecules, fixed with 4% paraformaldehyde in PBS, and then cell membranes were permeabilized with Perm-Buffer (PBS, 2% FCS, 5 mM EDTA, 0.
1% saponin, 0.2% NaN3) and stained with ��IFN��-FITC or ��TNF��-PE. Relative fluorescence intensities were measured with a FACScan? or BD? LSRII (BD, Mountain View, CA) and analyzed using FlowJo? software (Tree Star, Ashland, OR). Purification of LCMV-immune CD4+ T cells BL/6 mice were infected with 200 pfu LCMV-WE. 17 days later splenocytes were harvested and enriched for CD4+ T cells by positive selection with MACS (Miltenyi Biotec, Bergisch Gladbach, Germany) and were adoptively transferred i.v. to recipient mice. The purity of CD4+ T cells was ��90%. Isolation of hepatic lymphocytes To isolate hepatic lymphocytes, liver was removed, and cut in small pieces. Afterwards liver was digested with BSS containing 2% FCS, 0.
6% BSA, 5 mM CaCl2, 5 mM MgCl2, 1 mg/ml collagenase Type IA (Sigma-Aldrich), 20 ��g/ml DNase I (Roche) for 15 min at 37��C. In a next step, liver was filtered through a sterile 40-��m nylon cell strainer (BD Biosciences) and washed once with BSS before erythrocytes were removed by Puregene red blood cell lysis solution (Qiagen). Finally, lymphocytes were isolated by Ficoll gradient centrifugation (Ficoll-PaquePlus, Amersham Biosciences, Uppsala, Sweden). Real time RT PCR of chemokines Spleens were shock frozen and disrupted in Trizol. RNA was isolated according to manufacture guidelines (Invitrogen) and real time PCR analysis was performed using QuantiFast SYBR Green (Eurogentech) PCR kit.
The following primer combination was used: GAPDH forward 5�� CCA CCC CAG CAA GGA GAC T and GAPDH reverse 5�� GAA ATT GTG AGG GAG ATG CT; Ccl19-atg 5�� -CTG CCT CAG ATT ATC TGC CAT- 3�� and 5�� -AGG TAG CGG AAG GCT TTC AC- 3; Ccl21-ser 5�� -ATC CCG GCA ATC CTG TTC TC- 3�� and GSK-3 5�� -GGT TCT GCA CCC AGC CTT C- 3��; CXCL13 5��- GAG GCT CAG CAC AGC AAC-3�� and 5��-TTG AAA TCA CTC CAG AAC ACC TAC A- 3��. Alanine aminotransferase We measured ALT with a serum multiple biochemical analyzer (cobas? 8000 modular analyser, Roche, Switzerland). Statistical evaluation For statistical analysis Graph Pad Prism Version 5 (GraphPad Software) was used. Significances were tested using an unpaired, two-tailed student’s t-test.