Anti cd4 mab

Spleens were harvested from mice and purified CD4 T cells (>95% CD4 as determined by flow cytometry analysis) were obtained by positive selection using magnetic beads coated with anti-CD4 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
The CD4 cells were resuspended in alpha modification of Eagle’s medium supplemented with 20 mM HEPES buffer, 100 U/ml benzyl penicillin, 100 μg/ml streptomycin sulfate, 4 mM l-glutamine and 50 μM 2-mercaptoethanol. Antigen-presenting cells (APCs) were produced by irradiation of the non-CD4+ cells with 1500 rads (caesium source) to prevent cell division. APCs were mixed with CD4+ cells to reach a final concentration of 0.6 × 10⁶ cells/ml for APCs and 1.25 × 10⁶ cells/ml for CD4+ cells. 1% mouse serum was added as a protein source. Band 3 peptides were added at a final concentration of 20 or 200 μg/ml and the cells were incubated in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C. Dominant band 3 peptide 861–874 and its soluble analog as well as concanavalin A (ConA) were used as positive controls.

Inguinal lymph nodes were collected as draining LNs, and used for the experiments. CD4⁺ cells in draining LNs and spleen were isolated by positive selection using a magnetic-activated cell sorting (MACS) system with anti-CD4 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany). CXC chemokine receptor 5⁺ (CXCR5) follicular helper T (Tfh) cells were isolated with Moflo cell sorter (DakoCytomation, Glostrup, Denmark) from MACS-isolated CD4⁺ T cells. For the isolation of Foxp3⁺ Treg cells and Foxp3⁻ non-Treg cells, CD4⁺GFP⁺ and CD4⁺GFP⁻ cells were further purified using a Moflo cell sorter from MACS-isolated CD4⁺ T cells in C57BL/6-Foxp3^GFP and RORγt Tg-Foxp3^GFP mice. The isolated cells were used for the experiment of adoptive cell transfer and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) (see below).

To compare the immunogenicities of wild-type Tokyo172 with Tokyo172Δurease, each harboring plasmid bearing the SIV gag gene, five C57BL/6J mice were primed by inoculation with 0.5 mg of either strain. Five weeks later, the mice were boosted with 1 × 10⁷ PFU m8Δ-SIV-Gag by scarification, and then 4 weeks later, splenocytes were prepared. CD4⁺ T and CD8⁺ T cells were isolated with anti-CD4 MAb- or anti-CD8 MAb-coated microbeads (Miltenyi Biotec). For the ELISPOT assay, CD4⁺ and CD8⁺ T cells were suspended at a concentration of 1 × 10⁶ cells/well in culture medium and stimulated with 2 µg/ml peptide 25 (CD4⁺ T-cell epitope of Ag85B, a major protein of BCG, FQDAYNAAGGHNAVF) or GM10 peptide (CD8⁺ T-cell epitope of mycobacterial Mtb32a protein, GAPINSATAM), DD13 peptide (CD4⁺ T-cell epitope of SIV Gag, DRFYKSLRAEQTD), or AL11 peptide (CD8⁺ T-cell epitope of SIV Gag, AAVKNWMTQTL) on ELISPOT plates (Mabtech, Nacka Strand, Sweden). After incubation at 37°C for 24 h, the plates were washed thoroughly with PBS and incubated for 2 h at room temperature with a solution of 1 µg/ml biotinylated anti-mouse IFN-γ MAb R4-6A2 (Mabtech) in PBS containing 0.5% FBS. After the plates had been washed with PBS, 100 µl streptavidin-alkaline phosphatase (Mabtech) at a 1/500 dilution was added to each well. Spots were visualized and counted using a KS ELISPOT reader (Zeiss, Oberkochen, Germany).