e. i.n. or i.vag., mice were first anesthetized with ketamine and xylazine chloride given i.p. Five days before i.vag. immunization, mice were i.p. injected with 3 mg of medroxiprogesterone-acetate (Sigma-Aldrich). For prime-boost experiments, mice were boosted click here i.n., i.vag. or i.m. 6 wk after the first immunization. Lymphocytes were isolated as described previously 36. Briefly, blood was collected in heparin and red blood cells were lysed using ACK Lysing Buffer (Invitrogen). Spleens, ILN and NALT were dissociated against metal screens and washed with Leibovitch’s L-15-modified medium (Mediatech). For isolation of lymphocytes from the GT, the vagina, cervix, uterus, uterine horns and ovaries were removed and

cut into fragments. Tissue segments were submitted to constant shaking at 130 rpm for 1 h in RPMI 1640 (Mediatech) containing 5% FBS and 1% penicillin–streptomycin (Sigma-Aldrich). Fragments were enzymatically digested with 1.4 mg/mL of collagenase type

I (Gibco) for 15 min. Cells from the two cycles were pooled and lymphocytes purified by a discontinuous Percoll gradient (Sigma-Aldrich) consisting of 40% fraction containing cells overlaid over a 70% fraction. BALB/c mice were primed i.m. with AdC6gag and boosted 6 wk later with AdC68gag given i.m. Splenocytes were isolated 14 days later, and 5×107 splenocytes were injected i.v. into naïve Thy1.1 recipient mice. Tissues were analyzed for the presence of tet+CD8+ donor cells 7 days after the transfer. Staining was performed using a Gag peptide- (AMQMLKETI) and H-2Kd-specific tetramer (NIAID Tetramer Facility). For phenotyping, BGJ398 mouse cells were incubated with the tetramer, and Ab to CD8α, α4β7, CD27, CD103 (BD Pharmingen), CD44, CD62L, PD-1 (Biolegend), CD69, CD127 and NKG2D Glutamate dehydrogenase (eBioscience). Cells were permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (BD Bioscience) and stained for granzyme B, Ki-67 (BD Pharmingen), CTLA-4 (RD Systems) and perforin (eBioscience). For transfer experiments, cells were also stained for CD90.1 (Thy 1.1) (BD Pharmingen). Prior to analysis, cells were fixed with BD Stabilizing Fixative (BD Bioscience). Flow cytometric analyses of cells were performed with a

BD LSR II (Becton-Dickinson) flow cytometer. Data were analyzed using FlowJo V8.8 software (Tree Star). BD CompBeads Compensation Particles (Becton-Dickinson) were used to set distinct negative- and positive-stained populations for the fluorochrome-labeled Ab used in the experiments. For the assessment of background and nonspecific activation, we immunized animals with AdC vectors expressing an unrelated transgene; phenotypes for those cells mirrored those from naïve groups, as did frequencies of tetramer+CD8+ T cells (data not shown). Samples were gated on live lymphoid cells, and then on CD8+ cells versus side scatter, followed by gating on CD8+ cells versus forward scatter. The remaining cells were then plotted as CD44 cells versus tetramer for further analysis.