Private specificities of CD8 T cell responses control patterns of heterologous immunity

Kim, Sung-Kwon; Cornberg, Markus; Wang, Xiaoting Z.; Chen, Hong D.; Selin, Liisa K.; Welsh, Raymond M.

Flow cytometry and intracellular IFN-␥ staining. Single cell lymphocyte suspensions were prepared from peripheral blood and spleens. The erythrocytes were lysed using a 0.84% NH 4 Cl solution. LCMV peptidespecific, IFN-␥-secreting CD8 ϩ T cells were detected using the Cytofix/ Cytoperm Kit Plus (with GolgiPlug; BD Biosciences) as described previously (37) . In brief, cells were incubated with 5 M of synthetic peptide, 10 U/ml of human recombinant IL-2 (BD Biosciences), and 0.2 l GolgiPlug for 5 h at 37ЊC. After preincubation with 1 l of Fc block (2.4 G2) in 96well plates containing 100 l of FACS buffer (HBBS, 2% FCS, 0.1% NaN 3 ), the cells were stained for 30 min at 4ЊC with combinations of fluorescently labeled mAbs specific for IL-7R (CD127, A7R34 biotin), CD44 (IM7, FITC), CD8␣ (53-6.7 per CP) and, after cells were permeabilized with Cytofix/Cytoperm, stained with mAb to IFN-␥ (XMG1.2, allophycocyanin), all purchased from BD Biosciences except for Ab specific for IL-7R␣, which was purchased from eBioscience. For the staining of donor cells in adoptive transfer experiments, mAb specific for Thy1.1 (OX-7, PE) was additionally included. Freshly stained samples were analyzed using a Becton Dickinson FACSCalibur and CELLQuest software.