Cytometric Analysis of T Cell Subsets

Single-cell suspensions were prepared from lymph nodes, spleen, and lungs, as described (47 , 55 (link)). Unspecific staining was blocked with unconjugated anti-FcγRII/III antibody (anti-CD16/CD32, clone 2.4G2; BD Biosciences). Cells were stained with the following antibodies for multi-color cytofluorometric analyses: ECD-conjugated anti-CD8α (clone 53-6.7, Beckman Coulter), PE-conjugated anti-TCR Vβ5.1/5.2 (clone MR9-4, BD Biosciences), and APC-conjugated anti-TCR Vα2 (clone B20.1, BD Biosciences). Peptide/epitope-specific CD8⁺ T cells were identified with APC-conjugated MHC-I dextramer H-2Kb/SIINFEKL (Immudex, Copenhagen, Denmark). For the analyses, a “live gate” was routinely set on leukocytes in the forward scatter (FSC) versus sideward scatter (SSC) plot. All cytofluorometric analyses were performed with flow cytometer FC500 and CXP analysis software (Beckman Coulter).