Tetramer staining of epitope specific CD8+ T cells

Tetramers to detect epitope-specific CD8⁺ lymphocytes were generated via coupling of monomers (made at the University of Melbourne) to streptavidin-labeled APC or PE fluorochromes at a final concentration of 1 μg/μl. Lymphocytes were incubated in 50 μl of MACS buffer containing both H-2D^bNA_181-190-PE and H-2D^bNA_180-191-APC tetramers (final dilution 1:100, or as indicated in the individual experiment) and incubated at RT in the dark for 1 hour. Following tetramer staining, cells were stained for CD8α and either analysed or single, live, CD8⁺ tetramer⁺ cells individually sorted into a 96-well plate for multipex RT-PCR analysis. Gating strategy for spleen shown in Supplemental Fig. 1A, along with tetramer staining on control spleen and BAL samples stained with all antibodies but not H-2D^bNA_181-190-PE and H-2D^bNA_180-191-APC tetramers (Supplemental Fig. 1B) and tetramer staining on CD4+ T cell subset from spleen and BAL (Supplemental Fig. 1C). For analysis of tetramer dissociation kinetics, after tetramer staining, cells were washed and incubated for various times at 37°C with anti-H-2D^b/K^b Ab (28-8-6, BD Pharmingen) (25 μg/ml) to prevent tetramer rebinding. Cells were then washed and stained with anti-CD8α-BUV395 Ab for flow cytometric analysis.