Determination of N3A 366 + CD8 + T cell precursor frequency Naïve N3A 366 -specific CD8 + T cells were identified as described [33, 53] . Briefly, processed lymph nodes and spleen samples were resuspended in 100 ml of Sorter buffer, 100 ml FcR block (24G2 and CD16 culture supernatant, 1% mouse and 1% rat serum) was added, and clumps of dead cells were discarded. Tetramers at optimal staining concentrations (D b N3A 366 -PE at 40mg/ml and D b NP 366 -PE at 10mg/ml) were added to the cell mix and incubated for 1 hour at room temperature in the dark. Cells were washed and resuspended in 400 ml buffer with 100 ml anti-PE microbeads (Miltenyi Biotec), and incubated at 4uC for 25 mins. Following two washes, cells were resuspended in 3 ml of buffer and cells that had bound the microbeads were purified on a magnetic LS column according to manufacturers instructions (Miltenyi Biotec). Cells eluted from the column were centrifuged (5156g, 6 min, 4uC), supernatant carefully aspirated to leave 90 ml buffer remaining, and 10 ml antibody cocktail was added for 30 min at 4uC. The antibody cocktail contained anti-CD8-APC Cy7 (Pharmingen, BD), anti-CD4-PE Cy7 (eBiosciences), anti-B220-FITC (Pharmingen, BD), anti-CD11b-FITC (eBiosciences), anti-CD11c-FITC (eBiosciences), anti-F4/80-FITC (eBiosciences), anti-CD62L-APC (Pharmingen, BD), and anti-CD3-PerCP Cy5.5 (Pharmingen, BD) mAbs. Cells were washed in 2 ml buffer, centrifuged (5156g, 6 min, 4uC), and supernatant aspirated leaving 100 ml. Resuspended cells were passed through 45 mm sieve, and data acquired by flow cytometry on the LSR II (Becton Dickinson) and analysed with FlowJo (Treestar Inc.) software. In selected experiments, naïve NPN3A + Vb8.3 + CD8 + T cells were single-cell sorted for TCRb analysis. Experimental details of the single cell RT-PCR designed to amplify naive NPN3A + Vb8.3 + CD8 + T cells using 2 different sorting strategies are listed in Table S3 .