Il 2 488

CD44^hi 2W1S:I‐A^b+ CD4⁺ T cells were enumerated from spleen at 7 dpi and a pool of spleen and LN for all memory responses. Staining with PE conjugated 2W1S:I‐A^b was carried out as described previously 14. For in vivo restimulation experiments 10 μg brefeldin A was added. Cell surface staining was done at 4°C for 30 min, except for CXCR5 that was stained for 1 h at room temperature. Enrichment for 2W1S‐specific memory T cells was performed, as described previously, using anti PE MicroBeads (Miltenyi Biotech) and MACS enrichment 51. Samples were run using a Fortessa X20 (BD Biosciences) and analyzed using FlowJo software (Tree Star). For intracellular cytoplasmic staining, cells were fixed and permeabilized with Cytofix/Cytoperm Plus (BD). Intracellular cytokines were stained with IL‐2 488 and IFN‐γ PECy7 (BD Biosciences). Ten microliters of Spherotech Accucount blank particles was used to calculate cell frequency.

For tetramer staining, cells from secondary lymphoid tissue were pooled and stained for 1 hour at room temperature with 10 nm PE-conjugated 2W1S:I-A^b. For in vivo peptide experiments, 10 μg/mL brefeldin A was added at this stage. All cell surface staining was done at 4°C for 30 min, with the exception of CXCR5 that was stained for 1 hour at room temperature. Enrichment for 2W1S:I-A^b-specific T cells was performed at d2, d3, d4 and d28, as described previously, using anti-PE MicroBeads (Miltenyi Biotech) and MACS enrichment 29 (link). Enriched and run-through fractions were stained with the same cocktail of surface Abs to enable calculation of cell frequencies. Samples were run using an LSR11 (BD Biosciences) and analysed using FlowJo software (Tree Star). For intracellular cytoplasmic staining, cells were fixed and permeabilised with Cytofix/Cytoperm Plus (BD Biosciences) according to manufacturer's instructions. Intracellular cytokines were stained with IL-2 488 and IFN- γ PECy7 (BD Bioscience).