Clone fn50

Flow data were collected on an LSRII from BD Biosciences. Zombie UV Fixable Viability Kit (BioLegend) was used as live/dead stain for reactivation experiments. We had a range of 250 to 6000 events per data point and a minimum cut-off of 250 events in Fig 5B. The mean number of all events was 1364 and the median number was 678. All cells were washed and fixed in a final concentration of 2% paraformaldehyde prior to analysis. Cell sorting was performed on a MoFlo Astrios from Beckman Coulter. All flow experiments performed at Boston University School of Medicine Flow Cytometry Core Facility.
Cell activation and phenotypes were determined by CD69 expression (Brilliant Violet 421 anti-human CD69 antibody; Clone FN50, BioLegend) and CCR7 and CD45RA expression (Pe/Cy7 anti-human CCR7 antibody; Clone G043H7, BioLegend and PerCP/Cy5.5 anti-human CD45RA antibody; Clone HI100, BioLegend). We had a minimum of 1000 events to be included as a data point in Fig 2.

Peripheral blood lymphocytes were isolated from whole blood and stained as described previously^{22 (link)}. Single-cell suspensions of splenocytes were made by manual digestion as described elsewhere^{68 (link)} followed by lymphocyte isolation with Ficoll Paque Plus gradient (GE Healthcare). Lamina propria lymphocyte single-cell suspensions were isolated from fresh female reproductive tract tissue with collagenase IV^{68 (link)}. Single-cell suspensions were prepared and stained with Gag-CM9 tetramer and a cocktail of antibodies mentioned in T cell assays and an additional antibody to stain CD69 (clone FN50, BioLegend). ICS assay was also performed as described in T cell assays with the following modifications. The stimulants were no peptide or Gag-CM9 peptide at a concentration of 1 µg ml⁻¹ in complete RPMI medium with 10 µg ml⁻¹ brefeldin A for 16–18 h. Following stimulation, the cells were stained and analyzed as above. The ICS antibody cocktail included MIP-1β clone D21-1351, BD Biosciences). In situ tetramer staining and immunohistochemistry was performed on tissue sections as described previously^{22 (link)}.