Lymphocyte Isolation and Cytokine Assays

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)}.