Hla dr pe cy5

7 weeks before the first viral challenge, an open laparotomy was performed on each of the vaccinated and adjuvanted animals to obtain a colorectal wedge. For the naive animals, 10 rectal pinches were collected. The colonic wedge was rinsed with HBSS to remove the mucus, and then cut into 2″ pieces. The colonic tissue pieces/rectal pinches were incubated with HBSS containing 5 mM EDTA and 2 mM DTT at room temperature for 15 min twice. The supernatants, which contain intraepithelial lymphocytes (IEL) cells, were collected, pooled and filtered through 100 μM cell strainers. The collected colorectal IELs were washed and subjected to FACS staining with viability dye and antibody mixtures including anti-CD45 to exclude the dead and CD45 negative epithelial cells. The frequencies of ki67, CD69, CD38 and HLA-DR within CD4⁺ and CD8⁺ T cells were further assessed. About 8000, 6000, and 2000 CD4⁺T cells (mean) were analyzed for vaccinated, adjuvanted and naive animals (Supplementary Fig. 1). The following antibodies were purchased: CD3-PE-Cy7, CD8-APC-Cy7, Ki67-APC, HLA-DR PE-Cy5, and CCR5-PE (from BD Pharmingen); CD69-Alexa Fluor 700 (from Biolegend); CD38-FITC (from STEMCELL Technologies); and CD4-qdot 605 (from eBioscience).