Ricerc@Sapienza

Although clonal expansion is a hallmark of adaptive immunity, the location(s) where antigen-responding T cells enter cell cycle and complete it have been poorly explored. This lack of knowledge stems partially from the limited experimental approaches available. By using Ki67 plus DNA staining and a novel strategy for flow cytometry analysis, we distinguished antigen-specific CD8 T cells in G0 , in G1 and in S-G2 /M phases of cell cycle after intramuscular vaccination of BALB/c mice with antigen-expressing viral vectors. Antigen-specific cells in S-G2 /M were present at early times after vaccination in lymph nodes (LNs), spleen and, surprisingly, also in the blood, which is an unexpected site for cycling of normal non-leukaemic cells. Most proliferating cells had high scatter profile and were undetected by current criteria of analysis, which under-estimated up to 6 times antigen-specific cell frequency in LNs. Our discovery of cycling antigen-specific CD8 T cells in the blood opens promising translational perspectives.