Different cell cycle stages characterize early and late phases of antigen-specific CD8 T cell response after vaccination

After infection, or vaccination, naïve CD8 T cells are activated by antigen presenting cells (APCs) in secondary lymphoid organs. Activated antigen-specific CD8 T cells undergo a strong proliferation (so-called clonal expansion) and differentiate, generating a progeny composed by short-lived effectors and long-lived memory cells. Once antigen is eliminated, most cells die in the contraction phase and only few cells persist as memory CD8 T cells. These cells are able to respond to a second antigenic challenge in a more effective and faster way than in the primary response. Several phases of T cell response are regulated by responding T cell entry and exit from cell cycle. Indeed, naïve T cells are quiescent cells in G0 and the acute phase of response is characterized by their fast entry in cell cycle (from G0 to G1) and progression into subsequent phases till cell division (S-G2/M). Several cell cycles characterize the clonal expansion of activated T cells. During the early phases of immune response, changes in cell cycle regulation of activated T cells could deeply affect the response, for example reduced clonal expansion could lead to decreased number of effector and memory cells. Moreover, it has been proposed that memory T cells are maintained over time by a fine balance between different cell cycle states, including a fine regulation of quiescence. According to this hypothesis quiescence represents an actively regulated process like other cell cycle phases and not a passive mechanism of cell persistence over time. Any perturbation of this balance could affect the ability to mount an efficient secondary response with consequent loss of protection. In this scenario, our hypothesis is that a fine balance between different cell cycle phases regulates T cell response both in the acute and in the memory phase of response. Thus, in this project we aimed to investigate the kinetic of cell cycle phases of antigen-specific CD8 T cells responding to heterologous prime/boost vaccination in a mouse model. By using a combination of DNA and Ki67 staining together with a novel strategy for analysis of flow cytometry data, we were able to discriminate antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of cell cycle. At early times after vaccination we found a previously missed population of cycling cells characterized by high Forward and Side Scatter (FSC-SSC) parameters. Cells with these characteristics are usually excluded from the analysis of normal lymphocytes ex vivo. By including them, we discovered an “extra” population of cycling antigen-specific CD8 T cell in spleen, lymph nodes and also in the blood which is not expected to be a site for antigen-responding CD8 T cells proliferation. We found that antigen-specific CD8 T cells accumulated in lymph nodes and the bone marrow during memory phase. These cells switched to a quiescent phenotype and a few of them acquired a central memory phenotype at late times after priming. Interestingly, boosting when quiescent state was established resulted in a much higher frequency of antigen-specific CD8 T cells that persisted in different lymphoid organs, and accumulated in high numbers in the bone marrow. Our results have implications for prior and future immunological studies in animal models and in humans. Indeed, our results will be instrumental to track CD8 T cell response in humans after infections or vaccination, as well as in cancers, and will improve the design of new therapeutic approaches to cancer and immune-mediated diseases.