After infection, or vaccination, naïve CD8 T cells are activated by antigen presenting cells (APCs) in secondary lymphoid organs. Activated 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 and create the pool of the 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. Tracking the kinetics of CD8 T cell response in different clinical conditions is important for the development of new therapeutic approaches to many life-threatening diseases (such as HIV, tuberculosis, cancers). To date most research has been focused on the phenotypical discrimination between effector and memory CD8 T cells subsets, based on the expression of membrane markers. However, membrane phenotype is transient and can be modulated by several factors in the tissue microenvironment, and indeed it does not depict the presence of antigen-specific CD8 T cells in a specific phase of the response. In this project, we aim to investigate the antigen-specific CD8 T cell response after vaccination in mice. Our attention will be focused on tracking the transition from acute to memory phase by cell cycle analysis of antigen-specific CD8 T cells. Given that our recent results in vaccinated mice have shown that clonal expansion occurs not only in draining lymph-nodes but also in spleen, blood and bone marrow, we will evaluate if acute to memory phase transition is synchronized in different lymphoid organs. 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.
CD8 T cell response is crucial in many diseases such as infections, cancer and autoimmune disorders. Current methods to track CD8 T cell response are mostly based on the expression of surface markers that are not able to follow the antigen-specific CD8 T cell transition from acute to memory phases. Indeed, many features of the acute to memory phase transition are still unclear, for example whether it proceeds with a synchronized kinetics in different organs is still unknown. Our recent data in vaccinated mice demonstrated that clonal expansion during early phase of the response is not a local process, but occurs in the whole body. Indeed, we found antigen-specific CD8 T cells in S-G2-M phase of cell cycle in lymph node, blood, spleen and bone marrow after intramuscular vaccination in rear limbs (Simonetti S, Natalini A et al., Scand J Immunol, 2019; Natalini A et al., manuscript in preparation). Thus, although it is likely that expansion initiated in lymph-node draining the site of vaccine injection, soon after this event cycling antigen-specific CD8 T cells entered the circulation reaching other lymphoid sites (spleen and bone marrow).
Here we propose that cell cycle state analysis of antigen specific CD8 T cells after their clonal expansion can be used to follow the transition from acute to memory phase in vaccinated mice. Our results will be instrumental to: i) understand the kinetics of the transition from acute to memory phase; ii) lead to a better knowledge of memory generation; iii) design strategies to induce long-lasting memory. Thus, our results could have important implications in human diseases.
For example, in the context of vaccine design, an unsolved issue is how to shorten the time between the first (prime) and the second immunization (boost). This is important especially in case of sudden epidemics (such as Ebola) or in highly progressive diseases (Sallusto F et al., Immunity, 2010). Our analysis could be used to identify the most appropriate time for boosting. By measuring the proportion of quiescent and slowly proliferating cells in the memory phase, and by achieving a better understanding of the connection between cell cycle and response to boost, we could be also able to predict the efficiency of the secondary response.
Moreover, in both melanoma and non-small-cell-lung cancer (NSCLC) patients it has been shown that unleashed anti-tumor CD8 T cells increase Ki67 expression after treatment with checkpoint inhibitors (CI) (Kamphorst AO et al., Proc Natl Acad Sci U S A, 2017; Wieland A et al., Cancer Immunol Immunother, 2018). These findings suggest that upon CI treatment anti-tumor CD8 T cells undergo a proliferative expansion simultaneously to their re-activation. However, Ki67 expression does not discriminate between cells in different cell cycle states and is not a marker of dividing cells. In contrast, cell cycle analysis of anti-tumor CD8 T cells according to our method, can be essential to test the efficacy of the therapy. Furthermore, it is known that after a first phase of anti-tumor response, anti-tumor CD8 T cells become exhausted and are inhibited by several mechanisms, for example by inhibitory interactions in the immune-suppressive tumor microenvironment (Zuazo M et al., Ann Transl Med, 2017). Our analysis could be helpful to acquire a better knowledge on protective memory, and distinguish it from disfunctional anti-tumor CD8 T cell response.
Our findings will contribute to advancement in T cell memory field, with relevant implications for immuno-mediated treatments in human diseases.