Moreover, the intranuclear protein, Ki67, is only able to distinguish cells in G 0 (Ki67-negative cells) from those in any other cell cycle phase (Ki67-positive cells).
However, cell-labeling dyes, such as CFSE and BrdU, do not allow the determination as to whether cells found in a particular organ proliferated locally or rather migrated to this site after division 8, 9. These include monoclonal antibodies (mAbs) specific for the nuclear marker, Ki67, and cell dyes that either identify cells that have undergone the S-phase of the cell cycle (e.g., Bromodeoxyuridine (BrdU)) or discriminate among daughter cells and their ancestors (e.g., carboxyfluorescein succinimidyl ester (CFSE)). This lack of knowledge is due to the properties of available tools for cell cycle analysis. T cell-APC interaction is finely regulated by concentration and persistence of antigen, co-stimulatory signals, and soluble factors (cytokines and chemokines) that influence the quantity and quality of the T cell clonal progeny 4, 5, 6, 7.ĭespite intensive studies of T cell clonal expansion, it is still not known whether antigen-primed T cells complete their entire cell cycle at the site of antigen recognition, or whether they migrate to other organs during cell cycle progression. During the first steps of the primary response, naïve T cell interaction with antigen-presenting cells (APCs) within specialized niches in lymphoid organs is critical to induce the huge T cell proliferation that characterizes the clonal expansion phase 1, 2, 3. Differentiated T cells display effector functions that are essential for antigen clearance and for the maintenance of antigen-specific memory, which is key for long-lasting protection. Naïve T cells undergo clonal expansion and differentiation upon antigen-priming. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G 0/G 1 and G 2/M DNA peaks, while preserving membrane and intracellular staining. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G 0, in G 1 and in S-G 2/M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.Ĭritical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G 1 that can remain in this phase, proceed into cycling, or move into G 0. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G 0. However, these dyes do not allow identification of the cell cycle phase at the time of analysis.
Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations.
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