To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis

Abstract

This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker per se, and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G2/M phases of the cell-cycle in the peripheral blood (collectively termed “T Double S” for T cells in S-phase in Sanguine: in short “TDS” cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of TDS cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67/DNA dual staining, or TDS assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated “-omics” capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the TDS assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.

Low-dose IL-2 reduces IL-21+ T cell frequency and induces anti-inflammatory gene expression in type 1 diabetes

“…Both MKI67 (mRNA) and Ki-67 protein are synthesised and accumulate during the S and G2/M phases of the cell cycle and show very good correlation in synchronised cells 27 . However, in contrast to MKI67 , Ki-67 protein is more long-lived, and can still be detected in G1 after cell division 28 . The levels of Ki-67 protein in G1 depend on the rate of protein accumulation during the S and G2/M phases, leading to the much higher rate of detection of Ki-67 + cells compared to the frequency of cells in the S-G/M phase (e.g.…”

Low-dose IL-2 reduces IL-21+T cells and induces a long-lived anti-inflammatory gene expression signature inversely modulated in COVID-19 patients

“…Both MKI67 (mRNA) and Ki-67 protein are synthesised and accumulate during the S and G2/M phases of the cell cycle and show very good correlation in synchronised cells 30 . However, in contrast to MKI67 , Ki-67 protein is more long-lived, and can still be detected in G1 after cell division 31 . The levels of Ki-67 protein in G1 depend on the rate of protein accumulation during the S and G2/M phases, leading to the much higher rate of detection of Ki-67 + cells compared to the frequency of cells in the S-G/M phase (eg.…”

Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature

“…having a T CM phenotype (Sallusto et al, 2004; Ivetic et al, 2019), or lacking intracellular Ki-67, i.e. being in the quiescent phase G 0 (Di Rosa et al, 2021). Ki-67 and CD62L analysis were also combined, as explained in the next paragraph.…”