Tracking immunodynamics by identification of S-G2/M-phase T cells in human peripheral blood

Abstract

The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G 2 /M phases of the cell-cycle (collectively termed “T Double S” for T cells in S -phase in S anguine : in short “T DS ” cells). Without protocol refinement, T DS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of T DS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV + carriers; and recently-diagnosed T1D patients. T DS were reproducibly more abundant among CD8 + T cells and a defined subset of T-regulatory CD4 + T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive T DS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking T DS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.

A consensus Covid-19 immune signature combines immuno-protection with discrete sepsis-like traits associated with poor prognosis

“…Consistent with their activation, there were ∼10-fold increases in the percentage of blood CD4 + and CD8 + T cells in either G or S-G /M phases of the cycle (so-called T DS cells 32 by contrast to HC in which ≥98% of cells comprise G 0 cells in transit. These changes, illustrated for T EM cells ( Fig 6c ), were strongly severity-related, and applied to all states of CD4 + and CD8 + cells, excepting T N cells of which ≥99% remained in G 0 in almost all patients (Supp Fig 5d).…”

“…The study is termed Covid-IP (Covid-ImmunoPhenotyping), and the hospitalised Covid-IP patient cohort is termed CP. Although Covid-19 is mostly defined by respiratory failure contributed to by local inflammation, and frequently combined with coagulopathies and other organ failures, peripheral blood sampling has many merits, specifically: its practicality in multiple settings and locations thereby facilitating meta-analyses embracing scenarios such as vaccination and sepsis, and comparison with routine clinical blood measurements 29,31 ; and its established capacity to reflect, albeit incompletely, immune cells trafficking to and from tissues, particularly when appropriate experimental approaches are employed to maximise that capacity 32,33 .…”

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

“…The prospect that antigen-responding T cells proceeding into cell cycle share with blast cells traits including increasing size and modifications of internal organelles has been validated by image flow cytometry analysis of a TCR transgenic mouse CD8 T cell population following stimulation with its cognate antigen in vitro (16). In these studies, the combination of flow cytometry and microscopy permitted visualization and quantitative multiparameter characterization of T cells in different phases of the cell cycle, as identified by Ki-67/DNA dual staining: thus antigen-induced T cell cycle progression corresponded to quantitative increases in SSC, nuclear size (DNA area), cell size (brightfield area), and uptake of a mitochondrial marker (16).…”

“…Notably, T cells with such features of cycling blast cells were identified in the peripheral blood of some healthy donors (HD), without any in vitro stimulation (16). In this context, Ki-67 staining resolved an unexpected technical issue, i.e.…”

“…In this article, we will discuss how these considerations are brought into focus by recent findings on proliferating T cells in the peripheral blood of healthy subjects and those with diseases (15,16), including COVID-19 (17). We will briefly describe how a highly sensitive and effective flow cytometric method based on Ki-67/DNA dual staining, the T DS assay, provided a practical and reliable approach to distinguish between T cells in G 1 and those in S-G 2 /M phases of cell cycle (15)(16)(17). Finally, we will advocate incorporating the T DS assay into routine immuno-monitoring.…”