How Clinical Flow Cytometry Rebooted Sepsis Immunology

Abstract

On May 2017, the World Health Organization (WHO) recognized sepsis as a global health priority by adopting a resolution to improve the prevention, diagnosis, and management of this deadly disease. While it has long been known that sepsis deeply perturbs immune homeostasis by inducing a tremendous systemic inflammatory response, pivotal observations based on clinical flow cytometry indicate that sepsis indeed initiates a more complex immune response that varies over time, with the concomitant occurrence of both pro- and anti-inflammatory mechanisms. As a resultant, some septic patients enter a stage of protracted immunosuppression. This paved the way for immunostimulation approaches in sepsis. Clinical flow cytometry permitted this evolution by drawing a new picture of pathophysiology and reshaping immune trajectories in patients. Additional information from cytometry by time of flight mass cytometry and other high-dimensional flow cytometry platform should rapidly enrich our understanding of this complex disease. This review reports on landmarks of clinical flow cytometry in sepsis and how this single-cell analysis technique permitted to breach the wall of decades of unfruitful anti-inflammatory-based clinical trials in sepsis. © 2019 International Society for Advancement of Cytometry.

Keywords: HLA-DR; IL-7; PD-1; flow cytometry; monocyte; sepsis; time of flight mass spectrometry.

Figure 1.. “HLA-DR and sepsis” citations over years.

Yearly number of citations of articles matching terms HLA-DR and sepsis from 1997 to present time (publication year of landmark paper of Docke et al. is marked by a red arrow). Data from Web of Science.

Figure 2.. Immune trajectories in sepsis.

Theoretical evolutions over time of immune status of different septic patients are presented. Net inflammatory/suppressive immune responses are very heterogeneous in kinetics, duration and depth. Patients with dashed line return spontaneously to immune homeostasis overtime. Patients with continuous lines present with dysregulated immune responses. These patients should be identified to benefit from host directed immunointerventions.

Figure 3.. Illustrative example of unsupervised analysis of CyTOF results in healthy volunteers and septic patients.

These results have been obtained from reference (70) (A) SPADE clustering analysis was performed in order to group peripheral blood mononuclear cell sub-populations into “nodes” according to phenotype similarities. These nodes and their relationships are represented in a minimum spanning tree format (i.e. SPADE tree). Size of the nodes illustrates the number of cells within the clusters. In this analysis, CD4 expressing cells are highlighted as red nodes while CD19 expressing cells are represented as green nodes. (B) viSNE visualization tool was then performed on selected CD4+ cells from SPADE nodes. Based on the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm, viSNE determines the two dimensional representation of single-cell data sets that best matches their respective local and global geometry. Cells get unique x- and y-coordinates (tSNE1 and tSNE2) according to their expression of parameters from CyTOF panel. Their relative positions on the two-dimensional plot indicate similarities in terms of expression pattern. In the current analysis, we used color as a third dimension to visualize specific marker expression levels (PD-1 and Tim-3) in one representative example of a septic patient. Red and blue color intensities respectively represent high and low expression levels of the indicated marker. In this illustrative septic shock patient, PD-1 and Tim-3 are clearly not co-expressed by same CD4 T cell subpopulations (red squares). (C) Alternatively, viSNE algorithm was performed on selected CD19+ B cells to compare sepsis vs healthy volunteers. Expression level of CD38, a marker of B cell maturation was visualized on the viSNE plots in one representative septic shock patient and one healthy donor. A depletion of cells from the center of the map (green arrows), but an enrichment within the upper right cell cluster (purple arrows) are noticed in the patient. This latter cluster was constituted of CD38hi cells, suggesting a shift towards differentiated B cells in septic shock patients.