Multiparameter flow cytometry for discovery of disease mechanisms in rheumatic diseases

Abstract

Flow cytometry has emerged as an essential tool for investigators in the study of the complexity of the immune system and the examination of its role in human health and disease. This technology has developed to the point where one can readily generate a large descriptive data set that details the levels of important immune cell subsets and defines an individual immune cell signature or “Immune-cellome”. This immune cell signature would clearly display individual variation but also would change in a manner reflective of disease state. Analysis of the “immune-cellome” may provide novel insight into disease pathophysiology, provide new biomarkers of disease activity and perhaps identify therapeutic targets. In this brief review we will cover current advances in complex flow cytometry and suggest ways this may be applied to the study of rheumatic diseases.

Figure 1. A Basic Flow Cytometer

Illustrated are the basic elements of a simple flow cytometer. This includes a laser light source used to interrogate the cells delivered via a stream of fluid. Reflected laser light or fluorescence emissions are collected using a series of mirrors (blue rectangles) and lenses (blue ovals) to detectors that convert light signals to electronic signatures that can be processed and stored.

Figure 2. A Flow Cytometric Analysis of Human PBMCs

Human whole blood was first treated to remove RBCs, reacted with fluorescent-tagged markers for CD3 (FITC) and CXCR5 (PE) and analyzed by flow cytometry. Displayed in panel A is a two (dual) parameter dot plot displaying the FSC and SSC signals on a linear scale. Each dot represents single cell (or event) and by displaying both signals relative to each other one debris, lymphocytes, monocytes and granulocytes can be easily resolved. Panel B displays the distribution of the markers CD3 and CXCR5 on the “gated” lymphocytes as separate single color histograms that clearly define positive and negative lymphocyte populations. Panel C displays the same data viewed as a dual (two) parameter polychromatic plots were color is used to define high (red) and low (blue) density of events. This plot illustrates the relationship between CD3 and CXCR5 expression on human lymphocytes.

Figure 3. Expression of CD45RA and CD45RO on CD4⁺ or CD8⁺ T Cells

Human PBMCs were purified using Ficoll Paque and stained with fluorescent-tagged markers for CD3 (Pac-Orange), CD4 (Pac-Blue), CD8 (APC-H7), CD45RA (PE-Texas Red) and CD45RO (FITC). Displayed are the levels of CD45RA vs. CD45RO on gated CD3⁺/CD4⁺ (left) or CD3⁺/CD8⁺ T cells. While CD45RA⁺/RO⁻ (group I) and CD45RA⁻/RO⁺ (group III) subsets can be identified, there is also a significant population of cell with intermediate phenotype (group II).