Fig. 4
Flow cytometry validation of differential expression. Flow cytometry was performed using standard cell surface staining protocol using fresh EDTA blood, following red cell lysis. a CD3+CD4+ T-cells (top left panels orange gate) were first gated. Naïve cells were then gated as CD45RA+/CD45RO- (red square) and memory cells as CD45RA−/CD45RO+ (green circle) in a representative HC and RA patient. b The expression of CD4, IL6R, IL2R and CXCR4, were measured using Mean fluorescence intensity (MFI). Results are shown as box plot for 11 HC and 35 RA patients. CD62L was either positive or negative and percentage of naïve CL62L− cells was recorded and displayed. Significant differences (Mann–Whitney U-test, p < 0.05) are highlighted by stars. c CD45RB and CD62L were further used to refine the phenotype of naïve CD4+ T-cells. CD45RB expression was consistently high in naïve cells but declined in experienced cells and was low in memory cells (green circles), with no major difference between HC and RA for this subset. CD62L expression is positive on naïve cells (red circle, consistently in HC) but was either positive (red circle) or negative (blue square) in RA defining an subpopulation of naïve CD62L− cells also expressing reduced levels of CD45RB (blue circle). d Differential levels of expression for CD4, IL6R and IL2R are shown in a RA patients with a raised CD62L− naïve cells subpopulation (best representative patient displayed) for naïve (red) memory (green) and IRC (blue) cells. Levels of CD4 were not significantly different (n = 3) but a lower MFI was observed for CD4 expression on CD62L-naïve cells (2250) compared to CD62L+ naïve cells (2850) and memory cells (2700). The expression of the IL-6R was lower on CD62L− (MFI 7300) compared to CD62L+ naive cells (17,600) as well as on memory cells (11,400). The IL2R expression was negative on CD62L+ naïve cells but presented 2 populations (negative < 1000 fluorescence units and + fractions > 1000) for CD62L− naïve cells. Memory cells were mainly positive (72% of cells)