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. 2012 Apr;97(4):599–607. doi: 10.3324/haematol.2011.047316

Figure 1.

Figure 1.

Increased absolute numbers of Th17 cells in CLL and CLL subgroups compared to normal PBMC. (A) Strategy to measure Th17 cells. After gating on CD3+ cells, CD3+CD4+ cells containing IL-17A were analyzed by flow cytometry. These plots illustrate gating of the terminally selected Th17 (CD3+ CD4+ IL-17+) population (rightmost plot, red highlighted cells) and subsequent back-gating, and show that the Th17 population (red highlighted cells in all plots) selected for analysis comprises singlet lymphocytes (two left plots – FSA-C versus SSC-A and FSC-A versus FSC-H plots) that are CD3+ (middle plot) and CD4+ (2nd plot from the right). (B) Absolute numbers of Th17 cells (CD3+CD4+IL 17A+ cells) among PBMC from CLL patients. PBMC from 66 patients and 15 healthy subjects were stimulated with PMA and ionomycin for 5 h in the presence of monensin and then incubated with fluo-rochrome-labeled antibodies as described in the Design and Methods section. Marked cells were measured by flow cytometry. Absolute numbers (per mm3) of Th17 cells are higher in CLL patients (P=0.008). The absolute number of Th17 cells in blood (per mm3) for each patient was determined according to the formula: [ALC (per mm3)] × [% CD3 positive MNC] × [% CD4 positive CD3 cells] × [%IL-17 positive CD4 cells]. ALC means absolute lymphocyte count and MNC means mononuclear cells. (C) Absolute numbers of circulating Th17 cells in M-CLL and U-CLL patients compared to healthy individuals. Absolute numbers of Th17 cells were higher in the blood of M-CLL (n=28) and U-CLL (n=37) patients than in healthy subjects (n=15) (P=0.008 and P=0.015, respectively). (D) Absolute numbers of circulating Th17 in CLL patients stratified by CD38 levels compared to healthy subjects. Absolute numbers of Th17 cells were significantly higher in CD38Low patients (n=30) than in healthy controls (n=15) (P=0.0008). (E) Absolute numbers of circulating Th17 cells in M-CLL/CD38Low and U-CLL/CD38High patients were higher than in healthy individuals. M-CLL/CD38Low CLL patients (n=20) and U-CLL/CD38High (n=27) patients had higher absolute numbers of Th17 cells as compared to controls (n=15) (P=0.0006) and (P=0.034). See Online Supplementary Table S1 for the patients’ features. All values determined by the Mann-Whitney test.