Further Analysis of Interleukin-2 Receptor Subunit Expression on the Different Human Peripheral Blood Mononuclear Cell Subsets by Denis David, Lynda Bani, Jean-Louis Moreau, Christophe Demaison, Karine Sun, Ombretta Salvucci, Takayuki Nakarai, Marianne de Montalembert, Salem Chouaı̈b, Marcel Joussemet, Jerome Ritz, and Jacques Thèze Blood Volume 91(1):165-172 January 1, 1998 ©1998 by American Society of Hematology

Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by CD4 T lymphocytes and monocytes. Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by CD4 T lymphocytes and monocytes. PBMC from blood collected on heparin were treated by MoAbs 33B3 (anti–IL-2Rα), CF1 (anti–IL-2Rβ), and 3B5 (anti–IL-2Rγ). Further characterization of the two populations was achieved by treatment with anti-CD14 and anti-CD28 MoAbs. FITC-labeled Fab fragment anti-IgG was used to stain the cells, followed by PE-conjugated anti-CD4 MoAb. Quadrant settings distinguishing positive immunofluorescence from background fluorescence were determined by staining with isotype-matched control MoAbs: solid horizontal line for lymphocytes and dotted horizontal line for monocytes. The vertical dotted line separates CD4 low (monocytes) from CD4 high (lymphocytes) cells. The percentage of positive cells for the different markers is indicated. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology

Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by B lymphocytes. Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by B lymphocytes. PBMC from blood collected on heparin were treated by MoAbs 33B3, CF1, and 3B5. Characterization of the population was achieved by treatment with anti-CD19 and anti-CD23 MoAbs. FITC-labeled Fab fragment anti-IgG was used, followed by PE-conjugated anti-CD4 + FL3-conjugated anti-CD14 MoAbs. CD14+ monocytes were excluded for easier analysis. Quadrant setting distinguishing positive immunofluorescence from background fluorescence was determined by staining with isotype-matched control MoAbs. The percentage of positive cells for the different markers is indicated. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology

Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by NK cells. Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by NK cells. (A) PBMC from blood collected on heparin were treated by MoAbs 33B3, CF1, and 3B5. Further characterization of the population was achieved by treatment with anti-CD8 and anti-CD16 MoAbs. FITC-labeled Fab fragment anti-IgG was used, followed by PE-conjugated anti-CD56 MoAb + FL3-conjugated anti-CD14 MoAbs. CD14+ cells were excluded for easier analysis. Quadrant setting distinguishing positive immunofluorescence from background fluorescence was determined by staining with isotype-matched control MoAbs. Vertical dotted line separates CD56 low from CD56 high cells. The percentage of positive cells for the different markers is indicated. (B) NK cells were highly purified from PBMC as previously described.47 The resulting CD56 population was treated by MoAbs 33B3, CF1, and 3B5 followed by FITC-labeled Fab fragment anti-IgG. The percentage of positive cells for the different markers is indicated. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology

Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by CD8 T lymphocytes. Expression of IL-2Rα, IL-2Rβ, and IL-2Rγ by CD8 T lymphocytes. (A) PBMC from blood collected on heparin were treated by MoAbs 33B3, CF1, and 3B5. Further characterization of the population was achieved by treatment with anti-CD3 and anti-CD28 MoAbs. FITC-labeled Fab fragment anti-IgG was used, followed by PE-conjugated anti-CD8 MoAb. Quadrant setting distinguishing positive immunofluorescence from background fluorescence was determined by staining with isotype-matched control MoAbs. The percentage of positive cells for the different markers is indicated. (B) Staining and analysis was performed as in (A). The percentage of positive cells for the different markers is shown for a group of nine individuals. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology

Expression of the IL-2R by cells from blood collected with different anticoagulants. Expression of the IL-2R by cells from blood collected with different anticoagulants. (A) Blood of one donor was collected on CPD and PBMC were isolated the same day as the blood sample. Staining and analysis of CD4 T lymphocytes was performed as indicated in Fig 1except that FL3-labeled anti-CD14 MoAb was used. (B) Blood was collected on sodique heparin (n = 9) or on CPD (n = 3) or on ACD (n = 3), and PBMC were isolated the same day as the blood sample (d = 0) or 1 day after (d = 1) in some cases with sodique heparin (n = 4). Staining and analysis was performed as in (A) for CD4 T lymphocytes and as indicated in Fig 4 for CD8 T lymphocytes. Significant differences are indicated as follows, a, P ≤ .0001; b, .0001 < P ≤ .001; c, .001 < P ≤ .01; and d, .01 < P < .05 (nonpaired t-test). (C) Blood was collected on sodique heparin (H) or on CPD (C). CD4 T lymphocytes and monocytes were purified from PBMC isolated at d=0. IL-2Rα and β specific mRNAs were measured as previously described.30HPRT detection is also shown as positive control; N, PCR negative control. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology

Expression of IL-2R subunits on the different PBMC subsets in healthy donors and hemochromatosis patients. Expression of IL-2R subunits on the different PBMC subsets in healthy donors and hemochromatosis patients. Blood was collected on sodique heparin and PBMC were isolated the same day as the blood sample, for healthy donors (HD, n = 9) and hemochromatosis patients (HHP, n = 5). Staining and analysis was performed as in Figs1-4. Significant differences are indicated as in Fig 5. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology

Intracellular expression of IL-2Rγ in the different lymphocyte populations. Intracellular expression of IL-2Rγ in the different lymphocyte populations. (A) After cell permeabilization, intracellular staining was performed against IL-2Rγ and Bcl-2 protein as positive control. Two-color flow cytometry was performed on PBMC isolated from blood collected on sodique heparin for the upper and lower left panels. Results were confirmed for NK cells on purified CD56 cells as indicated (lower middle panel). Lower right panel shows a positive (YT) and a negative B-EBV cell line (B) derived from a XSCID child; background controls are identical with these two cell lines. (B) Blood was collected on sodique heparin and PBMC were isolated at d = 0. PBL, CD4 T lymphocytes and monocytes were purified and Western blots performed on their lysates as explained in Materials and Methods. Lane 1, YT cell line (positive control); lane 2, B-EBV cell line from a XSCID patient (negative control); lane 3, PBMC; lane 4, PBL; lane 5, monocytes; and lane 6, CD4 T lymphocytes. Denis David et al. Blood 1998;91:165-172 ©1998 by American Society of Hematology