Article Figures & Data
Figures
- Figure 1.
MDSC express markers of activated PMN and suppress CD8+ T-cell proliferation and IFNγ production. A, a representative flow cytometry analysis of MDSC from RCC patients (n = 10) compared with PMN from normal controls (n = 8) showing increased expression of CD66b, CD11b, and VEGFR1 and low expression of CD16 and CD62L in MDSC. B, a representative cytospin of PMN and MDSC (sorted using anti-CD66b or anti-CD11b+/CD14−) from peripheral blood of RCC patients (n = 10). C, 5 × 105 PBMC labeled with CFSE were activated with immobilized anti-CD3 (1 μg/mL) and anti-CD28 (0.1 μg/mL) antibodies. After 96 h of culture, T cells were labeled using antibodies against CD4 and CD8 and proliferation was determined by flow cytometry. D, supernatants were also collected 72 h after stimulation with anti-CD3/CD28 and measured for IFNγ production by ELISA. C and D, experiments were repeated thrice using samples from individual RCC patients and controls.
- Figure 2.
Activation of PMN induces MDSC characteristics. A, MDSC were isolated from RCC patients (n = 15) by sorting of CD11b+/CD14− cells, whereas PMN (from patients and controls) were isolated over dextran. The expression of CD66b was measured as MFI by flow cytometry. B, resting PMN from normal individuals were activated with fMLP and PMA for 30 min, after which the cells were tested for CD66b expression, changes in density after centrifugation over Ficoll-Hypaque (C), and levels of arginase I in the tissue culture medium (D). B to D, columns, mean of three different experiments; bars, SD.
- Figure 3.
High arginase activity and arginase I protein are found in the plasma of RCC patients. Plasma obtained from RCC patients (n = 23) and controls (n = 13) was tested for arginase activity (A), arginase I protein by ELISA (B), and l-arginine levels by HPLC (C). D, arginase II protein expression in plasma of RCC patients and controls was tested by immunoprecipitation.
- Figure 4.
Degranulation and release of arginase I decreases intracellular arginase I in MDSC. Arginase activity (A) and arginase I mRNA expression (B) were determined in MDSC and autologous PMN from RCC patients (n = 19) and PMN from controls (n = 13). PMN from RCC patients were isolated from the fraction of cells that did not separate with PBMC over Ficoll-Hypaque. Plasma from RCC patients (n = 5) and controls (n = 6) was tested for the levels of MPO activity (C) and gelatinase B (D) by ELISA.
- Figure 5.
MDSC have decreased levels of Annexin V. A, apoptosis was tested by the expression of Annexin V in MDSC and autologous PMN from RCC patients (n = 11) and PMN from normal controls (n = 13). Positive controls were PMN from healthy donors activated in vitro with 20 ng/mL PMA for 1 h. B, 1 × 106 human RCC tumor cell line 786-O cells were cocultured in Transwells (0.4 μm pores) with 5 × 106 PMN and Annexin V expression was tested after 6, 12, and 24 h. Tumor cells were in the lower chamber, whereas PMN were in the upper chamber. Controls included PMN cultured alone for the same time period.
- Figure 6.
Treatment with anti-VEGF antibody bevacizumab does not prevent the accumulation of MDSC in peripheral blood of RCC patients. RCC patients (n = 23) receiving bevacizumab followed by bevacizumab/IL-2 therapy were tested for levels of VEGF (A), number of MDSC in peripheral blood (B), arginase I (C), and l-arginine concentrations in plasma (D). Normal control samples were included in each measurement (n = 12). A, levels of VEGF were tested by ELISA. B, the percentage of MDSC (CD66b+) in the PBMC of RCC patients was measured by flow cytometry. C, the levels of arginase I were determined by ELISA. D, the levels of l-arginine in plasma were determined by HPLC.
Additional Files
Supplementary Data, Rodriguez, et al.
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Volume 69, Issue 4
