CD14 + S100A9 + Monocytic Myeloid-Derived Suppressor Cells and Their Clinical Relevance in Non-Small Cell Lung Cancer
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1 CD14 + S1A9 + Monocytic Myeloid-Derived Suppressor Cells and Their Clinical Relevance in Non-Small Cell Lung Cancer Po-Hao, Feng M.D., Kang-Yun, Lee, M.D. Ph.D., Ya-Ling Chang, Yao-Fei Chan, Lu- Wei, Kuo,Ting-Yu Lin, M.D., Fu-Tsai, Chung, M.D., Chih-Shi Kuo, M.D., Chih-Teng Yu, M.D., Shu-Min Lin, M.D., Chun-Hua Wang, M.D., Chun-Liang Chou, M.D., Chien-Da, Huang M.D., Han-Pin, Kuo M.D. Ph.D Online Data Supplement
2 Material and Methods Oxidative stress measurement The intracellular oxidation levels were determined by using 5-(and-6)-chloromethyl-2,7 - dichlorodihydrofluoresceindiacetate-acetyl-ester (DCFDA, Invitrogen, Carlsbad, CA), which is metabolized to fluorescent (FL-1) 2,7 -dichlorofluorescein (DCF) upon oxidation. Briefly, PBMCs were incubated with.25 μmol DCFDA for 1 minutes at 37 C, washed twice in ice-cold PBS and stained for surface markers, and the mean fluorescence intensity (MFI) of intracellularly retained DCF was determined by flow cytometry. Activation of T cells T cells in the PBMCs with or without depletion of MDSCs were stimulated in 24-well flatbottomed plates with immobilized anti-cd3 antibody (prepared by pre-coating with 5 µg/ml Ab at 4 C overnight, BioLegend, San Diego, CA, USA) and anti-cd28 antibody (2 µg/ml, BioLegend, San Diego, CA, USA). The cells were pre-labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE, CellTrace TM CFSE Cell Proliferation Kit, 5 mm, Invitrogen, Eugene, Oregon, USA), and proliferation was determined by FACS analysis after 96 hours of stimulation. Fluorescence confocal microscopy The expression of and interaction between S1A9 and RAGE in freshly isolated CD11b+CD14+ cells were determined by immunofluorescence staining using an anti- S1A9-Rhodamine antibody and anti-rage-fitc antibody and visualized with a Leica TCS 4D confocal laser microscope (Leica Microsystem, Wetzlar, Germany). All cell images were obtained using a 4X dry objective lens on the confocal microscope with Leica Confocal Software.
3 Supplemental Table E1 Marker Clone Isotype Fluorochrome Manufacturer CD4 SK3 IgG1 PE BD CD8 RPA-T8 IgG1,kappa PE BioLegend CD11b ICRF44 IgG1,kappa PE/Cy5 BD CD11b 2LPM19c IgG1,kappa PE DAKO CD14 TUK4 IgG2a,kappa PE/Alexa Serotec CD14 M5E2 IgG2a,kappa FITC Santa Cruz CD15 TG-1 IgM FITC BD CD33 P67.6 IgG1 FITC BD IL-4Rα IgG2A PE R&D HLA-DR G46-6 IgG2a, kappa FITC BD RAGE (1 Ab) IgG2a, kappa Abcom inos N-2 IgG1 FITC Santa Cruz Arginase 1 (1 Ab) IgG Serotec S1A8 MAC387 IgG1 FITC GeneTex S1A9 (1 Ab) 4G9 IgG1 Abnova Isotype control MOPC-21 IgG1 kappa PE/Cy5 BD G IgG2a kappa FITC BD IgG2a PE/Alexa Serotec X4 IgG1 PE BD X4 IgG1 FITC BD Primary antibody, negative control, anti-mouse DAK-G1 IgG1 Dako Polyclonal rabbit anti mouse IgG PE Dako IgG PE Polyclonal rabbit anti-mouse IgG FITC Dako IgG FITC Primary antibody, negative IgG Dako control, anti-rabbit Polyclonal swine anti-rabbit IgG FITC Dako IgG FITC Goat anti-mouse IgG FITC IgM FITC Santa Cruz
4 Figure legend Supplement E1. A. Percentage of non-lymphocytic mononuclear cells (NLMNC) in the PBMCs of healthy donors (n=17) or NSCLC patients (n=36). NC vs. CA: 14±1.3 vs. 28±2.4, p<.5 B. Percentage of CD11b+CD14+ and CD11b+CD14- cells in NLMNC of NC (n=17) and CA patients (n=36). CD14+ in NC vs. CA: 79.3±1.8 vs ±1.1, p<.5; CD14- in NC vs. CA: 9.8±.6 vs. 21.8±2.7, p<.5. E2. Column bar graph analysis of the data in Figure 2A. The data are the mean fluorescence intensity (MFI). Empty bars, normal CD11b + /CD14 + cells; black bars, NSCLC CD11b + /CD14 + cells; gray bars, NSCLC CD11b + /CD14 - cells; n=4. The data are the mean ± SEM; p<.5. E3. Representative dot plots of CFSE-labeled T cell proliferation assay in response to CD3/CD28 co-stimulation in the PBMCs from healthy donors (NC) or NSCLC patients (CA). E4. Effects of CD11b + CD14 + cells from healthy donors on CD3/CD28-stimulated IFN-γ production in the cultured supernatants of effector cells analyzed by ELISA. n=3 independent experiments. The data are the mean ± SEM and are presented as the percentage of maximal IFN-γ production. E5. Representative dot plots of flow cytometry analysis of HLA-DR expression on CD11b + /CD14 + cells from normal subjects (left panel) and NSCLC patients (middle panel). The data from magnetic bead enriching HLA-DR -/low cells are also shown (right panel). E6. Column bar graph analysis of the mean fluorescence intensity (MFI) of designated intracellular staining. The data are presented as the mean ± SEM. n=5; p<.5 E7. A. Correlation of the percentage of S1A9 + cells in CD11b + CD14 + cells with the T cell suppression ability of CD11b + CD14 + cells from healthy donors. n=4, Spearman r=, p=1.8. B. Correlation of the MFI of S1A9 in CD11b + CD14 + cells to the T cell suppression ability from healthy donors. Spearman r=, n=4, p=1.8.
5 E8. Correlation of the percentage of CD11b + CD14 + IL-4Rα + cells in PBMCs with progression-free survival after platinum-based doublet chemotherapy. Spearman r=-.2, n=14, p=.93. E9. Fluorescence confocal microscopic analysis of CD11b+CD14+ cells from healthy donors or NSCLC patients that were stained with anti-rage-fitc (Green), anti-s1a9- Rhodamine (Red), and DAPI. Insets showed a magnification of the merged image. CD11b + CD14 + cells had significant yellow fluorescence in the merged image. E1. MTT assay for the cell viability of A549 cells (2x1 3 cells/ml) with or without CD14 + MDSC cells (5x1 5 cells/ml) co-cultured in 1 ml RPMI and 1% FBS medium in a 24-well plate. The cells were treated with 1 μm cisplatin in the presence or absence of anti-human RAGE blocking antibody (1 μg/ml) or IgG control (1 μg/ml) for 96 hr. n=4, p<.5, p<.1, p<.1.
6 E1 A 8 NLMNC B 1 CD11b+/CD14+ CD11b+/CD14- % PBMC NC Non Lym M % of NC CA NC CA NC CA
7 E NC CD11b+/CD14+ CA CD11b+/CD14+ CA CD11b+/CD14- Mean MFI CD15 CD33 IL-4Rα HLA-DR
8 E3
9 E4 25 % Ma ax IFN-γ Non 1:4 1:2 1:1 Ratio of CD11b + CD14 + : Effector cells
10 E5 A
11 E6 8 NC CD11b+CD14+ 6 CA CD11b+CD14+ CA CD11b+CD14- MFI 4 2 inos Arginase DCFH
12 E7 A B %M Max T cell proliferatio n Spearmn r= p= % of A9+ ( CD11b+/CD14+) Prolifera ation % Ma ax T cell Spearmn r= p= S1A9 MFI
13 E8 CD14+/ /IL-4Rα α% of PBMC Spearmen r= p = PFS (Months)
14 E9 NC CD14+ CA CD14+ RAGE RAGE S1A9 RAGE RAGE S1A9 DAPI Merge DAPI Merge
15 GE G Cis+M MDSC+aRAGE E Cis+MDSC Cis + MDSC + IgG Cis - 2 % Cell viability
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SUPPLEMENT Supplementary Figure 1: (A) (B)
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Supplementary Table 1
Supplementary Table 1 Flow Cytometry Antibodies Antibody Fluorochrome Clone Vendor CD45 PE-cyanine 7 30-F11 D ioscience CD3 Pacific lue 17A2 iolegend (San Diego, CA) CD11b APC M1/70 iolegend (San Diego,
<10. IL-1β IL-6 TNF + _ TGF-β + IL-23
3 ns 25 ns 2 IL-17 (pg/ml) 15 1 ns ns 5 IL-1β IL-6 TNF
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