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Figure 1. Phenotypic and functional characterization of nasopharyngeal carcinoma (NPC)– and healthy donor (HD)–derived exosomes. A) Electron microscopy.

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Presentation on theme: "Figure 1. Phenotypic and functional characterization of nasopharyngeal carcinoma (NPC)– and healthy donor (HD)–derived exosomes. A) Electron microscopy."— Presentation transcript:

1 Figure 1. Phenotypic and functional characterization of nasopharyngeal carcinoma (NPC)– and healthy donor (HD)–derived exosomes. A) Electron microscopy of a C15-Exo (1) and Patient-Exo (2) suspension contrasted with 2% phosphotungstic acid (PTA). In inset a, the diameter of two vesicles was measured. Scale bar represents 100nm. The images are representatives from four independent experiments for C15-Exo and two independent experiments for Patient-Exo. B) (1) Equal amounts of LCL proteins or LCL, C15, C17, or HD exosomes were analyzed by western blotting for HLA-DRA, CD63 and HSP90B1 (GRP94) as common markers and for LMP1 and galectin-9 as NPC-specific markers. 2) Extracts from patient-derived exosomes were analyzed by western blotting for CD63 in comparison with increasing amounts of extracts from the C15-Exo. C) CD4+ T cells were isolated from blood of healthy donors, then activated by anti-CD3 and anti-CD28 mAbs and incubated with increasing amounts of (1) C15-Exo or (2) HD-Exo. Proliferation was measured using [<sup>3</sup>H]-thymidine incorporation assay during the last 18 hours, and values were obtained as counts per minute (cpm). Assays were performed after 48 and 120 hours of culture. Results are means of normalized cpm values of triplicate wells of three independent experiments. Error bars represent standard deviations. P values from Student’s t-test. All statistical tests were two-sided. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

2 Figure 2. Impact of CCL20 on regulatory T cells (Treg) recruitment into nasopharyngeal carcinoma (NPC) tumor. A) (1) Gene expression analysis (real-time quantitative polymerase chain reaction) of several chemokines associated with Treg chemotaxis performed on C15 cells. Results are presented as means of three independent experiments expressed in 1/ΔCt ± SD bars using GADPH mRNA as housekeeping-gene. 2) Schematic representation of the established humanized NPC mouse model. Confocal microscopy (upper right image) was used to assess Treg labelling in the various cell mixes before their use in C15-SCID-Hu mice for CCL20 chemotactic cell migration assay. Scale bar = 30 µm. Four groups of C15 subcutaneously (s.c.) xenotransplanted SCID mice were established: (i) a control group only receiving IgG1 isotype control without mononuclear cells (PBMC) and anti-CCL20 (n = 5); (ii) a group only receiving PBMC enriched with 10% of Treg (n = 5); (iii) a group only receiving PBMC enriched with 10% of VT 680 labeled Treg (n = 5); and (iv) a group receiving both PBMC enriched with 10% of VT 680 labeled-Treg and anti-CCL20 (n = 5). B) (1) Fluorescence measurements in vivo (IVIS-LUMINA RX) at two levels (10 and 15mm) of four mouse groups, from left to right (i) nonreconstituted; (ii) reconstituted with PBMC + 10% of Treg; (iii) reconstituted with PBMC + 10% of VT 680 labeled Treg + anti-IgG1 mAb; and (iv) reconstituted with PBMC + 10% of VT 680 labeled Treg + anti-CCL20 mAb. 2) Confocal analysis of 5 µm tumor slices stained with DAPI (blue). White arrows on the left image indicate the presence of VT680 labeled-Treg. Scale bar = 10 µm C) FACS analysis of Treg in blood of C15 xenotransplanted mice treated with anti-IgG1 isotype or anti-CCL20 Ab. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

3 Figure 6. Impact of C15-Exo on regulatory T cells (Treg) phenotype
Figure 6. Impact of C15-Exo on regulatory T cells (Treg) phenotype. Purified CD4<sup>+</sup>CD25<sup>high</sup>FOXP3<sup>+</sup>CD127<sup>low</sup> Treg were cultured for 48 hours with increasing amounts of C15-Exo or healthy donor (HD)–Exo, after which their phenotypic changes were assessed by studying proliferative potential (A), surface markers (B) and intracellular markers (C). A) Effects of C15-Exo or HD-Exo on Treg cell proliferation. Treg were expanded polyclonaly using anti-CD3 (100ng/mL), anti-CD28 (100ng/mL) mAbs, high dose of interleukin (IL)-2 (300U/mL) and irradiated (4000 Gy) autologous mononuclear cells (2:1 ratio) for 48 hours in standard medium (0) or in contact of increasing concentration of C15-Exo (1) or HD-Exo (2). Results are means of triplicates wells of five independent experiments. Proliferation assays were determined by [<sup>3</sup>H]-thymidine incorporation and estimated in normalized values from cpm counts. Error bars are standard deviation. P values from Student’s t test. All statistical tests were two-sided. B) (1) Characterization of surface markers of isolated Treg after 48 hours culture in the absence (control) or presence of 10 µg/mL of C15-Exo or HD-Exo. Cells were analyzed by flow cytometry. The number in the upper-right quadrant indicates the percentage of CD4<sup>+</sup>CD25<sup>high</sup> Treg cells within the CD4<sup>+</sup>CD25<sup>+</sup> T cell population. 2) Frequency of CD4<sup>+</sup>CD25<sup>high</sup> fraction within C15-Exo or HD-Exo treated Treg ± SD bars. Results represent the means of five independent experiments. P values from Student’s t test. All statistical tests were two-sided. C) (1) FOXP3+ expression in untreated Treg (blue line), C15-Exo (10 µg/mL) treated Treg (orange line), and HD-Exo (10 µg/mL) treated Treg (red line). Representative results of three independent experiments. 2) Results are the means of normalized values of frequency of CD4<sup>+</sup>CD25<sup>+</sup>FOXP3<sup>high</sup> fraction within Treg of three independent experiments ± SD bars. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

4 Figure 3. Regulatory T cells (Treg) recruitment by nasopharyngeal carcinoma–derived CCL20<sup>+</sup> exosomes. A) (1) Semiquantitative western blot for detection of CCL20 in C15-Exo and healthy donor (HD)–Exo extracts using increasing concentrations of h-rec-CCL20 as positive controls. 2) Electron microscopy after immunogold staining of purified C15-Exo (0.05 to 0.15 µg/mL) (left panel), Patient-Exo (0.05 to 0.15 µg/mL) (center panel), and HD-Exo (0.015 to µg/mL) (right panel) preincubated with primary anti-CCL20 mAb. Scale bar = 100nm. 3) Gene expression analysis of CCR6 (CCL20 receptor) by real-time quantitative polymerase chain reaction in Treg ± 10 µg/mL of C15-derived exosomes after 48 hours of culture. Data are means of X independent experiments. Error bars are standard deviation. B) Chemotactic potential of C15-derived exosomes on CD4<sup>+</sup>CD25<sup>high</sup>CD127<sup>low</sup> T cells assessed in Boyden chamber, using crude RPMI medium as negative control and SDF1α and h-rec-CCL20 as positive controls. Results are presented as means of quadruplicates of four independent experiments expressed as index of migration related to crude medium ± SD. P values from Student’s t test. All statistical tests were two-sided. C) Chemotactic potential of Patient-derived exosomes on CD4<sup>+</sup>CD25<sup>high</sup>CD127<sup>low</sup> T cells assessed in Boyden chamber, using crude RPMI medium and h-rec-CCL20 as negative and positive controls, respectively Results are presented as means of quadruplicates of 2 independent experiments expressed as index of chemoattraction related to medium ± SD. P values from Student’s t test. All statistical tests were two-sided. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

5 Figure 4. Recruitment of CD4<sup>+</sup>CD25<sup>-</sup> T cells and conversion into regulatory T cells by C15-Exo. A) Chemotaxis assays were performed in Boyden chamber with increasing doses of C15-Exo (0.1, 1, and 10 µg/mL), RPMI 1640 medium as negative control and CXXL12 as positive control. Results are presented as means of quadruplicates wells of four independent experiments expressed in index of chemotaxis related to medium ± SD bars. P values from Student’s t test. All statistical tests were two-sided. B) Purified CD4<sup>+</sup>CD25<sup>-</sup> T cells were cultured ± C15-Exo or healthy donor (HD)–Exo (10 µg/mL) then analyzed by FACS after 72 hours and 120 hours of culture. Data represents the frequency of CD4<sup>+</sup>CD25<sup>high</sup> cells within CD4<sup>+</sup>CD25<sup>-</sup> initial cells and shows in (1) and (2) that C15-Exo statistically significantly increases the frequency of CD4<sup>+</sup>CD25<sup>high</sup> fraction after 72 hours and 120 hours of culture compared with control, and in (3) and (4) that HD exosome treatment has no statistically significant impact on it. (1) and (3) are representative of five independent experiments, while (2) and (4) results are presented as means of five independent experiments expressed in normalized values of frequency of CD4<sup>+</sup>CD25<sup>high</sup> cells ± SD bars. P values from Student’s t test. All statistical tests were two-sided. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

6 Figure 5. Functionality of converted CD4<sup>+</sup>CD25<sup>+</sup> T cells. A) TGF-β1 secretion after 48 hours or 72 hours treatment of CD4<sup>+</sup>CD25<sup>-</sup> T cells with increasing concentration of C15-Exo or HD-Exo. Results are the means in pg/mL of duplicates of three independent experiments ± SD bars. B) CD4<sup>+</sup>CD25<sup>-</sup> cells were preincubated for 48 hours with increasing amounts (0.01 to 10 µg/mL) of C15-Exo or with 1 µg/mL of the short h-rec-galectine-9 isoform (Gal9S) before being cocultured for 48 hours with autologous mononuclear cells (PBMC) at varying PBMC: CD4<sup>+</sup>CD25<sup>-</sup> ratios ranging from 1:0.5 to 1:3 and activated by anti-CD3 and anti-CD28 mAbs. Proliferation was determined by [<sup>3</sup>H]-thymidine incorporation for the last 18 hours. Results are means of triplicates of five independent experiments expressed in normalized values of proliferation from cpm ± SD bars. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please

7 Figure 7. Impact of nasopharyngeal carcinoma–derived exosomes on regulatory T cells (Treg) functions. A) Gene expression analysis (real-time quantitative polymerase chain reaction) of several markers associated with Treg immunosuppressive phenotype and function after 48 hours of incubation with 10 µg/mL C15-Exo. Results are expressed as relative gene expression (2^-ΔΔCt) and presented as means of four independent experiments ± SD bars. B) Coculture of mononuclear cells and Treg in the presence of increasing amounts of either C15-Exo (1), Patient-Exo (2) or healthy donor (HD)–Exo (3). Results represent the means of normalized values of proliferation of four independent experiments ± SD bars for C15- and HD-Exo and two independent experiments ± SD bars for Patient-Exo. P values from Student’s t test. All statistical tests were two-sided. C) IL10 amounts were determined by ELISA. Results represent the means of duplicate wells of three independent experiments expressed in pg/mL ± SD bars. P values from Student’s t test. All statistical tests were two-sided. D) TGFB1 amounts of supernatants of Treg cell cultured for 48 hours with C15-Exo or HD-Exo determined by ELISA. Results represent the means of duplicate wells of three independent experiments expressed in pg/mL ± SD bars. From: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells J Natl Cancer Inst. 2014;107(1). doi: /jnci/dju363 J Natl Cancer Inst | © The Author Published by Oxford University Press. All rights reserved. For Permissions, please


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