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Tumor-derived exosomes modulate PD-L1 expression in monocytes

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Science Immunology  28 Jul 2017:
Vol. 2, Issue 13, eaah5509
DOI: 10.1126/sciimmunol.aah5509
  • Fig. 1 Characterization and quantification of CLL-derived exosomes.

    (A to D) Characterization of MEC-1 exosomes (Exo). (A) Transmission electron microscopy (representative of seven independent measurements). Scale bar, 200 nm. (B) Size profile by NTA (representative of five independent measurements). %cum, cumulative percentage. (C) Western blot analysis of RAB5a, HSP70, and HLA-DR for MEC-1 Exo and parental cells (representative of two to three independent experiments). (D) Immunogold electron microscopy for HLA-DR (representative of six independent experiments). Scale bar, 200 nm. (E) Absolute Exo counts in plasma of CLL patients (n = 26) and healthy donors (HD; n = 27) acquired by NTA. P = 0.30. n.s., not significant. (F) CD20 detection on MEC-1 Exo via immunogold electron microscopy. Scale bar, 200 nm. (G) Flow cytometry analysis of CD20 on MEC-1 Exo coupled to latex beads (representative of three independent experiments). IgG, immunoglobulin G. (H) Quantification of CD20 in CLL (n = 18) and HD (n = 18) plasma via ELISA. **P = 0.008. For (E) and (H), horizontal lines represent mean values; P values were determined by unpaired t test.

  • Fig. 2 Small RNA sequencing of CLL plasma–derived exosomes and MEC-1 exosomes and cells.

    (A) Whole-RNA profiles of MEC-1 exosomes (Exo) and cells obtained by Agilent 2100 Bioanalyzer (representative of three independent measurements). FU, fluorescence units. (B to H) Sequencing of small RNA (<200 nt) of CLL plasma–derived Exo (n = 3), MEC-1 Exo (n = 5), and MEC-1 cells (n = 3). (B) Heat map showing Pearson correlations of the top 1000 most abundant small RNA transcripts of MEC-1 Exo and cells. (C) Volcano plot showing the differential abundance of noncoding RNA transcripts in MEC-1 Exo compared with cells. Bubble size corresponds to average read counts per 1 million reads; P values were calculated by DESeq2 and corrected for multiple testing using the Benjamini-Hochberg method. snoRNA, small nucleolar RNA. (D) Read distribution of noncoding RNA species for CLL plasma–derived Exo and MEC-1 Exo and cells. Mt/vault/sn/linc/misc RNA, mitochondrial/vault/small nuclear/long intergenic noncoding/miscellaneous RNA. (E) Top 15 abundant RNA transcripts. (F) Top 15 abundant miRNA transcripts. (G) Read distribution of four human Y RNAs. For (E) to (G), error bars represent the SEM. (H) Coverage plots of full-length hY4 transcript were visualized by Integrative Genomic Viewer; individual tracks from replicates were merged and visualized with the overlay option. Y axis is shown in log scale.

  • Fig. 3 Northern blot analysis of hY4 in CLL-derived exosomes and cells.

    Full-length (hY4 96 nt) and 5′ fragment hY4 (hY4 31 nt) were detected using an antisense DNA oligonucleotide probe against the 5′ end of hY4. Sample loading per lane: (A) 150 ng of RNA of MEC-1 exosomes (Exo) and cells (representative of five independent measurements), as well as total exosomal RNA of 3 ml of healthy donor (HD) and CLL plasma each; (B) 200 pg of RNA of PBMCs, as well as total exosomal RNA of 3 ml of HD and CLL plasma each; and (C) 150 ng of RNA of CD19-sorted B cells of three HD and three CLL patients.

  • Fig. 4 Uptake of MEC-1 exosomes in myeloid cells, induction of PD-L1 expression, and cytokine release.

    (A to C) J774 cells were treated with 10 μg of PKH67-labeled MEC-1 exosomes (Exo). (A) Confocal microscopy (representative of four independent experiments). Blue, nuclear staining with 4′,6-diamidino-2-phenylindole; green, membrane labeling of Exo with PKH67. 1h, 1 hour. Scale bars, 5 μm. (B) Quantification of uptake shown as mean fold changes (±SD) of Exo treated at 4° or 37°C, over untreated J774 cells at 37°C (n = 2). RelFluorescence, relative fluorescence. (C) Costaining of human HLA-DR (red) and PKH67+ MEC-1 Exo. Scale bar, 5 μm. (D) Uptake of 5 μg of PKH67-labeled MEC-1 Exo by human primary monocytes was analyzed by flow cytometry after 8 hours of Exo treatment (representative of three independent experiments). (E and F) Expression of PD-L1 (E) and CCR2 (F) in monocytes after 8 hours of treatment with 3 to 5 μg of MEC-1 Exo was analyzed by flow cytometry. Relative median fluorescence intensity (MFI) (RelMFI) was normalized to isotype control (n = 6 each) (right); representative histogram (left). (G) Cytokine concentrations in cell culture supernatant of monocytes after 8 hours of treatment with 5 μg of MEC-1 Exo were measured by ELISA (CCL2) and cytometric bead array (CCL4 and IL-6) (n = 3). (H and I) Expression of PD-L1 (H) and CCR2 (I) in monocytes after 8 hours of treatment with 5 μg of CLL or healthy donor (HD) plasma–derived Exo (n = 3) was analyzed by flow cytometry; RelMFI was normalized to isotype control. For (A) and (D) to (I), Ctrl denotes phosphate-buffered saline. For (E) to (I), mean values are depicted by horizontal lines. P values were determined by paired t test. *P = 0.02 (E); *P = 0.01 (H).

  • Fig. 5 Response of monocytes to hY4 delivery and cytokine levels in CLL serum.

    Human primary monocytes were treated with Effectene-packaged RNA for 8 hours. (A and B) Uptake of 50 nM Alexa 488–coupled hY4 fragment (hY4 31 nt) was analyzed by flow cytometry (A) and confocal microscopy (B) (representative of three independent experiments each). Blue, nuclear staining with Hoechst 33342; red, staining of acidified endosomes and lysosomes with LysoTracker DND-99; green, Alexa 488–coupled hY4 fragment. Scale bar, 10 µm. (C and D) Gene expression data of primary human monocytes treated with Effectene alone (Ctrl), 50 nM hY4 5′ fragment (hY4 31 nt) or 50 nM full-length hY4 (hY4 96 nt) were obtained by quantitative RT-PCR array (n = 3 each). P values were determined by moderated t test via empirical Bayes method and adjusted using the method of Benjamini and Hochberg to control the false discovery rate. (C) Heat map of log2 gene expression data of significantly deregulated genes with at least twofold change, sorted according to fold change of hY4 96 nt over Ctrl. (D) Comparison of expression levels for all analyzed genes. (E) Cytokine concentrations in cell culture supernatant of monocytes treated with 50 nM RNA were analyzed by ELISA (CCL2 and CCL3) and cytometric bead array (CCL4, CXCL10, and IL-6) (n = 3). (F and G) Expression of PD-L1 (F) and CCR2 (G) in monocytes treated with 50 nM RNA was analyzed by flow cytometry (n = 3). Relative median fluorescence intensity (RelMFI) was normalized to isotype control is depicted. (H) Expression of PD-L1 and CCR2, as well as cell culture cytokine concentrations upon monocyte treatment with 600 ng of Effectene-packaged MEC-1 exosomal RNA (Exo RNA), was analyzed by flow cytometry and cytometric bead array, respectively (n = 3). For (E) to (H), mean values are depicted by horizontal lines. P values were determined by paired t test. *P < 0.05; ***P < 0.001. (I) Comparison of serum cytokine ratios of 11 CLL patients versus 5 healthy donors (HD) analyzed by cytokine array (black bars), and gene expression data provided in (B) (gray bars show log2 fold change of hY4 96 nt versus Ctrl). Cytokines that are transcriptionally induced by hY4 in monocytes and elevated in CLL plasma are depicted. For (A) to (H), Ctrl denotes Effectene alone.

  • Fig. 6 Role of TLR signaling in hY4-induced changes and effects of TLR inhibition.

    (A and B) Response of bone marrow–derived myeloid cells of wild-type (WT), Mavs KO, and Tlr7 KO mice (n = 3 to 4 each) 8 hours after treatment with 50 nM Effectene-packaged full-length hY4 (hY4 96 nt) or the TLR agonist LPS or CL075. (A) Cytokine concentrations in culture supernatant were analyzed by cytometric bead array (CCL4) and ELISA (IL-6). (B) PD-L1 expression measured by flow cytometry. (C) Monocytes were pretreated with 50 μM chloroquine (CQ) for 30 min to inhibit TLR signaling, before treatment with 50 nM Effectene-packaged full-length hY4 (hY4 96 nt) (left) or 10 μg of MEC-1 exosomes (Exo) (right). Cytokine concentrations in cell culture supernatant were measured by cytometric bead array after 8 hours (n = 3). (A to C) Ctrl, Effectene alone; CL075 and LPS, Effectene + CL075 (3 μg/ml) (TLR7/8 agonist) or + LPS (10 ng/ml) (TLR4 agonist). (D to F) Therapeutic targeting of TLR signaling by CQ treatment in vivo. C57BL/6 mice were transplanted with 1.5 × 107 splenocytes from leukemic Eμ-TCL1 mice and randomized to treatment, with vehicle or CQ (46 mg/kg per day) added to drinking water. (D) Tumor load in blood at indicated time points of treatment as absolute counts of circulating CD5+CD19+ CLL cells was determined by flow cytometry. (E) Tumor load in spleen after 3.5 weeks of treatment as absolute counts of CD5+CD19+ CLL cells was determined by flow cytometry (F) and spleen weight. For (A) to (F), mean values are depicted by horizontal lines. For (A) to (C), P values were determined by paired t test. For (D) to (F), P values were determined by unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/13/eaah5509/DC1

    Materials and Methods

    Fig. S1. Plasma exosome quantification and correlation with clinical parameters.

    Fig. S2. Northern blot analysis of hY4 in non-CLL exosomes and cells.

    Fig. S3. Exosome uptake and triggered response in myeloid cells.

    Fig. S4. hY4-induced gene expression changes and functional effects in monocytes.

    Fig. S5. Relevance of TLR signaling in exosomal RNA–induced response.

    Table S1. Patient information.

    Table S2. Proteome of CLL and healthy donor plasma–derived exosomes.

    Table S3. Small RNA sequencing data of CLL-derived exosomes and cells.

    Table S4. Quality control data of small RNA sequencing libraries.

    Table S5. Abundance of RNA species.

    Table S6. Gene expression results of monocytes upon hY4 treatment.

    Table S7. Comparison of serum cytokine levels with hY4-induced genes.

    Table S8. Raw data.

    References (5054)

  • Supplementary Materials

    Supplementary Material for:

    Tumor-derived exosomes modulate PD-L1 expression in monocytes

    Franziska Haderk, Ralph Schulz, Murat Iskar, Laura Llaó Cid, Thomas Worst, Karolin V. Willmund, Angela Schulz, Uwe Warnken, Jana Seiler, Axel Benner, Michelle Nessling, Thorsten Zenz, Maria Göbel, Jan Dürig, Sven Diederichs, Jérôme Paggetti, Etienne Moussay, Stephan Stilgenbauer, Marc Zapatka, Peter Lichter, Martina Seiffert*

    *Corresponding authors. Email: m.seiffert{at}dkfz.de

    Published 28 July 2017, Sci. Immunol. 2, eaah5509 (2017)
    DOI: 10.1126/sciimmunol.aah5509

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Plasma exosome quantification and correlation with clinical parameters.
    • Fig. S2. Northern blot analysis of hY4 in non-CLL exosomes and cells.
    • Fig. S3. Exosome uptake and triggered response in myeloid cells.
    • Fig. S4. hY4-induced gene expression changes and functional effects in monocytes.
    • Fig. S5. Relevance of TLR signaling in exosomal RNA–induced response.
    • References (50?54)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Patient information.
    • Table S2 (Microsoft Excel format). Proteome of CLL and healthy donor plasma– derived exosomes.
    • Table S3 (Microsoft Excel format). Small RNA sequencing data of CLL-derived exosomes and cells.
    • Table S4 (Microsoft Excel format). Quality control data of small RNA sequencing libraries.
    • Table S5 (Microsoft Excel format). Abundance of RNA species.
    • Table S6 (Microsoft Excel format). Gene expression results of monocytes upon hY4 treatment.
    • Table S7 (Microsoft Excel format). Comparison of serum cytokine levels with hY4-induced genes.
    • Table S8 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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