ICAM-1-mediated adhesion is a prerequisite for exosome-induced T cell suppression

Abstract

Tumor-derived extracellular vesicles (TEVs) suppress the proliferation and cytotoxicity of CD8⁺ T cells, thereby contributing to tumor immune evasion. Here, we report that the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) co-localizes with programmed death ligand 1 (PD-L1) on the exosomes; both ICAM-1 and PD-L1 are upregulated by interferon-γ. Exosomal ICAM-1 interacts with LFA-1, which is upregulated in activated T cells. Blocking ICAM-1 on TEVs reduces the interaction of TEVs with CD8⁺ T cells and attenuates PD-L1-mediated suppressive effects of TEVs. During this study, we have established an extracellular vesicle-target cell interaction detection through SorTagging (ETIDS) system to assess the interaction between a TEV ligand and its target cell receptor. Using this system, we demonstrate that the interaction of TEV PD-L1 with programmed cell death 1 (PD-1) on T cells is significantly reduced in the absence of ICAM-1. Our study demonstrates that ICAM-1-LFA-1-mediated adhesion between TEVs and T cells is a prerequisite for exosomal PD-L1-mediated immune suppression.

Keywords: CD8(+) T cells; ICAM-1; PD-L1; exosome; extracellular vesicles; immune suppression.

Figure 1.. ICAM-1 is expressed on melanoma cell-derived exosomes and is up-regulated by IFN-γ.

(A) Characterization of exosomes purified from melanoma cells (WM9 and WM164) using nanoparticle tracking analysis (NTA). The X-axis represents diameters, and the Y-axis represents the concentration (particles/ml) of exosomes. (B) Western blot analysis of ICAM-1, PD-L1 and exosome markers (Hrs, Alix, Tsg101 and CD63) in the whole cell lysate (WCL), purified exosomes (EXO) and microvesicles (MV) from WM9 and WM164 cells. All lanes were loaded with equal amounts of total protein. (C) ICAM-1 co-fractionated with PD-L1 and exosome markers (Alix, CD63 and Tsg101) on density gradient centrifugation. (D) Immunoblot analysis of ICAM-1 and PD-L1 in control and IFN-γ-treated cells and their exosomes. All lanes were loaded with equal amounts of total protein. (E) Quantification of EV ICAM-1 expression in WM9 (left panel) and WM164 (right panel) cells with or without IFN-γ treatment. (F) Schematic of ELISA to measure ICAM-1 levels on the surface of exosomes isolated from human melanoma cell supernatants. TMB, 3, 3′, 5, 5′-tetramethylbenzidine; SA-HRP, streptavidin-horseradish peroxidase. See MATERIALS AND METHODS for details. ELISA of ICAM-1 on exosomes and microvesicles from WM9 (G) and WM164 (H) melanoma cells, with or without IFN-γ treatment. Exosomes and MVs from WM9 and WM164 cells were collected and added into the ELISA plate which was coated with anti-ICAM-1 antibodies. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using two-sided unpaired t-test (E, G, H).

Figure 2.. Hrs and Rab27 mediate the secretion of exosomal ICAM-1.

(A) Immunoblot analysis of ICAM-1, PD-L1 and exosome markers (CD63 and Tsg101) in Hrs-knockdown cells (left panel). Quantification of exosomal ICAM-1 is shown in the right panel. (B) Immunofluorescence staining showing the intracellular co-localization of ICAM-1 with Hrs in WM9 cells. Scale bars, 10 μm. (C) Co-immunoprecipitation of ICAM-1 with Flag-tagged Hrs from 293T cells expressing ICAM-1 and Hrs. (D) Immunoblot analysis of ICAM-1, PD-L1 and exosome markers (CD63 and Tsg101) in Rab27A-knockdown cells (left panel). Quantification of exosomal ICAM-1 is shown in the right panel. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using one-way ANOVA analysis with Dunnett’s multiple comparison tests (A, B).

Figure 3.. ICAM-1 and PD-L1 colocalize on the exosomes derived from melanoma cells.

(A) An EM image of a WM9 cell-derived exosome co-stained with anti-ICAM-1 antibodies (conjugated with 10 nm gold particles) and anti-PD-L1 antibodies (conjugated with 5 nm gold particles). Scale bar, 100 nm. (B) ExoView images of exosomes derived from control and IFN-γ-treated WM9 cells. The exosomes were captured by anti-PD-L1 antibodies and then incubated with fluorescence-labeled anti-PD-L1 (green), anti-ICAM-1 (red) and anti-pantetraspanin (Tetra, a mixture of anti-CD63, anti-CD81 and anti-CD9, blue) antibodies. (C) Quantification of PD-L1⁺ ICAM-1⁺ exosomes among total Tetra⁺ exosomes. (D) Schematic of ICAM-1⁺ exosome isolation from WM9-derived TEVs by magnetic beads. See MATERIALS AND METHODS for details. (E) Immunoblot analysis of ICAM-1, PD-L1 and other exosome markers in total, ICAM-1⁺ and void exosomes from WM9 cells. The void and ICAM-1⁺ exosomes were isolated from the WM9 cells-derived exosomes. The same amounts of proteins were loaded in each lane. See MATERIALS AND METHODS for details. (F) Quantification of exosomal ICAM-1 (left) and PD-L1 (right) expression in WM9-derived exosomes sorted as shown in (E). (G) Schematic of ELISA to measure ICAM-1 levels on the surface of PD-L1⁺ exosomes isolated from melanoma patient plasma. (H) ELISA of ICAM-1 expression levels on circulating PD-L1⁺ exosomes in healthy donors (“HD”, n = 10) and melanoma patients (“MP”, n =27). Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using two-sided unpaired t-test (C), one-way ANOVA analysis with Dunnett’s multiple comparison tests (F) and two-sided unpaired Welch’s t-test (H).

Figure 4.. ICAM-1/LFA-1 interaction mediates the adhesion between TEVs and stimulated CD8⁺ T lymphocytes.

(A) Representative histograms of human peripheral CD8⁺ T cells binding to CFSE-exosomes derived from WM9 cells with or without IFN-γ treatment. The proportions of CFSE positive CD8⁺ T cells (with or without stimulation with anti-CD3/CD28 antibodies) are shown on the right. (B) Representative histograms of human peripheral CD8⁺ T cells bound to CFSE-exosomes or microvesicles derived from WM9 cells with or without IFN-γ treatment. CD8⁺ T cells were stimulated with anti-CD3/CD28 antibodies. The proportions of CFSE positive CD8⁺ T cells are shown on the right. (C) Representative histograms of human peripheral CD8⁺ T cells bound to CFSE-exosomes that were pre-treated with IgG or anti-ICAM-1 antibodies. The proportions of CFSE positive CD8⁺ T cells are shown on the right. (D) Representative histograms of human peripheral CD8⁺ T cells treated with CFSE exosomes derived from control or ICAM-1 knockdown (KD) WM9 cells. The proportions of CFSE positive cells are shown on the right. (E) Representative histograms of human peripheral CD8⁺ T cells that bound to CFSE-exosomes pre-treated with anti-CD11a, anti-CD11b or anti-CD18 antibodies, then treated with exosomes from WM9 cells. The proportions of CFSE positive CD8⁺ T cells are shown on the right. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using unpaired t-test (A) or one-way ANOVA analysis with Dunnett’s (B, D) or Sidak’s (C, E) multiple comparison tests.

Figure 5.. ICAM-1 is required for T cell suppression by TEVs.

(A) Schematic of the exosomal ligand-receptor blockade assay. Genetically engineered Jurkat T cells with NFAT-mediated expression of luciferase activities were stimulated with anti-CD3/CD28 antibodies. TEVs derived from WM9 cells were pre-treated with anti-ICAM-1 antibodies and added into the system. Luminescence was measured to indicate the activation of effector T cells. (B) Effector T cell activation with indicated treatment. RLU, relative luminometer units. (C) Histogram of CFSE positive human peripheral CD8⁺ T cells treated with WM9-derived exosomes. The exosomes were pre-treated with IgG isotype or anti-ICAM-1 antibodies. The proportions of cells with diluted CFSE dye (proliferating cells) are shown on the right. (D) Representative contour plots of human peripheral CD8⁺ T cells with indicated treatment for the expression of granzyme B (GzmB). The proportions of GzmB positive CD8⁺ T cells are shown on the right. (E) Representative contour plots of human peripheral CD8⁺ T cells for the expression of Ki67 after indicated treatment. The proportions of Ki67 positive CD8⁺ T cells are shown on the right. (F) Representative contour plots of human peripheral CD8⁺ T cells for the expression of GzmB after indicated treatment. The proportions of GzmB positive CD8⁺ T cells are shown on the right. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using one-way ANOVA with Sidak’s multiple comparison tests (B, C, D, E, F).

Figure 6.. Exosomal PD-L1 is not necessary for the binding of TEVs to CD8⁺ lymphocytes.

(A) Immunoblot analysis of PD-L1, ICAM-1 and exosome markers (CD63, Hrs and CD9) in the whole cell lysate (WCL) and purified exosomes (EXO) from control and PD-L1 knockout (KO) WM9 cells. The same amounts of proteins were loaded in each lane. (B) Representative histograms of human peripheral CD8⁺ T cells bound to CFSE-exosomes from control and PD-L1 KO WM9 cells. The proportions of exosome-binding CD8⁺ T cells are shown on the right. (C) Representative histogram of CFSE-labelled human peripheral CD8⁺ T cells after indicated treatment. The proportions of cells with diluted CFSE dye (proliferating cells) are shown on the right. (D) Representative contour plots of human peripheral CD8⁺ T cells for the expression of Ki67 after indicated treatment. The proportions of Ki67 positive CD8⁺ T cells are shown on the right. (E) Representative contour plots of human peripheral CD8⁺ T cells for the expression of granzyme B (GzmB) after indicated treatment. The proportions of GzmB positive CD8⁺ T cells are shown on the right. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using two-sided unpaired t-test (B) or one-way ANOVA analysis with Sidak’s multiple comparison tests (C, D, E).

Figure 7.. Blocking exosomal ICAM-1 inhibits T cells and promotes tumor progression in vivo.

(A) Immunoblot analysis of PD-L1, ICAM-1 and exosome-associated markers (Tsg101 and CD9) in the whole cell lysate (WCL) and purified exosomes (EXO) from control and ICAM-1 knockout (KO) YUMM1.7 mouse cell line. The same amounts of proteins was loaded in each lane. (B) Representative histograms of splenic CD8⁺ T cells (stimulated with anti-CD3/CD28 antibodies) bound to CFSE-labeled exosomes derived from control and ICAM-1 KO YUMM1.7 cells. The proportions of exosome-bound CD8⁺ T cells are shown on the right. (C) Representative histograms of splenic CD8⁺ T cells bound to YUMM1.7-derived exosomes pre-treated with IgG isotype or anti-ICAM-1 antibodies. The proportions of exosome-bound CD8⁺ T cells are shown on the right. (D) Representative contour plots of Ki-67 and granzyme B (GzmB) expression in stimulated mouse splenic CD8⁺ T cells after treatment with exosomes from control or ICAM-1 KO YUMM1.7 cells, or from YUMM1.7 cell-derived exosomes with or without pre-treatment of IgG isotype or anti-ICAM-1 antibodies. The percentage of Ki67⁺GzmB⁺ CD8⁺ T cells among total is shown on the right. (E) Growth curve of YUMM1.7 tumors in mice with tail vein injections with indicated exosomes (n = 7 mice per group). (F) The number of tumor infiltrating CD8⁺ lymphocytes (TILs) from the YUMM1.7 tumors with indicated treatment were analyzed using flow cytometry 22 days post-implantation. The expression levels of Ki67 (G) and GzmB (H) by PD-1⁺ TILs as in (E) were analyzed using flow cytometry. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using two-sided unpaired t-test (B), one-way ANOVA analysis with Sidak’s multiple comparison tests (D), two-way ANOVA analysis with Tukey’s multiple comparison tests (E), or Welch ANOVA with Dunnett’s T3 multiple comparison tests (F, G, H).

Figure 8.. Establishing the ETIDS system and testing the necessity of ICAM-1 in exosomal PD-L1 interaction with PD-1 on T cell surface.

(A) Schematic representation of the ETIDS system. Murine PD-L1-SrtA and murine PD-1-G5 were stably expressed in WM9 and Jurkat T cells, respectively. PD-L1-SrtA⁺ exosomes were collected from WM9 cells and incubated with Jurkat PD-1-G5 cells in the presence of Biotin-LPETG. Biotin-LPETG are transferred to G5 tag on Jurkat cells that are catalyzed by SrtA on exosomes. The biotin signals were analyzed using flow cytometry. See MATERIALS AND METHODS for details. (B) Western blot analysis showing the expression of PD-L1-SrtA in whole cell lysates and exosomes derived from WM9 PD-L1-SrtA cells. (C) Western blot analysis showing PD-L1-SrtA on exosomes was conjugated with biotin-LPETG. (D) Flow cytometry analysis of Jurkat T cells after treatment with WM9 PD-L1-SrtA-derived exosomes. (E) Flow cytometry analysis of biotin positive G5-PD-1 Jurkat T cells after incubation with PD-L1-SrtA⁺ exosomes that were pre-treated with anti-human PD-L1 or anti-mouse PD-L1 antibodies. The proportions of biotin positive Jurkat T cells are shown on the right. (F) Flow cytometry analysis of biotin positive G5-PD-1 Jurkat T cells after incubation with PD-L1-SrtA⁺ exosomes that were pre-treated with IgG isotype, anti-human CD63 antibodies, or anti-human ICAM-1 antibodies. The proportions of biotin positive Jurkat T cells are shown on the right. (G) Flow cytometry analysis of biotin positive G5-PD-1 Jurkat T cells after treatment with PD-L1-SrtA⁺ exosomes that were harvested from ICAM-1 KD or control WM9 cells. The proportions of biotin positive Jurkat T cells are shown on the right. Data represent mean ± s.d. of three independent biological replicates. Statistical analysis is performed using one-way ANOVA analysis with Sidak’s (E, F) or Dunnett’s multiple comparison tests (G).