a, The log2-transformed RPPA data showing a higher level of exosomal PD-L1 secreted by metastatic melanoma cell lines compared with primary melanoma cell lines. Data represent mean?±?s.d. of four primary (WM1552C, WM35, WM793, WM902B) or metastatic (UACC-903, 1205Lu, WM9, WM164) melanoma lines. b, Density gradient centrifugation confirming that PD-L1 secreted by WM9 cells co-fractionated with exosome markers CD63, Hrs, Alix and TSG101. c, Immunoblots for PD-L1 in the whole cell lysate (W), purified exosomes (E) or microvesicles (M) from different metastatic melanoma cell lines. The same amount of protein was loaded in each lane. d, Levels of PD-L1 on the exosomes or microvesicles derived from melanoma cells as assayed by ELISA. e, The levels of exosomal PD-L1 and microvesicle PD-L1 produced by an equal number of melanoma cells. f, Immunoblots for PD-L1 in the whole cell lysate, purified exosomes or microvesicles from mouse melanoma B16-F10 cells. The same amount of protein was loaded in each lane. g, h, Western blot analysis of PD-L1 in Hrs knockdown cells without (g) or with (h) IFN-γ treatment. Quantification of the western blotting data (g, right; h, right). i, Co-immunoprecipitation of PD-L1 and Hrs from MEL624 cells expressing exogenous PD-L1 and Hrs. j, Immunofluorescence staining of intracellular PD-L1 and exosome marker Hrs in WM9 cells treated with IFN-γ. k, Immunofluorescence staining of intracellular PD-L1 and CD63 in WM9 cells treated with IFN-γ. l, western blotting analysis showing intracellular accumulation of PD-L1, and decreased exosomal secretion of PD-L1 in WM9 cells with RAB27A knockdown (left). The levels of exosomal PD-L1 were compared (right). Two experiments were repeated independently with similar results (b, c, f, i–k). Data represent mean?±?s.d. of four (d, e), or three (g, h, l) independent biological replicates. Statistical analysis is performed by two-sided unpaired t-test (a, d, e, g, h, l). For gel source data (b, c, f–i, l), see Supplementary Fig. 1. Source Data
a, The monoclonal antibodies against the extracellular domain of human PD-L1 specifically detect human exosomal PD-L1, but not mouse exosomal PD-L1 (n?=?3 biologically independent experiments). b, Levels of human PD-L1 in exosomes from the plasma of control nude mice (n?=?10) and human WM9 melanoma xenograft-bearing nude mice (n?=?10) per?mg of total circulating exosomal proteins. c, Characterization of circulating exosomes purified from the plasma of a patient with Stage IV melanoma using NanoSight nanoparticle tracking analysis. d, Characterization of circulating microvesicles purified from the plasma sample of a patient with Stage IV melanoma using NanoSight nanoparticle tracking analysis. e, Immunoblots for PD-L1 in the microvesicles purified from the plasma samples of 8 patients with Stage IV melanoma (denoted as P1–P8). f, Immunoblots for PD-L1 in the exosomes purified from the plasma samples of 5 healthy donors and 5 patients with stage IV melanoma (left panel). Quantification of the levels of exosomal PD-L1 by western blot analysis (right panel). Results are expressed as the percentage of the mean value of healthy donors. g, Standard density gradient centrifugation analysis showing that circulating PD-L1 co-fractionated with exosome markers Hrs and TSG101 and melanoma-specific marker TYRP-2. Three (c, d) or two (e, g) experiments were repeated independently with similar results. Data represent mean?±?s.d. (a, b, f). Statistical analyses were performed using two-sided unpaired t-test (b, f). For gel source data (e–g), see Supplementary Fig. 1. Source Data
a, ELISA showing the level of PD-L1 on circulating exosomes purified from healthy donors (HD, n?=?11) and melanoma patients (MP, n?=?44). The exosomes were purified using differential centrifugation. b, Pearson correlation between the ELISA-detected levels of PD-L1 on circulating exosomes purified by differential centrifugation or using the commercial exosome isolation kit (n?=?44). c, Comparison of the number of circulating exosomes between healthy donors (n?=?10) and melanoma patients (n?=?38). d, Comparison of the protein content of circulating exosomes between healthy donors (n?=?10) and melanoma patients (n?=?38). e, ELISA of the circulating level of microvesicle PD-L1 in healthy donors (HD, n?=?11) and melanoma patients (MP, n?=?44). f, Detailed data associated with the ROC curve analysis depicted in Fig. 2g. Data represent mean?±?s.d. Statistical analyses are performed by two-sided unpaired t-test (a, c-e). Source Data
a, Representative contour plots showing the general gating strategy used to identify the purified CD8 T cells (CD3+CD8+CD4?) from human peripheral blood. b, Confocal microscopy analysis of human peripheral CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after incubation with CFSE-labelled WM9 cell-derived exosomes for 2 h. The experiments were repeated three times independently with similar results. c, Representative histogram of human peripheral CD8 T cells with or without anti-CD3/CD28 antibody stimulation after incubation with CFSE-labelled WM9 cell-derived exosomes for 2 h (left). The proportion of exosome-bound cells is shown (right). d, Representative histogram of human peripheral CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after incubation with the same number of CFSE-labelled exosomes purified from control or IFN-γ-treated WM9 cells for 2 h (left panel). The proportion of EXO-bound cells is shown in the right panel. Data represent mean?±?s.d. of four (c) or three (d) independent biological replicates. Statistical analyses are performed using two-sided unpaired t-test (c, d). Source Data
a, The log2-transformed RPPA data showing the levels of PD-L1 in the exosomes secreted by control (MEL624) or PD-L1-expressing (PD-L1/MEL624) human melanoma MEL624 cells (Bottom). b, Immunoblots for PD-L1 in the whole cell lysate (W) or in the purified exosomes (E) from MEL624 or PD-L1/MEL624 cells. The same amount of protein was loaded in each lane. The experiments were repeated two times independently with similar results. For source data, see Supplementary Fig. 1. c, PD-L1 on the surface of exosomes secreted by MEL624 or PD-L1/MEL624 cells as determined by ELISA. d, Levels of PD-L1 on exosomes secreted by MEL624 or PD-L1/MEL624 cells, as measured by ELISA. e, qPCR analyses of IL-2, IFN-γ, and TNF in human peripheral CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after treatment with MEL624 cell-derived exosomes, PD-L1/MEL624 cell-derived exosomes or WM9-cell-derived exosomes with or without blocking by IgG isotype or the anti-PD-L1 antibodies. The relative mRNA expression level was calculated as the ratio to the control cells. f, ELISA of IL-2, IFN-γ, and TNF in human peripheral CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after treatment with MEL624 cell-derived exosomes, PD-L1/MEL624 cell-derived exosomes or WM9-cell-derived exosomes with or without blocking by IgG isotype or PD-L1 antibodies. g, Representative histogram of CFSE-labelled human peripheral CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after treatment with WM9 cell-derived exosomes with or without antibody blocking (left). The proportion of cells with diluted CFSE dye is shown (right). h, Representative contour plots of human peripheral CD8 T cells (stimulated with anti-CD3/CD28 antibodies) examined for the expression of granzyme B (GzmB) after treatment with WM9 cell-derived exosomes with or without antibody blocking (left). The percentage of GzmB+ CD8 T cells stimulated with anti-CD3/CD28 antibodies is shown at the right panel. Data represent mean?±?s.d. of three (a, c, e, f, h) or four (d, g) independent biological replicates. Statistical analyses are performed using two-sided unpaired t-test (d–h). Source Data
a, Representative contour plots showing the general gating strategy used to identify the purified CD8 T cells (CD3+CD8+CD4?) from mouse splenocytes. b, Representative histogram of CFSE-labelled mouse splenic CD8 T cells (stimulated with anti-CD3/CD28 antibodies) after treatment with B16-F10 cell-derived exosomes with or without blocking by IgG isotype or the anti-PD-L1 antibodies (left). The proportion of cells with diluted CFSE dye is shown at the right panel. c, Representative contour plots of mouse splenic CD8 T cells (stimulated with anti-CD3/CD28 antibodies) examined for the expression of Ki-67 and granzyme B (GzmB) after treatment with B16-F10 cell-derived exosomes with or without blocking by IgG isotype or the anti-PD-L1 antibodies (left). The percentage of Ki-67+GzmB+ CD8 T cells stimulated with anti-CD3/CD28 antibodies is shown (right). d, Representative contour plots of mouse splenic CD8 T cells (stimulated with anti-CD3/CD28 antibodies) examined for the expression of Ki-67 and GzmB after treatment with B16-F10 cell-derived exosomes in the presence or absence of anti-PD-1 blocking antibodies (left). The percentage of Ki-67+GzmB+ CD8 T cells stimulated with anti-CD3/CD28 antibodies is shown at the right panel. e, OT-I CD8 T cell-meditated tumour cell killing assay was performed in B16-OVA cells with PD-L1 knockdown, or B16-F10 cells with PD-L1 knockdown (negative control). Apoptosis of tumour cells was evaluated by flow cytometric analysis of intracellular cleaved caspase-3 (left), and the relative cytotoxicity was calculated (right). f, OT-I CD8 T cells, activated by OVA-pulsed bone marrow-derived dendritic cells and treated with PBS (as a control), exosomes derived from B16-F10 cells with or without IgG isotype or PD-L1 antibody blocking, were co-cultured with PD-L1 knockdown B16-OVA cells for 48 h. Tumour cell apoptosis was evaluated by flow cytometric analysis of intracellular cleaved caspase-3 (left), and the relative cytotoxicity was calculated (right). Data represent mean?±?s.d. of three (b–f) independent biological replicates. Statistical analyses are performed using two-sided unpaired t-test (b–f). Source Data
a, Immunoblots for PD-L1 in the whole cell lysate (W), purified exosomes (E) or microvesicles (M) from different lung cancer cell lines. The same amounts of proteins were loaded for each fraction. b, Immunoblots for PD-L1 in the whole cell lysate, purified exosomes or microvesicles from the breast cancer cell line MDA-MB-231. The same amount of protein was loaded for each fraction. c, Immunoblots for PD-L1 in the whole cell lysate (WCL) or in the purified exosomes (EXO) from control (C) or IFN-γ-treated (IFN) lung cancer cells. The same amounts of exosome proteins from IFN-γ-treated and control cells were loaded (left). Quantification of the exosomal PD-L1 level determined by western blot analysis (right). d, Immunoblots for PD-L1 in the whole cell lysate or in the purified exosomes from control or IFN-γ-treated the breast cancer MDA-MB-231 cells. The same amounts of exosome proteins from IFN-γ-treated and control cells were loaded (left). Quantification of the exosomal PD-L1 level determined by western blot analysis (right). e, Representative contour plots of human peripheral CD8 T cells examined for the expression of Ki-67 and GzmB after treatment with H1264 cell-derived exosomes with or without blocking by IgG isotype or PD-L1 antibodies (left). The percentage of Ki-67+ or GzmB+ CD8 T cells is shown (right). The experiments were repeated twice independently with similar results (a, b). Data represent mean?±?s.d. of three (c–e) independent biological replicates. Statistical analyses are performed using two-sided unpaired t-test (c–e). For source data (a–d), see Supplementary Fig. 1. Source Data
a, Representative flow cytometric histograms of B16-F10 cells examined for the expression of PD-L1 with or without PD-L1 knockdown. B16-F10 cells were stably depleted of PD-L1 using lentiviral shRNA against PD-L1 (shPD-L1) or the scrambled control shRNA (shCTL). The experiment was repeated twice independently with similar results. b, Representative images showing the growth of PD-L1 knockdown B16-F10 tumours in C57BL/6 mice after indicated treatments. Experiments were performed using 7 mice for each group. c, The weights of PD-L1 knockdown B16-F10 tumours from C57BL/6 mice with indicated treatments (n?=?7 mice per group). Data represent mean?±?s.d. d, Representative contour plot of CD8 TILs or splenic or lymph node CD8 T cells examined for the expression of Ki-67 after indicated treatments. Experiments were performed using 7 mice for each group. See Fig. 3c for quantification data. e, Representative immunofluorescence images of CD8 TILs in tumour tissues (left). The number of CD8 TILs for each mouse (n?=?7 mice per group) were quantified from 5 high-power fields (HPF) (right). Statistical analysis is performed using two-sided unpaired t-test (c, e). Source Data
a, The levels of PD-L1 on circulating microvesicles at serial time points pre- and on-treatment (n?=?39). b, The frequency of PD-1+ Ki-67+ CD8 T cells and the level of circulating exosomal PD-L1 in clinical responders at serial time points pre- and on-treatment (n?=?8). c, Pearson correlation of the maximum level of circulating exosomal PD-L1 at week 3–6 to the maximum frequency of PD-1+Ki-67+ CD8 T cells at week 3–6 in clinical responders (n?=?8) and non-responders (n?=?11). d, Pearson correlation of the maximum fold change of circulating exosomal PD-L1 level at week 3–6 to the maximum fold change of PD-1+Ki-67+ CD8 T cells at week 3–6 in clinical responders (n?=?8) and non-responders (n?=?11). e, Kaplan–Meier progression-free and overall survival of patients with high (n?=?11) and low (n?=?12) fold changes of circulating exosomal PD-L1 at 3–6 weeks. f, Comparison of the maximum fold change of total circulating PD-L1 at week 3–6 between the clinical responders and non-responders. R, responders, n?=?19; NR, non-responders, n?=?20. g, Comparison of the maximum fold change of circulating microvesicle PD-L1 at week 3–6 between the clinical responders (n?=?19) and non-responders (n?=?20). h, Comparison of the maximum fold change of extracellular-excluded PD-L1 at week 3–6 between the clinical responders (n?=?19) and non-responders (n?=?20). Data represent mean?±?s.d. Statistical analyses were performed using two-sided paired t-test (a), log-rank test (e), or two-sided unpaired t-test (f–h). Source Data
a, Tracking the levels of circulating exosomal PD-L1 before and during anti-PD-1 treatment may stratify responders to anti-PD-1 therapy (green) from non-responders (red) as early as 3–6 weeks into the treatment. b, Potential application of circulating exosomal PD-L1 to predict patients’ response to anti-PD-1 therapy. The pre-treatment level of circulating exosomal PD-L1 is lower in metastatic melanoma patients with clinical response to anti-PD-1 therapy. After 3–6 weeks of anti-PD-1 treatment, the level of circulating exosomal PD-L1 increases significantly in clinical responders but not in non-responders. c, Tracking both the pre-treatment and on-treatment levels of circulating exosomal PD-L1 may help identify the possible reasons for success (green) or failure (red) of the therapy. Source Data
