Tumor-derived exosomes induce PD1+ macrophage population in human gastric cancer that promotes disease progression

Abstract

Macrophages constitute a major component of tumor-infiltrating immune cells. M2 macrophages have been reported to promote tumor progression through promoting tumor angiogenesis and metastasis and regulating T-cell function. Here, we identified a protumorigenic subset of macrophages that constitutively expressed programmed cell death 1 (PD1) and accumulated in advanced-stage gastric cancer (GC). These PD1⁺ tumor-associated macrophages (TAMs) exhibited an M2-like surface profile, with a significant increase in the expression of CD206, IL-10, and CCL1, and a clear decrease in the expression of MHC class II, CD64, and IL-12 and the ability to phagocytose ovalbumin. Moreover, PD1⁺ TAMs can suppress CD8⁺ T-cell function and this immunosuppressive activity can effectively be enhanced upon triggering PD1 signal. GC-derived exosomes effectively educated monocytes to differentiate into PD1⁺ TAMs with M2 phenotypic and functional characteristics. Together, our results are the first to show that GC-derived exosomes can effectively induce PD1⁺ TAM generation, and these cells can produce a large number of IL-10, impair CD8⁺ T-cell function, and thereby create conditions that promote GC progression. Thus, methods in which immunotherapy is combined with targeting PD1⁺ TAMs and tumor-derived exosomes should be used to restore immune function in GC patients.

Fig. 1. PD1 expression on GC-infiltrating macrophages and its clinical significance.

a Flow cytometry gating strategy for GC-infiltrating macrophages. Debris and doublets were removed, and then TAMs were assessed as Hoechst−CD45+CD11b+F4/80+. b, c Analysis of PD1 expression on macrophages from the samples as follows: paired blood, non-tumoral gastric tissue, and tumor tissue from 26 patients. Representative FACS plots of macrophage PD1 expression are shown in (b). Frequencies of PD1⁺ macrophages relative to total macrophage frequencies are shown in (d). d, e Immunofluorescence analysis of the PD1 expression levels (red); and the CD68 expression levels (green) in GC tissue from 15 patients. Representative micrographs are shown in d. The density of tumor-infiltrating PD1⁺ macrophages is shown in (e). Associations of tumor PD1⁺ macrophages with GC patients’ TNM stage are shown in (f, g). f The proportions of PD1⁺ macrophages were determined by flow cytometry. g The number of PD1⁺ macrophages was determined by immunofluorescence staining. h Association of tumor PD1⁺ macrophages with early recurrence. Using 1 year as the cutoff, 20 GC were divided into two groups: no recurrence (n = 12) and recurrence (n = 8). *P < 0.01, **P < 0.001 (Student's t test)

Fig. 2. Phenotypic and functional characteristics of PD1⁺ macrophages in GC tissue.

a–d Flow cytometry analysis was performed to analyze the phenotypic characteristics of PD1⁻, and PD1⁺ macrophages from GC tissues. a Analysis of representative markers expressed by tumor-infiltrating PD1⁻, and PD1⁺ macrophages. b, c Results represent the mean of four independent experiments. e, f Analysis of IL-10, IL-12, and CCL1 expression in PD1⁻, and PD1⁺ macrophages derived from GC tissue by q-PCR (e) and ELISA (f). g, h Phagocytic ability analysis of PD1⁻, and PD1⁺ macrophages derived from GC tissue by flow cytometry. Representative flow cytometry plots are shown in (g). h Results represent the mean of five independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student's t test)

Fig. 3. PD1⁺ macrophages exhibit strong immunosuppressive activity.

a, b CFSE-labeled CD8⁺ T cells were activated by anti-CD3/CD28 beads, and PD1⁻, and PD1⁺ macrophages derived from GC tissue were added at 1:1 ratio. After 3 days, these cells were stained with anti-CD8 Ab, and the proliferation of CD8⁺ T cells in NO TAM (CD8⁺ T cells were cultured alone), PD1⁺ TAM (CD8⁺ T cells were cocultured with PD1⁺ macrophages), and PD1⁻ TAM (CD8⁺ T cells were cocultured with PD1^– macrophages) was analyzed. Representative flow cytometry plots are shown in (a). b Results represent the mean of three independent experiments. c, d Isolated CD8⁺T cells were activated by anti-CD3/CD28 beads, and PD1^–, and PD1⁺ macrophages derived from GC tissue were added at 1:1 ratio. After 3 days, these cells were labeled with anti-CD8 and IFN-γAbs, and the expression of IFN-γ in CD8⁺ T cells in NO TAM, PD1⁺ TAM, and PD1^– TAM was analyzed. Representative flow cytometry plots are shown in c. d Results represent the mean of three independent experiments. e, f Isolated CD8⁺T cells were activated by anti-CD3/CD28 beads, and PD1^–, and PD1⁺ macrophages derived from GC tissue were added at 1:1 ratio. After 3 days, these cells were collected and stained with anti-CD8 and perforin antibody, and the expression of perforin in CD8⁺ T cells in NO TAM, PD1⁺ TAM, and PD1^– TAM was analyzed. Representative flow cytometry plots are shown in (e). f Results represent the mean of three independent experiments. g, l Analysis of suppressive activity by PD1⁺ macrophages left untreated or incubated with 10 μg/ml anti-PD1 antibody, or polyclonal goat IgG. g CD8⁺T-cell proliferation was analyzed, and the data indicate the mean ±  standard error of the mean of three independent experiments. h The expression of Perforin and IFNγ in CD8⁺T cells was analyzed, and the data indicate the mean  ± standard error of the mean of three independent experiments. l the expression of IL10 in PD1⁺ macrophages was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments. m,n analysis of suppressive activity by PD1⁺ macrophages treated with NA-IL10 in the presence of 10 μg/ml anti-PD1antibody, or polyclonal goat IgG. m CD8⁺T-cell proliferation was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments. n the expression of Perforin and IFNγ inCD8⁺T cells was analyzed, and the data indicate the mean ± standard error of the mean of three independent experiments

Fig. 4. PD1⁺ macrophages are negatively correlated with CD8 T cells in GC tissue.

a, b Flow cytometry analysis of PD1 expression on macrophages and the ratio of CD4⁺ or CD8⁺ T cells in CD45⁺ immune cells from the GC tissue samples. a, b Quantification indicating the associations between PD1⁺ macrophages and CD4⁺ or CD8⁺ T cells in GC tissue

Fig. 5. Tumor-derived exosomes promote PD1⁺ macrophages expansion from monocytes in vitro and in vivo.

a Isolated exosomes were characterized by Nano-sight, western blot, and flow cytometry. b–d On day 3, monocytes cultured with or without exosomes were evaluated by flow cytometry to assess the frequency of PD1, and CD206 in monocytes. Representative FACS plots are shown in (c). b, d Results represent the mean of three independent experiments. e–g Analysis of IL-10 (e), IL-12p70 (g), and CCL1 (f) expression in monocytes cultured with or without exosomes by ELISA. Results represent the mean of three independent experiments. h Phagocytic ability analysis of monocytes cultured with or without exosomes by flow cytometry. Results represent the mean of three independent experiments. i–l Suppressive ability analysis of monocytes cultured with or without exosomes by flow cytometry. Results represent the mean of three independent experiments. m On day 3, monocytes cultured in the presence of exosomes that were collected from SGC7901 with or without the exosome release inhibitor spiroepoxide were evaluated by flow cytometry to assess the frequency of PD1. n, o FACS analysis of PD1 expression on macrophages from the samples as follows: non-tumoral tissue and tumor tissue from tumor-bearing mouse. Representative FACS plots of macrophage PD1 expression are shown in (n). Frequencies of PD1⁺ macrophages relative to total macrophage frequencies are shown in (o)