Negative regulation of tumor-infiltrating NK cell in clear cell renal cell carcinoma patients through the exosomal pathway

Abstract

Natural killer cells are the key components in tumor immunity and defects in function are necessary for tumor immune escape. Emerging studies on tumor cell-derived exosomes have shown the biological significance in tumor microenvironment, but the underlying role of exosomes in regulating natural killer cells functions in clear cell renal cell carcinoma patients remains unknown. Firstly, we precisely characterized the phenotype and function of natural killer cells in clear cell renal cell carcinoma patients vs healthy controls. With an inhibitory phenotype, tumor-infiltrating natural killer cells exhibited poor cytotoxic capacity and deficient potential to produce cytokines compared with natural killer cells from tumor margin tissue and non-tumor tissue. Next, we revealed that primary tumor cells trigged natural killer cell dysfunction in an exosome-dependent manner. Interestingly, exosomes from primary tumor cells were preferentially enriched with TGF-β1 which acted as important mediator of natural killer cell functional deficiency. In vitro culture of exosomes induced natural killer cell dysfunction mediated by activation of the TGF-β/SMAD signaling pathway, and abrogated by knockdown TGF-β. Our data indicate that exosomes from clear cell renal cell carcinoma induce natural killer cells dysfunction by regulating the TGF-β/SMAD pathway to evade innate immune surveillance.

Keywords: clear cell renal cell carcinoma; exosome; natural killer; transforming growth factor-β1; tumor immune evasion.

Conflict of interest statement

CONFLICTS OF INTEREST

All authors declare that they have no competing interests.

Figures

Figure 1. TINK cells in RCC display an abnormal phenotype

(A) Lymphocyte gate of freshly purified mononuclear cells (MNCs) and NK cells. (B) Percentage of NK cells from tumor tissue (T, n = 36), tumor margin tissue (M, n = 36), and non-tumor tissue (NT, n = 16, mean±sd). (C) Percentage of NK cells in PBMCs from ccRCC patients (ccRCC, n = 36) and healthy individuals (HC, n = 36, mean±sd). (D) The percentage of NK-cell activation receptors NKp46, NKG2D, NKG2C and inhibitory receptors NKG2A, CD158a, CD158b expressed on NK cells from tumor tissue (T, n = 36), tumor margin tissue (M, n = 36), and non-tumor tissue (NT, n = 16, mean±sd). (E) Pooled percentage of NK receptors expressed on NK cells from tumor tissue, tumor margin tissue, and non-tumor tissue (T, n = 36; M, n = 36; NT, n = 16, mean±sd), *p < 0.05, **p < 0.01 and ***p < 0.001vs corresponding control.

Figure 2. TINK cells in RCC exhibit a functional deficiency and are associated with tumor progression

(A) The percentage of CD107a, IFN-γ and TNF-α expressed on NK cells from tumor tissue (T, n = 36), tumor margin tissue (M, n = 36), and non-tumor tissue (NT, n = 16, mean±sd). (B) The cytolytic activity of NK cells from different tissues cocultured with K562. (C) NK functional markers (CD107a, IFN-γ and TNF-α) expressed on TINK cells in patients with tumor diameters ≥7 cm (n = 13) VS patients with tumor diameters < 7 cm (n =23). (D) The percentage of CD107a, IFN-γ and TNF-α expressed on TINK cells decreases as tumors progress (n = 36). *p < 0.05, **p < 0.01 and ***p < 0.001vs corresponding control.

Figure 3. NK cell function was impaired via exosome

(A) Transwell coculture model was used for the co-culture of primary NK cells and ccRCC cells. (B) Representative micrographs of immunofluorescence staining of cytokeratin and CAIX. DAPI counterstains nuclei in blue. 200× magnification. (C, D) NK cell functions (CD107a degranulation, IFN-γ and TNF-α production) in NT, M, T+GW4869, T+transwell and T groups. (E) Cytotoxicity of NK cells coculture with NT, M, T+GW4869, T+transwell, T and anti-TGFβ1 groups. All data shown were from three independent experiments. Error bars indicate ±SD, *p < 0.05, **p < 0.01 and ***p < 0.001vs corresponding control.

Figure 4. Exosome characterization

(A) Transmission Electron Microscopy (TEM). (B) Western Blot analysis for exosomal marker in exosomes and cell lysate samples. (C) The quantification and size distribution analysis of exosomes. Size distribution of exosomes derived from tumor and non-tumor tissues were measured by qNano system. Bar Chart showing the mean diameter (D) and concentration for exosomes from NT and T (E). Statistical analysis of the correlations between exosomal protein concentrations and clinical parameters, including tumor size (F) and TNM stage (G). All data shown were from three independent experiments. Error bars indicate ±SD, *p < 0.05, **p < 0.01 and ***p < 0.001vs corresponding control.

Figure 5. Texs treatment suppresses the cytolytic activity of NK cells in vitro

(A) Cytotoxicity of NK cells coculture with Texs at different concentrations. (B, C) The level of CD107a, IFN-γ and TNF-α production of NK cells in coculture with Texs from different stages. (D) Cytotoxicity of NK cells coculture with Texs from different stages. All data shown were from three independent experiments. Error bars indicate ±SD, *p < 0.05, **p < 0.01 and ***p < 0.001vs corresponding control.

Figure 6. Texs interacted with NK cells through TGFβ1/Smad signaling

(A) TGFβ1 were measured by ELISA in NT-exos, T-exos, M-exos, 786-O exos and HK-2 exos. High expressions of TGFβ1 protein were found in T-exos and 786-O. (B) Western blot of TGF-β1 protein. (C) Exos and NK cells were cocultured together to access the cytotoxicity of NK cells. Exos could impair the cytotoxicity of NK cell, and TGF-β1 knockdown could reverse it. (D) Knockdown of TGF-β1 could inhibit the smad signaling expression. All data shown were from three independent experiments. Error bars indicate ±SD, *p < 0.05, **p < 0.01 and ***p < 0.001vs corresponding control.

Figure 7. A schematic illustration showed that clear cell renal carcinoma cell-derived exosomes could induce NK cells dysfunction by activating Smad signaling through exosomal surface TGF-β1