Abstract
BackgroundPrevious evidence demonstrated that restoration of wild type VHL in human renal cancer cells decreased in vitro NK susceptibility. To investigate on the role of tumoral VHL status versus NK capability in renal cancer patients, 51 RCC patients were characterized for VHL mutational status and NK function.MethodsVHL mutational status was determined by direct DNA sequencing on tumor tissue. NK cytotoxicity was measured against specific target cells K562, VHL-wild type (CAKI-1) and VHL-mutated (A498) human renal cancer cells through externalization of CD107a and IFN-γ production. Activating NK receptors, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NCAM-1 and FcγRIIIa were evaluated through quantitative RT-PCR. RCC tumoral Tregs were characterized as CD4+CD25+CD127lowFoxp3+ and Treg function was evaluated as inhibition of T-effector proliferation.ResultsVHL mutations were detected in 26/55 (47%) RCC patients. IL-2 activated whole-blood samples (28 VHL-WT-RCC and 23 VHL-MUT-RCC) were evaluated for NK cytotoxicity toward human renal cancer cells A498, VHL-MUT and CAKI-1, VHL-WT. Efficient NK degranulation and increase in IFN-γ production was detected when IL-2 activated whole-blood from VHL-MUT-RCC patients were tested toward A498 as compared to CAKI-1 cells (CD107a+NK: 7 ± 2% vs 1 ± 0.41%, p = 0.015; IFN-γ+NK: 6.26 ± 3.4% vs 1.78 ± 0.9% respectively). In addition, IL-2 activated NKs induced higher CD107a exposure in the presence of RCC autologous tumor cells or A498 as compared to SN12C (average CD107a+NK: 4.7 and 2.7% vs 0.3% respectively at 10E:1 T ratio).VHL-MUT-RCC tumors were NKp46+ cells infiltrated and expressed high NKp30 and NKp46 receptors as compared to VHL-WT-RCC tumors. A significant lower number of Tregs was detected in the tumor microenvironment of 13 VHL-MUT-RCC as compared to 13 VHL-WT-RCC tumors (1.84 ± 0.36% vs 3.79 ± 0.74% respectively, p = 0.04). Tregs isolated from VHL-MUT-RCC patients were less suppressive of patients T effector proliferation compared to Tregs from VHL-WT-RCC patients (Teff proliferation: 6.7 ± 3.9% vs 2.8 ± 1.1%).ConclusionsVHL tumoral mutations improve NKs effectiveness in RCC patients and need to be considered in the evaluation of immune response. Moreover therapeutic strategies designed to target NK cells could be beneficial in VHL-mutated-RCCs alone or in association with immune checkpoints inhibitors.
Highlights
Previous evidence demonstrated that restoration of wild type Von Hippel-Lindau (VHL) in human renal cancer cells decreased in vitro Natural killer cells (NKs) susceptibility
VHL mutated (VHL-MUT)-Renal cell carcinomas (RCCs) patients whole blood cytotoxicity was more efficient toward A498-VHL-MUT than CAKI-1-VHLWT cells interleukin 2 (IL-2) activated whole-blood-NK derived cytotoxicity was evaluated in 51 available RCC patients (28 VHL wild-type (VHL-WT) and 23 VHL-MUT) and 12 healthy donors (HD)
IL-2 activated whole-blood samples were co-cultured with target cells K562, A498-VHL-MUT [26] and CAKI-1-VHL-WT cell lines
Summary
Previous evidence demonstrated that restoration of wild type VHL in human renal cancer cells decreased in vitro NK susceptibility. To investigate on the role of tumoral VHL status versus NK capability in renal cancer patients, 51 RCC patients were characterized for VHL mutational status and NK function. Renal cell carcinoma (RCC) is the most common kidney tumor in adults. Certain VHL mutations correlate with low expression of classical HLA-I molecules and HLA-E expression that determine higher NK susceptibility reducing the engagement of KIR and NKG2A inhibitory receptors, respectively. Multiple mechanisms concur in determining the NK sensitivity of cancer cells such as NK receptors surface ligands, LFA-1/ICAM-1 and HLA-I/NKR interactions, HLA-E molecules, soluble MICA A-B ligands, HLA-G, TGF beta secretion [15]. Mechanisms affecting NK sensitivity may regulate gene transcription such as autophagy genes [16]
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