HIF-1 stabilization in T cells hampers the control of Mycobacterium tuberculosis infection.

The hypoxia-inducible factors (HIFs) regulate the main transcriptional pathway of response to hypoxia in T cells and are negatively regulated by von Hippel-Lindau factor (VHL). But the role of HIFs in the regulation of CD4 T cell responses during infection with M. tuberculosis isn't well understood. Here we show that mice lacking VHL in T cells (Vhl cKO) are highly susceptible to infection with M. tuberculosis, which is associated with a low accumulation of mycobacteria-specific T cells in the lungs that display reduced proliferation, altered differentiation and enhanced expression of inhibitory receptors. In contrast, HIF-1 deficiency in T cells is redundant for M. tuberculosis control. Vhl cKO mice also show reduced responses to vaccination. Further, VHL promotes proper MYC-activation, cell-growth responses, DNA synthesis, proliferation and survival of CD4 T cells after TCR activation. The VHL-deficient T cell responses are rescued by the loss of HIF-1α, indicating that the increased susceptibility to M. tuberculosis infection and the impaired responses of Vhl-deficient T cells are HIF-1-dependent.

Hypoxia-inducible factor activity promotes antitumor effector function and tissue residency by CD8+ T cells.

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8+ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8+ T cells induced HIF-1α/HIF-2α-dependent differentiation of tissue-resident memory-like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8+ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.

Mutated Von Hippel-Lindau-renal cell carcinoma (RCC) promotes patients specific natural killer (NK) cytotoxicity.

BACKGROUND: 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. METHODS: VHL 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. RESULTS: VHL 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%). CONCLUSIONS: VHL 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.

The E3 ligase VHL controls alveolar macrophage function via metabolic-epigenetic regulation.

Metabolic pathways such as glycolysis or oxidative phosphorylation play a key role in regulating macrophage function during inflammation and tissue repair. However, how exactly the VHL-HIF-glycolysis axis is involved in the function of tissue-resident macrophages remains unclear. Here we demonstrate that loss of VHL in myeloid cells resulted in attenuated pulmonary type 2 and fibrotic responses, accompanied by reduced eosinophil infiltration, decreased IL-5 and IL-13 concentrations, and ameliorated fiber deposition upon challenge. VHL deficiency uplifted glycolytic metabolism, decreased respiratory capacity, and reduced osteopontin expression in alveolar macrophages, which impaired the function of type 2 innate lymphoid cells but was significantly reversed by HIF1α inhibition or ablation. The up-regulated glycolysis altered the epigenetic modification of osteopontin gene, with the metabolic intermediate 3-phosphoglyceric acid as a key checkpoint controller. Thus, our results indicate that VHL acts as a crucial regulatory factor in lung inflammation and fibrosis by regulating alveolar macrophages.

Molecular characterization and gene expression analysis of hypoxia-inducible factor and its inhibitory factors in kuruma shrimp Marsupenaeus japonicus.

In shrimp aquaculture, overcrowded farming causes fluctuations in dissolved oxygen concentrations. Low-oxygen conditions (hypoxia) affect shrimp growth. Hypoxia-inducible factor (HIF) is a transcriptional factor in the basic helix-loop-helix/PAS family and is activated in response to hypoxic stress. However, little is known about HIF and other inhibitors of the HIF pathway in crustaceans. In this study, we cloned MjHIF-1α, an inhibitory factor, MjFIH-1 (factor inhibiting HIF-1α), and MjVHL (Von Hippel-Lindau tumor suppressor) from kuruma shrimp (Marsupenaeus japonicus). MjVHL is the first crustacean VHL ortholog to be cloned. MjHIF-1α, MjFIH-1, and MjVHL exhibit significant sequence similarity and share key functional domains with previously described vertebrate and invertebrate genes. As a result of gene expression analysis in various tissues, MjHIF-1α and MjVHL were more highly expressed in the intestine than in any other organ tissues. In hypoxia experiments, HIF-induced expression levels of MjHIF-1α in the hypoxic group increased significantly for 24 h after initiating hypoxia stimulation and expression of MjVHL decreased significantly for 6 h after hypoxia stimulation (P < 0.05).

Hypoxia and hypoxia-inducible factor (HIF) downregulate antigen-presenting MHC class I molecules limiting tumor cell recognition by T cells.

Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME in vivo and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures in vitro; (ii) decreased MHC in human renal cell carcinomas with constitutive expression of HIF due to genetic loss of von Hippel-Lindau (VHL) function as compared with isogenically paired cells with restored VHL function, and iii) increased MHC in tumor cells with siRNA-mediated knockdown of HIF. In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME.

VHL promotes immune response against renal cell carcinoma via NF-κB-dependent regulation of VCAM-1.

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4ß1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression.

Hypoxia-inducible factors enhance the effector responses of CD8(+) T cells to persistent antigen.

Cytolytic activity by CD8(+) cytotoxic T lymphocytes (CTLs) is a powerful strategy for the elimination of intracellular pathogens and tumor cells. The destructive capacity of CTLs is progressively dampened during chronic infection, yet the environmental cues and molecular pathways that influence immunological 'exhaustion' remain unclear. Here we found that CTL immunity was regulated by the central transcriptional response to hypoxia, which is controlled in part by hypoxia-inducible factors (HIFs) and the von Hippel-Lindau tumor suppressor VHL. Loss of VHL, the main negative regulator of HIFs, led to lethal CTL-mediated immunopathology during chronic infection, and VHL-deficient CTLs displayed enhanced control of persistent viral infection and neoplastic growth. We found that HIFs and oxygen influenced the expression of pivotal transcription, effector and costimulatory-inhibitory molecules of CTLs, which was relevant to strategies that promote the clearance of viruses and tumors.

Inhibiting autophagy: a novel approach for the treatment of renal cell carcinoma.

The common clear cell subtype of renal cell carcinoma is associated with hereditary or acquired loss of function of the von Hippel-Lindau tumor suppressor, a key component in oxygen sensing, perpetuating a stressed state. Autophagy is primarily a highly conserved, catabolic process by which stressed cells shuttle damaged or effete organelles and proteins into autophagosomes for sequestration and digestion after fusion with lysosomes. Autophagy is directed by autophagy-related genes and is divided into 4 discrete steps: initiation, nucleation, maturation, and degradation. During early tumorigenesis, apoptosis is enhanced and autophagy is suppressed, allowing accumulation of mutations and emergence of genomic instability. Late, an "autophagic switch" occurs, promoting survival and limiting apoptosis. Compounds such as chloroquine and hydroxychloroquine that prevent acidification of the lysosomal compartment are the sole clinically available inhibitors of autophagy. Currently, there are more than 30 trials examining combinations of hydroxychloroquine with anticancer agents. The intricate effects of autophagy on the immune response complicate manipulation of autophagy as part of the antitumor strategy. Further understanding of basic mechanisms of renal cell carcinoma pathogenesis and of autophagy will enable development of the next generation of pharmacologic modulators of autophagy.

Expression of VHL and HIF-1α and Their Clinicopathologic Significance in Benign and Malignant Lesions of the Gallbladder.

BACKGROUND: The Von Hippel-Lindau (VHL) gene is a tumor-suppressor gene. Recent studies have shown that low expression of VHL has a close relationship with tumor formation, progression, and prognosis. Hypoxia-inducible factor (HIF)-1α is a transcription factor, which exists universally in mammals including humans under states of hypoxia. Some studies have also shown that expression of HIF-1α closely correlates with tumor progression, angiogenesis, metastasis, and invasion. This molecule is also an important cytokine, which may be used to evaluate the prognosis of some malignant tumors. In this study, we studied the expression of VHL and HIF-1α and evaluated their clinicopathologic significance and relationship in benign and malignant lesions of the gallbladder. METHODS: EnVision immunohistochemistry was used for detecting the expression level of VHL and HIF-1α in routinely paraffin-embedded sections from specimens of gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyps (n=15), and chronic cholecystitis (n=35). RESULTS: The frequency of positive VHL expression was significantly lower in adenocarcinoma of gallbladder (48.1%) than that in peritumoral tissues (80.4%), adenomatous polyps (80.0%), and chronic cholecystitis (88.6%) (P<0.05 or P<0.01). In contrast, the positive HIF-1α expression was significantly higher in adenocarcinoma (53.7%) than that in peritumoral tissues (34.8%), adenomatous polyps (26.7%), and chronic cholecystitis (14.3%) (P<0.05 or P<0.01). The benign lesions with positive VHL and/or negative HIF-1α expression showed atypical hyperplasia in gallbladder epithelium. The positive expression of VHL and negative expression of HIF-1α were significantly associated with differentiation, tumor mass, lymph node metastasis, and invasion of adenocarcinoma (P<0.05 or P<0.01). The highly inconsistent expression of VHL and HIF-1α in gallbladder adenocarcinoma was found (P<0.01). Univariate Kaplan-Meier analysis showed that elevated expression of VHL (P=0.023) or lowered expression of HIF-1α (P=0.020) was closely associated with decreased overall survival. Multivariate Cox regression analysis showed that positive expression of VHL (P=0.013) and/or negative expression of HIF-1α (P=0.005) was an independent poor-prognostic predictor in gallbladder adenocarcinoma. CONCLUSIONS: The lowered expression of HIF-1α and elevated expression of VHL in gallbladder adenocarcinoma are important markers for the progression, clinical biological behavior, and prognosis. Measurement of VHL and HIF-1α expression could be a tool for early detection of gallbladder cancer in benign lesions and in population screening. The highly inconsistent expression of VHL and HIF-1α in gallbladder may require further study to see whether they are intrinsically related.

A pilot clinical trial testing mutant von Hippel-Lindau peptide as a novel immune therapy in metastatic renal cell carcinoma.

BACKGROUND: Due to the lack of specific tumor antigens, the majority of tested cancer vaccines for renal cell carcinoma (RCC) are based on tumor cell lysate. The identification of the von Hippel-Lindau (VHL) gene mutations in RCC patients provided the potential for developing a novel targeted vaccine for RCC. In this pilot study, we tested the feasibility of vaccinating advanced RCC patients with the corresponding mutant VHL peptides. METHODS: Six patients with advanced RCC and mutated VHL genes were vaccinated with the relevant VHL peptides. Patients were injected with the peptide mixed with Montanide subcutaneously (SQ) every 4 weeks until disease progression or until the utilization of all available peptide stock. RESULTS: Four out of five evaluable patients (80%) generated specific immune responses against the corresponding mutant VHL peptides. The vaccine was well tolerated. No grade III or IV toxicities occurred. The median overall survival (OS) and median progression-free survival (PFS) were 30.5 and 6.5 months, respectively. CONCLUSIONS: The vaccine demonstrated safety and proved efficacy in generating specific immune response to the mutant VHL peptide. Despite the fact that the preparation of these custom-made vaccines is time consuming, the utilization of VHL as a vaccine target presents a promising approach because of the lack of other specific targets for RCC. Accordingly, developing mutant VHL peptides as vaccines for RCC warrants further investigation in larger trials. TRIAL REGISTRATION: 98C0139.

Sunitinib inhibition of Stat3 induces renal cell carcinoma tumor cell apoptosis and reduces immunosuppressive cells.

The novel multitargeted tyrosine kinase inhibitor sunitinib is used as an antiangiogenic agent for the treatment of several types of cancer, including metastatic renal cell carcinoma (RCC). Sunitinib was shown to positively change the immunosuppressive phenotype in RCC patients. To improve its antitumor efficacy, and offer strategies for its combination with other approaches, it is critical to fully elucidate its mechanisms of action. We show that sunitinib induces tumor cell apoptosis and growth arrest in RCC tumor cells, which correlates with signal transducer and activator of transcription 3 (Stat3) activity inhibition. Sunitinib-mediated direct effects on tumor cells occur regardless of von Hippel-Lindau tumor suppressor gene status and hypoxia-inducible transcription factor-2alpha levels. Reduction of Stat3 activity enhances the antitumor effects of sunitinib, whereas expression of a constitutively activated Stat3 mutant rescues tumor cell death. Intravital multiphoton microscopy data show that sunitinib induces mouse Renca tumor cell apoptosis in vivo before tumor vasculature collapse. Sunitinib also inhibits Stat3 in Renca tumor-associated myeloid-derived suppressor cells (MDSC), down-regulates angiogenic gene expression, and reduces MDSCs and tumor T regulatory cells. These results suggest that Stat3 activity is important for RCC response to sunitinib, and Stat3 inhibition permits the direct proapoptotic activity of sunitinib on tumor cells and positive effects on tumor immunologic microenvironment.

TGF-alpha as a candidate tumor antigen for renal cell carcinomas.

OBJECTIVES: Patients with renal cell carcinomas (RCC) have few treatment options, underscoring the importance of developing new approaches such as immunotherapy. However, few tumor associated antigens (TAA), which can be targeted by immunotherapy, have been identified for this type of cancer. von Hippel-Lindau clear cell RCC (VHL(-/-)RCC) are characterized by mutations in the VHL tumor suppressor gene. Loss of VHL function causes the overexpression of transforming growth factor (TGF)-alpha, leading us to hypothesize that TGF-alpha could be a potential TAA for immunotherapy of kidney cancer, which was evaluated in this study. METHODS AND RESULTS: We first confirmed the absent or weak expression of TGF-alpha in important normal tissues as well as its overexpression in 61% of renal tumors in comparison to autologous normal kidney tissues. In addition, we demonstrated the immunogenicity of TGF-alpha, by expanding many T cell lines specific for certain TGF-alpha peptides or the mature TGF-alpha protein, when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. Interestingly, some of these TGF-alpha-specific T cells were polyfunctionals and secreted IFN-gamma, TNF-alpha and IL-2. CONCLUSION: We have shown that TGF-alpha is a valid candidate TAA, which should allow the development of a targeted immunotherapy.