STUB1 is an intracellular checkpoint for interferon gamma sensing.

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models-with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies.

Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1(125-133), LMP2A(426-434) or EBNA1(562-570) in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1(562-570) in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.

Identification of a subpopulation of nasopharyngeal carcinoma cells with cancer stem-like cell properties by high aldehyde dehydrogenase activity.

OBJECTIVES/HYPOTHESIS: Cancer stem cells have been reported as a new therapeutic target in many cancers, but their existence in nasopharyngeal carcinoma (NPC) is largely unknown. This study was conducted to determine cancer stem-like cells in NPC cell line. STUDY DESIGN: Basic science experimental study. METHODS: Aldehyde dehydrogenase (ALDH) activity, a putative functional marker for cancer stem cells, was assessed in Epstein-Barr virus-associated NPC cell line C666-1 cells. The ability of cells with high and low ALDH activity to proliferate, resist therapy, and initiate tumor formation was compared. RESULTS: Enrichment of cancer stem-like cells (with high ALDH activity) in C666-1 was associated with a significantly greater ability to proliferate, be clonogenic, resist chemotherapy drugs and radiation, reconstitute a heterogeneous population, and express pluripotent markers. Furthermore, subcutaneous injection of these cells into immunodeficient nude mice resulted in a tendency of tumor formation at a higher rate as compared to cells with low ALDH activity. CONCLUSIONS: These results provide evidence for the existence of cancer stem-like cells in the NPC cell line C666-1 cells.

Eosinophils in human oral squamous carcinoma; role of prostaglandin D2.

Eosinophils are often predominant inflammatory leukocytes infiltrating oral squamous carcinoma (OSC) sites. Prostaglandins are secreted by oral carcinomas and may be involved in eosinophil infiltration. The objective of this study was to determine the factors contributing to eosinophil migration and potential anti-neoplastic effects on OSC. Eosinophil degranulation was evaluated by measuring release of eosinophil peroxidase (EPO). Eosinophil chemotaxis towards OSC cells was assessed using artificial basement membrane. Eosinophil infiltration was prominent within the tissue surrounding the OSC tumor mass. We observed growth inhibition of the OSC cell line, SCC-9, during co-culture with human eosinophils, in vitro, which correlated with EPO activity that possesses growth inhibitory activity. The PGD2 synthase inhibitor, HQL-79, abrogated migration towards SCC-9. Our data suggest that OSC-derived PGD2 may play an important role via CRTH2 (the PGD2 receptor on eosinophils) in eosinophil recruitment and subsequent anti-tumor activity through the action of eosinophil cationic proteins.

CD137L-stimulated dendritic cells are more potent than conventional dendritic cells at eliciting cytotoxic T-cell responses.

Dendritic cells (DCs) are highly potent initiators of adaptive immune responses and, as such, represent promising tools for immunotherapeutic applications. Despite their potential, the current efficacy of DC-based immunotherapies is poor. CD137 ligand (CD137L) signaling has been used to derive a novel type of DCs from human peripheral blood monocytes, termed CD137L-DCs. Here, we report that CD137L-DCs induce more potent cytotoxic T-cell responses than classical DCs (cDCs). Furthermore, in exploring several DC maturation factors for their ability to enhance the potency of CD137L-DCs, we found the combination of interferon γ (IFNγ) and the mixed Toll-like receptor (TLR)7/8 agonist R848, to display the highest efficacy in potentiating the T-cell co-stimulatory activity of CD137L-DCs. Of particular importance, CD137L-DCs were found to be more efficient than cDCs in activating autologous T cells targeting the cytomegalovirus (CMV)-derived protein pp65. Specifically, CD137L-DC-stimulated T cells were found to secrete higher levels of IFNγ and killed 2-3 times more HLA-matched, pp65-pulsed target cells than T cells activated by cDCs. Finally, in addition to stimulating CD8+ T cells, CD137L-DCs efficiently activated CD4+ T cells. Taken together, these findings demonstrate the superior potency of CD137L-stimulated DCs in activating CMV-specific, autologous T cells, and encourage the further development of CD137L-DCs for antitumor immunotherapy.

Induction of neutralizing antibodies against dengue virus type 2 upon mucosal administration of a recombinant Lactococcus lactis strain expressing envelope domain III antigen.

Mucosal vaccines present several advantages over conventional parenteral vaccines including their ease of administration and low cost, both criteria being priorities for developing countries plagued by infectious diseases. A recombinant Lactococcus lactis strain producing the envelope domain III (EDIII) antigen from dengue virus serotype 2 was engineered, and the ability of the live recombinant bacteria to trigger a systemic anti-EDIII IgG antibody response upon nasal or oral administration to BALB/c and C57BL/6 mice was investigated. Results showed that the antibody response depended on the route of administration and on the mouse strain inoculated. Out of six, two and three C57BL/6 mice orally and nasally inoculated with the recombinant bacteria, respectively, displayed anti-EDIII antibody responses higher than that obtained in the mouse group intraperitoneally (i.p.) immunized with heat-inactivated dengue 2 virus. The protective potential of the immune sera was measured using the plaque reduction neutralizing test (PRNT) and results indicated that high anti-EDIII antibody levels did not correlate directly with high neutralizing activities. Immune sera from orally inoculated mice were found the most potent to neutralize in vitro dengue infection with neutralizing antibody activities in some cases higher than that obtained with the immune sera from mice i.p. injected with heat-inactivated virus.

RNA interference against enterovirus 71 infection.

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.

UV induces GADD45 in a p53-dependent and -independent manner in human keratinocytes.

BACKGROUND: GADD45 is a multifunctional protein involved in DNA repair and in cell cycle checkpoint control. p53 plays an important role in regulating DNA repair and in response to UVB in keratinocytes. OBJECTIVE: GADD45 and p53 expression was examined and compared at the mRNA and protein level after exposure to UV irradiation. METHODS: Human keratinocytes were exposed to increasing doses of UVB, and an RNA protection assay and a Western blot analysis were performed. RESULTS: The RNase protection assays using human keratinocytes showed that GADD45 mRNA increases after 4 h and remains elevated for 24 h in cells irradiated at 100, 300, or 600 J/m2 UVB. The level of GADD45 protein increases after 8 h and remains elevated for 48 h, with maximal induction at 300 J/m2. p53 mRNA did not rise in concert with GADD45 at any dose used, and p53 protein was not up-regulated at the lower dose of 100 J/m2. CONCLUSION: GADD45 is regulated in both a p53-dependent and a p53-independent manner in keratinocytes after UV exposure.

GADD45 regulates G2/M arrest, DNA repair, and cell death in keratinocytes following ultraviolet exposure.

GADD45 is a multifunctional protein that is regulated by p53. p53 plays an important role in regulating DNA repair and in the response to ultraviolet light in keratinocytes. This study investigates the role of GADD45 in the response to ultraviolet B. Cell cycle analysis demonstrated that wild-type and Gadd45-deficient cells have transient G2/M arrest, but only in the wild-type cells was arrest sustained. Cdc2 kinase activity in immunoprecipitates from normal and Gadd45-deficient cells decreases after irradiation in normal cells but not in Gadd45-deficient cells. An immunofluorescent study with Cdc2 antibody demonstrated diffuse cellular fluorescence before ultraviolet irradiation in both Gadd45-deficient and wild-type cells, but upon ultraviolet irradiation only Gadd45-proficient cells showed Cdc2 sequestration in the cytoplasm. Gadd45-deficient cells also have a slower rate of nucleotide excision repair. The lack of G2/M arrest coupled with reduced DNA repair leads to a higher ultraviolet sensitivity of Gadd45-deficient cells. These results reveal that GADD45 promotes G2/M arrest via nuclear export and kinase activity of Cdc2, increases global genomic DNA repair, and inhibits cell death in keratinocytes. Thus, GADD45 plays an important role in maintaining genomic integrity in ultraviolet-exposed skin.