A nuclear NKRF interacting long noncoding RNA controls EBV eradication and suppresses tumor progression in natural killer/T-cell lymphoma.

Long intergenic noncoding RNAs (lincRNAs) are differentially expressed in EBV-infected cells and play an essential role in tumor progression. Molecular pathogenesis of lincRNAs in EBV-driven natural killer T cell lymphoma (NKTCL) remains unclear. Here we investigated the ncRNA profile using high-throughput RNA sequencing data of 439 lymphoma samples and screened out LINC00486, whose downregulation was further validated by quantitative real-time polymerase chain reaction in EBV-encoded RNA (EBER)-positive lymphoma, particularly NKTCL. Both in vitro and in vivo studies revealed the tumor suppressive function of LINC00486 through inhibiting tumor cell growth and inducing G0/G1 cell cycle arrest. As mechanism of action, LINC00486 specifically interacted with NKRF to abrogate its binding with phosphorylated p65, activated NF-κB/TNF-α signaling and subsequently enhanced EBV eradication. Solute carrier family 1 member 1 (SLC1A1), upregulated and mediated the glutamine-addiction and tumor progression in NKTCL, was negatively correlated with the expression of NKRF. NKRF specifically bound to the promoter and transcriptionally downregulated the expression of SLC1A1, as evidenced by Chromatin Immunoprecipitation (ChIP) and luciferase assay. Collectively, LINC00486 functioned as a tumor suppressor and counteracted EBV infection in NKTCL. Our study improved the knowledge of EBV-driven oncogenesis in NKTCL and provided the clinical rationale of EBV eradication in anti-cancer treatment.

Screening and identifying a novel M-MDSCs-related gene signature for predicting prognostic risk and immunotherapeutic responses in patients with lung adenocarcinoma.

Background: Lung adenocarcinoma (LUAD) shows intratumoral heterogeneity, a highly complex phenomenon that known to be a challenge during cancer therapy. Considering the key role of monocytic myeloid-derived suppressor cells (M-MDSCs) in the tumor microenvironment (TME), we aimed to build a prognostic risk model using M-MDSCs-related genes. Methods: M-MDSCs-related genes were extracted from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Utilized univariate survival analysis and random forest algorithm to screen candidate genes. A least absolute shrinkage and selection operator (LASSO) Cox regression analysis was selected to build the risk model. Patients were scored and classified into high- and low-risk groups based on the median risk scores. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis along with R packages "estimate" and "ssGSEA" were performed to reveal the mechanism of risk difference. Prognostic biomarkers and tumor mutation burden (TMB) were combined to predict the prognosis. Nomogram was carried out to predict the survival probability of patients in 1, 3, and 5 years. Results: 8 genes (VPREB3, TPBG, LRFN4, CD83, GIMAP6, PRMT8, WASF1, and F12) were identified as prognostic biomarkers. The GEO validation dataset demonstrated the risk model had good generalization effect. Significantly enrichment level of cell cycle-related pathway and lower content of CD8+ T cells infiltration in the high-risk group when compared to low-risk group. Morever, the patients were from the intersection of high-TMB and low-risk groups showed the best prognosis. The nomogram demonstrated good consistency with practical outcomes in predicting the survival rate over 1, 3, and 5 years. Conclusion: The risk model demonstrate good prognostic predictive ability. The patients from the intersection of low-risk and high-TMB groups are not only more sensitive response to but also more likely to benefit from immune-checkpoint-inhibitors (ICIs) treatment.

Vaccine targeting TNF epitope 1-14 do not suppress host defense against Mycobacterium bovis Bacillus Calmette-Guérin infection.

Anti-TNF inhibitors are efficacious in the treatment of chronic inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease (CD), juvenile idiopathic arthritis (JIA), and ankylosing spondylitis (AS). However, more and more clinical case reports revealed that anti-TNF inhibitors could increase the risk of viral, fungal, and bacterial (especially intracellular) infection. In this study, based on Immune Epitope Database (IEDB) online B cell epitope prediction and the knowledge of TNF three dimensional (3D) structure we developed a novel vaccine (DTNF114-TNF114) that targeting TNF epitope 1-14, which produced antibodies only partially binding to trans-membrane TNF (tmTNF), therefore partially sparing tmTNF-TNFR1/2 interaction. Immunization with DTNF114-TNF114 significantly protected and prolonged the survival rate of mice challenged with lipopolysaccharide (LPS); and in the mCherry expressing Mycobacterium bovis Bacillus Calmette-Guérin (mCherry-BCG) infection model, DTNF114-TNF114 immunization significantly decreased soluble TNF (solTNF) level in serum, meanwhile did not suppress host immunity against infection. Thus, this novel and infection concern-free vaccine provides a potential alternative or supplement to currently clinically used anti-TNF inhibitors.

Recombinant KRAS G12D Protein Vaccines Elicit Significant Anti-Tumor Effects in Mouse CT26 Tumor Models.

Drug development targeting the most frequently mutation G12D of KRAS has great significance. As an attractive immunotherapy, cancer vaccines can overcome binding difficulties of small molecules; however, the weak immunogenicity and production difficulties of reported KRAS mutation vaccines limit their clinical application. To improve antigen-specific immune responses and Anti-Tumor effects on tumors expressing KRAS G12D mutation, we designed recombinant proteins containing KRAS peptide (amino acids 5-21) with G12D (called SP) in two forms: DTT-SP4 and DTSP. DTT-SP4 was constructed by fusing four copies of SP to the C-terminal of the translocation domain of diphtheria toxin (DTT), and DTSP was constructed by grafting SP onto DTT. The two vaccines in combination with aluminum hydroxide (Alum) and cytosine phosphoguanine (CpG) successfully induced conspicuous SP-specific humoral and cellular immune responses, and displayed prominent protective and therapeutic Anti-Tumor effects in mouse CT26 tumor models. Surprisingly, the DTSP-treated group displayed better Anti-Tumor effects in vivo compared with the DTT-SP4-treated and control groups. Moreover, 87.5 and 50% of DTSP-treated mice in the preventive and therapeutic models were tumor free, respectively. Notably, in the DTSP-treated group, the interferon-γ (IFN-γ) expression of T cells in vitro and the T-helper 1 (Th1)-related cytokine expression in tumor tissues indicated that the activated Th1 immune response may be involved in Anti-Tumor activity. Furthermore, DTSP treatment remarkably altered the subpopulation of T cells in splenocytes and tumor-infiltrating lymphocytes. The percentage of effector CD8+ T cells increased, whereas that of immunosuppressive CD4+Foxp3+ T cells remained reduced in the DTSP group. Dramatic tumor-inhibitory effects of DTSP, which is easily prepared, make it a more attractive strategy against KRAS G12D tumors.

Recombinant Costimulatory Fusion Proteins as Functional Immunomodulators Enhance Antitumor Activity in Murine B16F10 Melanoma.

Blocking inhibitory signaling and engaging stimulatory signaling have emerged as important therapeutic modalities for cancer immunotherapy. This study aimed to investigate immunomodulatory features of three recombinant costimulatory ligand proteins in a mouse model, which are extracellular domains of OX40-ligand (OX40L), 4-1BB-ligand (4-1BBL), or two domains in tandem, fused with the transmembrane domain of diphtheria toxin (DTT), named DTT-COS1, DTT-COS2, and DTT-COS12, respectively. In vitro study showed that DTT-COS1 and DTT-COS12 had immunological activity increasing the ratio of CD8/CD4 T cells. Treatments with DTT-COS1 and DTT-COS12 dramatically generated immune protection against the B16F10 tumor challenge in both prophylactic and therapeutic efficacy. Furthermore, regarding tumor microenvironment (TME) immunomodulation, DTT-COS1 treatment increased the proportion of CD4+ effector T cells (Teff) and decreased the expression of a suppressive cytokine. Meanwhile, DTT-COS12 reduced regulatory T cells (Treg) and improved the level of stimulatory cytokines. In addition, endogenous antibodies against OX40L/4-1BBL were generated, which may help with antitumor responses. Unexpectedly, DTT-COS2 lacked antitumor effects in vitro and in vivo. Importantly, serum analysis of liver-function associated factors and pro-inflammatory cytokines demonstrated that treatments were safe formulations in mice without signs of systemic toxicity. Remarkably, DTT-COS1 and DTT-COS12 are functional immunomodulators for mouse B16F10 melanoma, creating practical preclinical value in cancer immunotherapy.

A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model.

Engagement of programmed death 1 receptor (PD-1) and its ligand PD-L1/2 induces a signal transduction pathway that inhibits the activity of tumor-infiltrating cytotoxic T lymphocytes and promotes tumor growth and metastasis. Antibodies blocking PD-1 or PD-L1 can restore antitumor T cell responses and cause long-term remission in a subset of cancer patients with advanced or refractory tumors. In this study, we asked whether PD-L1 vaccination could confer tumor control in mouse tumor models. To address the central tolerance toward self-molecules, we fused the extracellular domain of PD-L1 (PD-L1E) to the C-terminal of the translocation domain of diphtheria toxin (DTT). DTT is able to elicit CD4+ T cell responses required for inducing robust immune responses against self-molecules. The fusion molecule is called DPDL1E. When formulated with incomplete Freund's adjuvant (IFA), DPDL1E elicited robust immune responses biased toward the Th1 type and inhibited tumor growth in both preventive and therapeutic mouse tumor models. We further showed that the anti-DPDL1E sera blocked PD-L1 binding to PD-1 in vitro. The DPDL1E vaccination increased the levels of tumor-infiltrating T lymphocytes (TILs) and reduced the levels of myeloid-derived suppressor cells (MDSCs) as well as exhausted LAG3+PD-1+ CD8+ T cells. All of these data suggest that DPDL1E vaccination reverses the suppressive phenotype of the tumor microenvironment and that it is a promising strategy for cancer therapy.

The Immunogenicity and Anti-tumor Efficacy of a Rationally Designed Neoantigen Vaccine for B16F10 Mouse Melanoma.

Tumor neoantigens are ideal targets for cancer immunotherapy as they are recognized by host immune system as foreigners and can elicit tumor-specific immune responses. However, existing strategies utilizing RNA or long peptides for the neoantigen vaccines render limited immune responses since only 20-30% of neoantigens predicted in silico to bind MHC I molecules are capable of eliciting immune responses with the majority of responding T cells are CD4+. Therefore, it warrants further exploration to enhance neoantigen-specific CD8+ T cells responses. Since neoantigens are naturally weak antigens, we asked whether foreign T help epitopes could enhance their immunogenicity. In present study, we chose 4 weak B16F10 neoantigens as vaccine targets, and fused them to the transmembrane domain of diphtheria toxin, namely DTT-neoAg. Strikingly, the vaccine elicited anti-tumor CD8+ T cells responses and enhanced tumor infiltration of both T cells and NK cells. Impressively, DTT-neoAg vaccine significantly deterred tumor growth with the inhibition rate reached 88% in the preventive model and 100% in the therapeutic model at low dose of tumor challenge. Furthermore, after second challenge with higher dose of tumor cells, 33.3% of the immunized mice remained tumor-free for 6 months in the therapeutic model. Because DTT is a non-toxic domain of diphtheria toxin, it may be not of great concern in terms of safety as a Th epitope provider. Thus, the fusion strategy employed by this study may become a feasible and powerful approach for development of personalized cancer vaccines.