TAP1 Expression Research Articles

Abstract B111: Vaccination against TAP downregulation-induced neoantigens to prevent future tumor development in the setting of recurrence or premalignancy

Abstract Development of therapeutic strategies to prevent recurrence in cancer patients, or tumor progression in individuals at high risk of developing cancer, has been challenging given the long and often unpredictable time to the emergence of the malignant tumors. Mutation-generated neoantigens represent the most potent antigens to induce antitumor immunity, yet the ability to predict which neoantigens will be expressed in future tumors is at present not an option.To overcome the limitations of targeting mutated neoantigens, we have developed a simple and broadly applicable approach to induce neoantigens in tumor cells in situ by reducing the expression of peptide transporter TAP, whereby a TAP-specific siRNA is targeted to tumor cells in mice by conjugation to a broad-range nucleolin-binding aptamer. Nucleolin, normally expressed in the cytoplasm and nucleolus of all somatic cells, is translocated to the cell surface of most murine and human tumors and hence could serve as an almost universal target to deliver therapeutics to tumors in vivo. Previous results have demonstrated that genetic ablation of TAP not only inhibits the canonical antigen processing pathway but also upregulates alternative pathways presenting new epitopes, essentially neoantigens, that can be recognized by the immune system to elicit effective CD8+ T-cell responses. Such epitopes are shared among all (tumor) cells in which TAP is downregulated, corresponding to the functional equivalent of clonal mutation-generated neoantigens. Our study shows that transiently increasing the neoantigen burden of tumor cells in situ by targeted downregulation of TAP represent a potent way of generating antitumor immunity in the absence of measurable toxicity.Exploiting the ability to induce neoantigens in situ, we are developing a novel vaccination strategy targeting potent neoantigens to control the growth of the future tumors—whereby mice in remission or with premalignant lesions are first vaccinated against TAP downregulation-induced neoantigens, and when or if tumor develops the same antigens are induced in the tumor, termed prorapeutic vaccination (prophylactic + therapeutic). To induce an immune response against TAP downregulation-induced neoantigens, mice were vaccinated with TAP siRNAs that are targeted to DC in situ by conjugation to a short CpG oligonucleotide leading to the downregulation of TAP, expression of neoantigens, as well as activation of the DC, and thereby priming of a potent T-cell response. To induce neoantigens in the future tumors, the TAP siRNA is targeted to the developing tumor lesion by conjugation to the nucleolin (Nucl) aptamer. Both Nucl and CpG targeted siRNA are administered systemically via intraperitoneal injection to reach the disseminated tumor lesions and resident DC present throughout the body. In murine models of recurrence and premalignant disease, prorapeutic vaccination elicited an adaptive and innate antitumor immune response and inhibited tumor growth. The prorapeutic vaccination approach was more effective than vaccinating against prototypic mutated neoantigens, and did not elicit measurable autoimmune toxicity, in stark contrast to a comparatively therapeutic dose of CTLA-4.The ability to vaccinate against experimentally induced neoantigens using a broadly applicable procedure with two chemically synthesized reagents, introduces a new paradigm in cancer immunotherapy of vaccinating against neoantigens induced in future tumors, to prevent recurrence in patients in remission or preventing tumor development in individuals at high risk of developing cancer. Citation Format: Greta Garrido Hidalgo, Brett Schrand, Ailem Rabasa, Agata Levay, Tal Gefen, Giri Bhuwan Bhuwan, Anthony R. Ferrantella, Vikas Dudeja, Koen Marijt, Thorbald T. van Hall, Eli Gilboa. Vaccination against TAP downregulation-induced neoantigens to prevent future tumor development in the setting of recurrence or premalignancy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B111.

Functional analysis of prv-miR-LLT11a encoded by pseudorabies virus.

Viral-encoded microRNAs (miRNAs) have vital roles in the regulation of virus replications and host immune responses. The results of previous studies have indicated that miRNA clusters are involved in the replication and virulence of the pseudorabies virus (PRV), which may potentially lead to immune escape or facilitation of PRV replication. This study's previous research revealed that prv-miR-LLT11a was differentially expressed during PRV infection. The present study's results have demonstrated that prv-miR-LLT11a could significantly inhibit PRV replication. It was further determined that SLA-1 was the target gene of prv-miR-LLT11a, and simultaneously, that overexpression of prv-miR-LLT11a could downregulate the mRNA and protein levels of SLA-1 in a dose-independent manner. Furthermore, the present study also observed that prv-miR-LLT11a can downregulate TAP1 expression. Our findings provide a better understanding of the molecular mechanism involved in the effects of prv-miR-LLT11a on SLA-1 and TAP1 as well as its involvement in immune system evasion of PRV.

Human preprocalcitonin self-antigen generates TAP-dependent and -independent epitopes triggering optimised T-cell responses toward immune-escaped tumours

Tumours often evade CD8 T-cell immunity by downregulating TAP. T-cell epitopes associated with impaired peptide processing are immunogenic non-mutated neoantigens that emerge during tumour immune evasion. The preprocalcitonin (ppCT)16–25 neoepitope belongs to this category of antigens. Here we show that most human lung tumours display altered expression of TAP and frequently express ppCT self-antigen. We also show that ppCT includes HLA-A2-restricted epitopes that are processed by TAP-independent and -dependent pathways. Processing occurs in either the endoplasmic reticulum, by signal peptidase and signal peptide peptidase, or in the cytosol after release of a signal peptide precursor or retrotranslocation of a procalcitonin substrate by endoplasmic-reticulum-associated degradation. Remarkably, ppCT peptide-based immunotherapy induces efficient T-cell responses toward antigen processing and presenting machinery-impaired tumours transplanted into HLA-A*0201-transgenic mice and in NOD-scid-Il2rγnull mice adoptively transferred with human PBMC. Thus, ppCT-specific T lymphocytes are promising effectors for treatment of tumours that have escaped immune recognition.

An update meta-analysis and systematic review of TAP polymorphisms as potential biomarkers for judging cancer risk

ObjectiveTransporter associated with antigen processing protein (TAP) is a heterodimer protein consist of TAP1 and TAP2, act a pivotal part in the immune surveillance. In recent days, controversial relationships were reported between TAP polymorphisms and cancer risk, thus, a systematic meta-analysis was performed to resolve this discrepancy. MethodsWe searched the PubMed, EMbase, Web of Science, CNKI and Wanfang databases, the cited references were also manually searched again, covering all the papers published until March 25, 2018. Quality assessment was conducted using the Newcastle–Ottawa Scale. All the meta-analysis was conducted with Stata version 12.0 software to assess the strength of the association. Results4719 cases and 4215 controls from 24 case-control studies related to TAP polymorphisms were enrolled. There was no significant association between TAP1-rs1135216, TAP1-rs4148873, TAP2-rs2228396, TAP2-rs241447 and TAP2-rs4148873 and cancer sensibility. Interestingly, a significant positive association was observed between TAP2 rs4148876 C/T polymorphism and increase cancer risk in homozygote and recessive models. Further in-silico results indicated the expression of TAP2 in cancer tissue is higher than that in normal tissue (cervical cancer, TPM = 70.2 vs. 24.0 respectively, P < 0.01; acute myeloid leukemia, TPM = 52.5 vs. 8.8 respectively, P < 0.01), and influence the survival time of acute myeloid leukemia patients (Log-rank P < 0.05). ConclusionsOur finding suggested that TAP1-rs1135216, TAP1-rs4148873, TAP2-rs2228396, TAP2-rs241447 and TAP2-rs4148873 might not be involved in cancer risk, but the T allele of TAP2-rs4148876 might be a potential biomarker for judging cancer risk, and larger-scale studies are required to confirm our findings.

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83.

Porcine reproductive and respiratory syndrome virus (PRRSV), a virulent pathogen of swine, suppresses the innate immune response and induces persistent infection. One mechanism used by viruses to evade the immune system is to cripple the antigen-processing machinery in monocyte-derived dendritic cells (MoDCs). In this study, we show that MoDCs infected by PRRSV express lower levels of the major histocompatibility complex (MHC)-peptide complex proteins TAP1 and ERp57 and are impaired in their ability to stimulate T cell proliferation and increase their production of CD83. Neutralization of sCD83 removes the inhibitory effects of PRRSV on MoDCs. When MoDCs are incubated with exogenously added sCD83 protein, TAP1 and ERp57 expression decreases and T lymphocyte activation is impaired. PRRSV nonstructural protein 1α (Nsp1α) enhances CD83 promoter activity. Mutations in the ZF domain of Nsp1α abolish its ability to activate the CD83 promoter. We generated recombinant PRRSVs with mutations in Nsp1α and the corresponding repaired PRRSVs. Viruses with Nsp1α mutations did not decrease levels of TAP1 and ERp57, impair the ability of MoDCs to stimulate T cell proliferation, or increase levels of sCD83. We show that the ZF domain of Nsp1α stimulates the secretion of CD83, which in turn inhibits MoDC function. Our study provides new insights into the mechanisms of immune suppression by PRRSV.IMPORTANCE PRRSV has a severe impact on the swine industry throughout the world. Understanding the mechanisms by which PRRSV infection suppresses the immune system is essential for a robust and sustainable swine industry. Here, we demonstrated that PRRSV infection manipulates MoDCs by interfering with their ability to produce proteins in the MHC-peptide complex. The virus also impairs the ability of MoDCs to stimulate cell proliferation, due in large part to the enhanced release of soluble CD83 from PRRSV-infected MoDCs. The viral nonstructural protein 1 (Nsp1) is responsible for upregulating CD83 promoter activity. Amino acids in the ZF domain of Nsp1α (L5-2A, rG45A, G48A, and L61-6A) are essential for CD83 promoter activation. Viruses with mutations at these sites no longer inhibit MoDC-mediated T cell proliferation. These findings provide novel insights into the mechanism by which the adaptive immune response is suppressed during PRRSV infection.

MicroRNA 125a Regulates MHC-I Expression on Esophageal Adenocarcinoma Cells, Associated With Suppression of Antitumor Immune Response and Poor Outcomes of Patients

Immune checkpoint inhibition may affect growth or progression of highly aggressive cancers, such as esophageal adenocarcinoma (EAC). We investigated the regulation of expression of major histocompatibility complex, class 1 (MHC-I) proteins (encoded by HLA-A, HLA-B, and HLA-C) and the immune response to EACs in patient samples. We performed quantitative polymerase chain reaction array analyses of OE33 cells and OE19 cells, which express different levels of the ATP binding cassette subfamily B member 1 (TAP1) and TAP2, required for antigen presentation by MHC-I, to identify microRNAs (miRNAs) that regulate their expression. We performed luciferase assays to validate interactions between miRNAs and potential targets. We overexpressed candidate miRNAs in OE33, FLO-1, and OACP4 C cell lines and performed quantitative polymerase chain reaction, immunoblot, and flow cytometry analyses to identify changes in messenger RNA (mRNA) and protein expression; we studied the effects of cytotoxic T cells. We performed miRNA in situ hybridization, RNA-sequencing, and immunohistochemical analyses of tumor tissues from 51 untreated patients with EAC inthe Netherlands. Clinical and survival data were collected for patients, and EAC subtypes were determined. We found OE19 cells to have increased levels of 7 miRNAs. Of these, we found binding sites for miRNA 125a (MIR125a)-5p in the 3' untranslated region of the TAP2 mRNA and binding sites for MIR148a-3p in 3' untranslated regions of HLA-A, HLA-B, and HLA-C mRNAs. Overexpression of these miRNAs reduced expression of TAP2 in OE33, FLO-1, and OACP4 C cells, and reduced cell-surface levels of MHC-I. OE33 cells that expressed the viral peptide BZLF1 were killed by cytotoxic T cells, whereas OE33 that overexpressed MIR125a-5p or MIR 148a along with BZLF1 were not. In EAC and nontumor tissues, levels of MIR125a-5p correlated inversely with levels of TAP2 protein. High expression of TAP1 by EAC correlated with significantly shorter overall survival times of patients. EACs that expressed high levels of TAP1 and genes involved in antigen presentation also expressed high levels of genes that regulate the adaptive immune response, PD-L1, PD-L2, and IDO1; these EACs had a poor response to neoadjuvant chemoradiotherapy and associated with shorter overall survival times of patients. In studies of EAC cell lines andtumor tissues, we found increased levels of MIR125a-5p and MIR148a-3p to reduce levels of TAP2 and MHC-I, required for antigen presentation. High expression of MHC-I molecules by EAC correlated with markers of an adaptive immune response and significantly shorter overall survival times of patients.

Impact of Mitochondrial Permeability on Endothelial Cell Immunogenicity in Transplantation.

Microvascular endothelial cells (ECs) are central to an allograft's immunogenicity. Cold ischemia and reperfusion injury associated with static cold storage and warm reperfusion activates ECs and increases the immunogenicity of the allograft. After reperfusion, mitochondrial permeability transition pore (mPTP) opening contributes to mitochondrial dysfunction in the allograft, which correlates to alloimmune rejection. Current understanding of this relationship, however, centers on the whole allograft instead of ECs. This study aimed to elucidate the relationship between EC mPTP opening and their immunophenotype. Mitochondrial metabolic fitness and glycolysis in ECs were assessed in parallel with metabolic gene microarray postreperfusion. NIM811 was used to inhibit mPTP opening to rescue mitochondrial fitness. The immunogenicity of NIM811-treated ECs was determined via levels of EC's proinflammatory cytokines and allogeneic CD8 T cell cocultures. Finally, EC surface expression of adhesion, costimulatory, coinhibitory, MHC-I molecules, and MHC-I machinery protein levels were characterized. Genes for glycolysis, tricarboxylic acid cycle, fatty acid synthesis, gluconeogenesis were upregulated at 6 hours postreperfusion but either normalized or downregulated at 24 hours postreperfusion. As mitochondrial fitness was reduced, glycolysis increased during the first 6 hours postreperfusion. Endothelial cell treatment with NIM811 during the early postreperfusion period rescued mitochondrial fitness and reduced EC immunogenicity by decreasing CCL2, KC release, and VCAM-1, MHC-I, TAP1 expression. Static cold storage and warm reperfusion leads to a reduction in mitochondrial fitness in microvascular ECs due to mPTP opening. Further, mPTP opening promotes increased EC immunogenicity that can be prevented by NIM811 treatment.

Regulation Of Antigen-Presenting Machinery In Melanoma After Plasma Treatment

Cold physical plasmas have been shown to eliminate multiple types of cancers in vitro and in vivo [1] Although being multi-component systems, evidence suggest plasmas to be active primarily via release of a plethora of reactive oxygen and nitrogen species (ROS/RNS). These species not only damage cells via oxidation of proteins and lipids but also trigger signaling cascades via redox relays. This redox signaling is vital for a number of processes, e.g. the formation and loading of major histocompatibility class I complexes (MHC-I). Once loaded with peptides and translocated to the cell surface, MHC-I molecules are recognized by cytotoxic T lymphocytes. In cancer, high numbers of these lymphocytes often correlate with a good prognosis due to their ability to specifically recognize and kill tumor cells. Understanding whether and how MHCI loading and expression is regulated via exogenous oxidants such as produced by cold plasmas is key to delineate possible immune-modulating effects of plasmas in onco-therapy. Intracellular proteins get degraded to peptides by the proteasome before they get transported to the endoplasmatic reticulum by antigen peptide transporter TAP1 and TAP2. In endoplasmatic reticulum the peptides get associated with MHC-class I molecules via a number of proteins, such as Tapasin, ERp57 and PDI [2, 3]. Histone deacetylase inhibitors, interferones and cytokines are able to enhance the expression of TAP1, TAP2, LMP2, LMP7, and Tapasin in association with increased expression of MHC class I [4-6]. Here, we treated melanoma cells in vitro with cold plasma and investigated subsequent effects on the antigen processing machinery. We confirmed cytotoxic effects of the plasma treatment, and measured an upregulation of MHC-I on the cell surface using flow cytometry. Subsequently, we investigated quantity and activity of several proteins crucial in the antigen processing machinery, such as TAP1, TAP2, Tapasin, and ERp57, and we observed a modulation of protein targets. Trichostatin A, Interferon γ, and IL-10 served as positive controls as they promote antigen processing and presentation [5]. Our results suggest a role of plasma-derived, exogenous reactive species in the regulation of antigen presentation of tumor cells important for mounting antitumor immune responses..

A functional SNP in the 3'-UTR of TAP2 gene interacts with microRNA hsa-miR-1270 to suppress the gene expression.

The transporter associated with antigen processing 2 (TAP2) is involved in the development of multidrug resistance and the etiology of immunological diseases. In this study, we investigated whether the expression of TAP2 can be perturbed by single nucleotide polymorphisms (SNPs) located in 3'-untranslated region (3'-UTR) of the gene via interactions with microRNAs. Using a series of in silico assays, we selected the candidate microRNAs (miRNAs) with the potential to interact with functional SNPs of TAP2. The SNP rs241456-located in the 3'-UTR of TAP2-resides in a potential binding site for hsa-miR-1270 and hsa-miR-620. HEK 293 cells, from a human kidney cell line, were used to characterize the extent of binding of miRNAs to each polymorphic allele of the SNP by a luciferase reporter gene assay. RNA electrophoretic mobility shift assays were used to evaluate the interaction between the miRNAs and each allele sequence of the SNP. We found that hsa-miR-1270 inhibited luciferase activity by binding to the T allele of the SNP in an allele-specific manner. A negative correlation was also found between the expression of hsa-miR-1270 and the T allele of the SNP in kidney tissues. Our findings support the hypothesis that hsa-miR-1270 suppresses the production of TAP2 by binding to this SNP in the 3'-UTR of this gene. Environ. Mol. Mutagen. 59:134-143, 2018. © 2017 Wiley Periodicals, Inc.

Downregulation of TAP1 and TAP2 in early stage breast cancer.

TAP1-TAP2 heterodimeric complexes are recognized as the transporter associated with antigen processing of major histocompatibility complex class I peptides for recognition by tumor-specific cytotoxic T lymphocytes. In this study, we investigated the immunohistochemical expression of TAP1 and TAP2 in 160 patients with breast cancer and correlated their expression levels with clinicopathologic parameters. The median age of the patient cohort was 52.5 years (range, 30–86 years). Both TAP1 and TAP2 immunohistochemical expression levels correlated significantly with breast cancer characteristics (P < .001). TAP1 expression levels were low to negative in stage I breast tumors. TAP1 and TAP2 levels were significantly higher in grade 3 tumors than low-grade (grade 1 and 2) tumors. TAP1 and TAP2 expression levels were not significantly different among different levels of HER2-expressing tumors and did not vary by estrogen and progesterone receptor status or patient age. Both TAP1 and TAP2 overexpression in breast cancer might be an indicator of an aggressive breast tumor.

TAP1 down-regulation elicits immune escape and poor prognosis in colorectal cancer

ABSTRACTThe anti-tumor immune response has been shown to be of great prognostic importance in colorectal cancer (CRC) and so has the tumors ability for immune evasion. Our aim of this study was to investigate tumor factors that influence immunity. We used a gene expression array to search for potential mechanisms of tumor immune escape.One candidate gene identified was TAP1, involved in antigen presentation by MHC class I. TAP1 protein expression was evaluated by immunohistochemistry in 436 CRC patients of the Colorectal Cancer in Umeå Study cohort. We found a significant association between a downregulated expression of TAP1 and low infiltration of various subtypes of lymphocytes as well as macrophages. A downregulated expression of TAP1 was further found to be independent of molecular characteristics, suggesting TAP1 down-regulation to reach beyond the well described highly immunogenic MSI CRCs. A low expression of TAP1 was also significantly associated with poor prognosis in patients with CRC, a result that stayed significant in tumor front of early stage tumors (stage I-II) through multivariable analyses. Furthermore, we found that TAP1 expression was inversely correlated with methylation at sites in close proximity to the promoter region.In summary, our results show down-regulation of TAP1 to be a general mechanism of tumor immune escape in CRC and a poor prognostic factor in stage I-II CRC patients. We also suggest that methylation of the TAP1 gene may be a putative mechanism for TAP1 downregulation.

A case-control study on association of proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1) polymorphisms and their transcript levels in vitiligo from Gujarat.

BackgroundAutoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo.ObjectivesTo explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat.MethodsPSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis.ResultsThe frequency of ‘TT’ genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively).ConclusionsPSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The down-regulation of PSMB8 in patients with risk genotype ‘CC’ advocates the vital role of PSMB8 in the autoimmune basis of vitiligo.

Inducible TAP1 Negatively Regulates the Antiviral Innate Immune Response by Targeting the TAK1 Complex.

The innate immune response is critical for host defense and must be tightly controlled, but the molecular mechanisms responsible for its negative regulation are not yet completely understood. In this study, we report that transporter 1, ATP-binding cassette, subfamily B (TAP1), a virus-inducible endoplasmic reticulum-associated protein, negatively regulated the virus-triggered immune response. In this study, we observed upregulated expression of TAP1 following virus infection in human lung epithelial cells (A549), THP-1 monocytes, HeLa cells, and Vero cells. The overexpression of TAP1 enhanced virus replication by inhibiting the virus-triggered activation of NF-κB signaling and the production of IFNs, IFN-stimulated genes, and proinflammatory cytokines. TAP1 depletion had the opposite effect. In response to virus infection, TAP1 interacted with the TGF-β-activated kinase (TAK)1 complex and impaired the phosphorylation of TAK1, subsequently suppressing the phosphorylation of the IκB kinase complex and NF-κB inhibitor α (IκBα) as well as NF-κB nuclear translocation. Our findings collectively suggest that TAP1 plays a novel role in the negative regulation of virus-triggered NF-κB signaling and the innate immune response by targeting the TAK1 complex.

RNA Seq analysis for transcriptome profiling in response to classical swine fever vaccination in indigenous and crossbred pigs

In present investigation, differential expression of transcriptome after classical swine fever (CSF) vaccination has been explored at the cellular level in crossbred and indigenous (desi) piglets. RNA Sequencing by Expectation-Maximization (RSEM) package was used to quantify gene expression from RNA Sequencing data, and differentially expressed genes (DEGs) were identified using EBSeq, DESeq2, and edgeR softwares. After analysis, 5222, 6037, and 6210 common DEGs were identified in indigenous post-vaccinated verses pre-vaccinated, crossbred post-vaccinated verses pre-vaccinated, and post-vaccinated crossbred verses indigenous pigs, respectively. Functional annotation of these DEGs showed enrichment of antigen processing-cross presentation, B cell receptor signaling, T cell receptor signaling, NF-κB signaling, and TNF signaling pathways. The interaction network among the immune genes included more number of genes with greater connectivity in vaccinated crossbred than the indigenous piglets. Higher expression of IRF3, IL1β, TAP1, CSK, SLA2, SLADM, and NF-kB in crossbred piglets in comparison to indigenous explains the better humoral response observed in crossbred piglets. Here, we predicted that the processed CSFV antigen through the T cell receptor signaling cascade triggers the B cell receptor-signaling pathway to finally activate MAPK kinase and NF-κB signaling pathways in B cell. This activation results in expression of genes/transcription factors that lead to B cell ontogeny, auto immunity and immune response through antibody production. Further, immunologically important genes were validated by qRT-PCR.

Expression of β-defensins in bovine oviduct and uterus during follicular and luteal phases

This study was conducted to examine the expression of β-defensins of cow’s reproductive tract and their relationship with estrus cycle using quantitative PCR and ELISA methods. Six kinds of β-defensins and the receptors of estrogen and progesterone were studied in cows’ reproductive tract during the follicular and luteal phases. The results show that besides LAP, TAP, BNBD4, and BNBD5, BNBD1 and BNBD10 were also found in cow oviduct, uterine horn, and uterine body. During the follicular phase, the uterine body had the highest expressions of BNBDI, TAP, LAP1, BNBD4, and BNBD5. However, the highest expression of BNBD10 was observed in the uterine horn at the same phase. The highest expressions of BNBD1, TAP, LAP1, BNBD4, BNBD5, and BNBD10 were observed in the uterine horn during the luteal phase, while the oviduct had the least expression. A significant higher expression of BNBD1, TAP, LAP1, BNBD4, and BNBD5 was observed in the uterine horn in the luteal phase than in the follicular phase. However, BNBD10 expression of the follicular phase was significantly higher than luteal phase. Significant expression of estrogen receptor protein was observed in the uterine body, the oviduct, and the uterine horn during the follicular phase, while a significant progesterone receptor protein (PGR) expression was detected in same portions of the reproductive tract during the luteal phase. BNBD10 was found to perform key function in the uterine horn natural defense process during the follicular phase. The production of bovine uterus defensins did not influenced the expression of estrogen receptor α and progesterone receptor.

Single nucleotide polymorphisms in the FcγR3A and TAP1 genes impact ADCC in cynomolgus monkey PBMCs.

Phenotypic variability is often observed in cynomolgus monkeys on preclinical studies and may, in part, be driven by genetic variability. However, the role of monkey genetic variation remains largely unexplored in the context of drug response. This study evaluated genetic variation in cynomolgus monkey FcγR3A and TAP1 genes and the potential impact of identified polymorphisms on antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro. Studies in humans have demonstrated that a single nucleotide polymorphism (SNP), F158V, in FcγR3A can influence response to rituximab through altered ADCC and that SNPs in TAP1/2 decrease natural killer (NK) cell activity against major histocompatibility complex (MHC) class I deficient cells, potentially through altered ADCC. Monkeys were genotyped for FcγR3A and TAP1 SNPs, and ADCC was assessed in vitro using peripheral blood mononuclear cells (PBMCs) treated with trastuzumab in the presence of NCI-N87 cells. FcγR3A g.1134A>C (exonic S42R), FcγR3A g.5027A>G (intronic), and TAP1 g.1A>G (start codon loss) SNPs were all significantly associated with decreased ADCC for at least one trastuzumab concentration ≥0.0001μM when compared with wild type (WT). Regression analysis demonstrated significant association of the SNP-SNP pairs FcγR3A g.1134A>C/TAP1 g.1A>G and FcγR3A g.5027A>G/TAP1 g.1A>G with a combinatorial decrease on ADCC. Mechanisms underlying the decreased ADCC were investigated by measuring FcγR3A/IgG binding affinity and expression of FcγR3A and TAP1 in PBMCs; however, no functional associations were observed. These data demonstrate that genetic variation in cynomolgus monkeys is reflective of known human genetic variation and may potentially contribute to variable drug response in preclinical studies.

An Ancient Fecundability-Associated Polymorphism Switches a Repressor into an Enhancer of Endometrial TAP2 Expression

Variation in female reproductive traits, such as fertility, fecundity, and fecundability, is heritable in humans, but identifying and functionally characterizing genetic variants associated with these traits has been challenging. Here, we explore the functional significance and evolutionary history of a T/C polymorphism of SNP rs2071473, which we have previously shown is an eQTL for TAP2 and significantly associated with fecundability (time to pregnancy). We replicated the association between the rs2071473 genotype and TAP2 expression by using GTEx data and demonstrated that TAP2 is expressed by decidual stromal cells at the maternal-fetal interface. Next, we showed that rs2071473 is located within a progesterone-responsive cis-regulatory element that functions as a repressor with the T allele and an enhancer with the C allele. Remarkably, we found that this polymorphism arose before the divergence of modern and archaic humans, segregates at intermediate to high frequencies across human populations, and has genetic signatures of long-term balancing selection. This variant has also previously been identified in genome-wide association studies of immune-related disease, suggesting that both alleles are maintained as a result of antagonistic pleiotropy.

Differential expression of genes BPI, TAP1, SLA-1 and SLA-3 in Escherichia coli F18-resistant and sensitive Meishan post-weaning piglets

The genetic basis and mechanism of sensitivity or resistance to the significant pathogen Escherichia coli F18 is not clear in native Chinese pig breeds and may differ from that in non-native breeds. Our previous research showed that the genes BPI, TAP1, SLA-1 and SLA-3 may play important roles in resistance to E. coli F18 in post-weaning piglets. This study was based on success in selecting and identifying full sib Chinese native Meishan breed post-weaning E. coli F18-resistant and sensitive piglets. Real-time PCR was used to detect expression levels of the genes BPI, TAP1, SLA-1 and SLA-3 in liver, spleen, thymus, lymph node, duodenum, and jejunum tissues between E. coli F18-resistant and -sensitive Meishan piglets. Only the BPI gene was obviously tissue specific; it was highly expressed in the duodenum and jejunum, but expressed at a low level in other tissues. The other three genes were expressed in all the studied tissues, and particularly highly in immune organs and intestinal tissues. The expression level of BPI in the duodenum of the resistant group was significantly higher than that in the sensitive group (P < 0.01). The SLA-3 expression level in the thymus of the resistant group was significantly higher than that in the sensitive group (P < 0.05). TAP1 and SLA-1 gene expression levels were generally higher in the resistant group than the sensitive group, but there were no significant differences. Genes BPI and SLA-3 play an important role in the processes of resistance to E. coli F18 in Meishan weaned piglets. We speculate that BPI protein may have a direct killing effect on Gram-negative bacteria such as E. coli strain F18 in the intestine; the resistance of Meishan weaned piglets to E. coli F18 is likely to be related to the upregulation of intestinal BPI. The upregulation of SLA-3 may increase the resistance of weaned piglets to E. coli F18 by regulation of the immune response.

Molecular and epigenetic features of melanomas and tumor immune microenvironment linked to durable remission to ipilimumab-based immunotherapy in metastatic patients

BackgroundIpilimumab (Ipi) improves the survival of advanced melanoma patients with an incremental long-term benefit in 10–15 % of patients. A tumor signature that correlates with this survival benefit could help optimizing individualized treatment strategies.MethodsFreshly frozen melanoma metastases were collected from patients treated with either Ipi alone (n: 7) or Ipi combined with a dendritic cell vaccine (TriMixDC-MEL) (n: 11). Samples were profiled by immunohistochemistry (IHC), whole transcriptome (RNA-seq) and methyl-DNA sequencing (MBD-seq).ResultsPatients were divided in two groups according to clinical evolution: durable benefit (DB; 5 patients) and no clinical benefit (NB; 13 patients). 20 metastases were profiled by IHC and 12 were profiled by RNA- and MBD-seq. 325 genes were identified as differentially expressed between DB and NB. Many of these genes reflected a humoral and cellular immune response. MBD-seq revealed differences between DB and NB patients in the methylation of genes linked to nervous system development and neuron differentiation. DB tumors were more infiltrated by CD8+ and PD-L1+ cells than NB tumors. B cells (CD20+) and macrophages (CD163+) co-localized with T cells. Focal loss of HLA class I and TAP-1 expression was observed in several NB samples.ConclusionCombined analyses of melanoma metastases with IHC, gene expression and methylation profiling can potentially identify durable responders to Ipi-based immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0990-x) contains supplementary material, which is available to authorized users.

Oxidative metabolism drives inflammation-induced platinum resistance in human ovarian cancer.

Tumour cells have long been considered defective in mitochondrial respiration and mostly dependent on glycolytic metabolism. However, this assumption is currently challenged by several lines of evidence in a growing number of tumours. Ovarian cancer (OC) is one of the most lethal cancers worldwide, but it continues to be a poorly understood disease and its metabolic features are far to be elucidated. In this context, we investigated the role of tumour necrosis factor receptor-associated protein 1 (TRAP1), which is found upregulated in several cancer types and is a key modulator of tumour cell metabolism. Surprisingly, we found that TRAP1 expression inversely correlated with grade, stage and lower survival in a large cohort of OC patients. Accordingly, TRAP1 silencing induced resistance to cisplatin, resistant cells showed increased oxidative metabolism compared with their sensitive counterpart, and the bioenergetics cellular index of higher grade tumours indicated increased mitochondrial respiration. Strikingly, cisplatin resistance was reversible upon pharmacological inhibition of mitochondrial oxidative phosphorylation by metformin/oligomycin. At molecular level, increased oxidative metabolism in low TRAP1-expressing OC cells and tissues enhanced production of inflammatory mediators such as interleukin (IL)-6 and IL-8. Mechanistically, we identified members of the multidrug resistance complex (MDR) as key mediators of such metabolism-driven, inflammation-induced process. Indeed, treatment of OC cell lines with TNFα and IL6 induced a selective increase in the expression of TAP1 and multidrug resistance protein 1, whereas TAP1 silencing sensitized cells to cisplatin-induced apoptosis. Our results unveil a novel role for TRAP1 and oxidative metabolism in cancer progression and suggest the targeting of mitochondrial bioenergetics to increase cisplatin efficacy in human OC.