Generation and application of immortalized sheep fetal fibroblast cell line.

BACKGROUND: Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure. RESULTS: In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility. CONCLUSIONS: Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.

Proteomics Analysis of the Polyomavirus DNA Replication Initiation Complex Reveals Novel Functional Phosphorylated Residues and Associated Proteins.

Polyomavirus (PyV) Large T-antigen (LT) is the major viral regulatory protein that targets numerous cellular pathways for cellular transformation and viral replication. LT directly recruits the cellular replication factors involved in initiation of viral DNA replication through mutual interactions between LT, DNA polymerase alpha-primase (Polprim), and single-stranded DNA binding complex, (RPA). Activities and interactions of these complexes are known to be modulated by post-translational modifications; however, high-sensitivity proteomic analyses of the PTMs and proteins associated have been lacking. High-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) of the immunoprecipitated factors (IPMS) identified 479 novel phosphorylated amino acid residues (PAARs) on the three factors; the function of one has been validated. IPMS revealed 374, 453, and 183 novel proteins associated with the three, respectively. A significant transcription-related process network identified by Gene Ontology (GO) enrichment analysis was unique to LT. Although unidentified by IPMS, the ETS protooncogene 1, transcription factor (ETS1) was significantly overconnected to our dataset indicating its involvement in PyV processes. This result was validated by demonstrating that ETS1 coimmunoprecipitates with LT. Identification of a novel PAAR that regulates PyV replication and LT's association with the protooncogenic Ets1 transcription factor demonstrates the value of these results for studies in PyV biology.

Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

T antigen-specific CD8+ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Stool Glycoproteomics Signatures of Pre-Cancerous Lesions and Colorectal Cancer.

Colorectal cancer (CRC) screening relies primarily on stool analysis to identify occult blood. However, its sensitivity for detecting precancerous lesions is limited, requiring the development of new tools to improve CRC screening. Carcinogenesis involves significant alterations in mucosal epithelium glycocalyx that decisively contribute to disease progression. Building on this knowledge, we examined patient series comprehending premalignant lesions, colorectal tumors, and healthy controls for the T-antigen-a short-chain O-glycosylation of proteins considered a surrogate marker of malignancy in multiple solid cancers. We found the T-antigen in the secretions of dysplastic lesions as well as in cancer. In CRC, T-antigen expression was associated with the presence of distant metastases. In parallel, we analyzed a broad number of stools from individuals who underwent colonoscopy, which showed high T expressions in high-grade dysplasia and carcinomas. Employing mass spectrometry-based lectin-affinity enrichment, we identified a total of 262 proteins, 67% of which potentially exhibited altered glycosylation patterns associated with cancer and advanced pre-cancerous lesions. Also, we found that the stool (glyco)proteome of pre-cancerous lesions is enriched for protein species involved in key biological processes linked to humoral and innate immune responses. This study offers a thorough analysis of the stool glycoproteome, laying the groundwork for harnessing glycosylation alterations to improve non-invasive cancer detection.

Establishment and Characterization of SV40 T-Antigen Immortalized Porcine Muscle Satellite Cell.

Muscle satellite cells (MuSCs) are crucial for muscle development and regeneration. The primary pig MuSCs (pMuSCs) is an ideal in vitro cell model for studying the pig's muscle development and differentiation. However, the long-term in vitro culture of pMuSCs results in the gradual loss of their stemness, thereby limiting their application. To address this conundrum and maintain the normal function of pMuSCs during in vitro passaging, we generated an immortalized pMuSCs (SV40 T-pMuSCs) by stably expressing SV40 T-antigen (SV40 T) using a lentiviral-based vector system. The SV40 T-pMuSCs can be stably sub-cultured for over 40 generations in vitro. An evaluation of SV40 T-pMuSCs was conducted through immunofluorescence staining, quantitative real-time PCR, EdU assay, and SA-ß-gal activity. Their proliferation capacity was similar to that of primary pMuSCs at passage 1, and while their differentiation potential was slightly decreased. SiRNA-mediated interference of SV40 T-antigen expression restored the differentiation capability of SV40 T-pMuSCs. Taken together, our results provide a valuable tool for studying pig skeletal muscle development and differentiation.

Establishment of COS-BK cells persistently infected with archetype BK polyomavirus.

BK polyomavirus (BKPyV) was the first human polyomavirus to be isolated from an immunosuppressed kidney transplant recipient in 1971. BKPyV reactivation causes BKPyV-associated nephropathy and hemorrhagic cystitis. However, the mechanisms underlying BKPyV replication remain unclear. In the present study, we performed the long-term cultivation of COS-7 cells transfected with archetype KOM-5 DNA, which were designated as COS-BK cells. BKPyV derived from COS-BK cells was characterized by analyzing the amount of the virus based on hemagglutination, viral replication, and the production of viral protein 1 (VP1). Immunostaining showed that VP1-positive cells accounted for a small percentage of COS-BK cells. The nucleotide sequences encompassing the origin of the DNA replication of BKPyV derived from COS-BK cells were generated from KOM-5 by the deletion of an 8-bp sequence, which did not involve T antigen binding sites. BKPyV replicated most efficiently in COS-BK cells in DMEM containing 2% fetal bovine serum. These results indicate that COS-BK cells are a suitable culture system for studying the persistent infection of archetype BKPyV.

Molecular cloning, characterisation and molecular modelling of two novel T-synthases from mollusc origin.

The glycoprotein-N-acetylgalactosamine ß1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 ß1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.

LT and SOX9 expression are associated with gene sets that distinguish Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive malignant neuroendocrine tumour. There are two subsets of MCC, one related to Merkel cell polyomavirus (MCPyV) and the other to ultraviolet radiation (UVR). MCPyV-positive and MCPyV-negative MCCs have been considered to be different tumours, as the former harbour few DNA mutations and are not related to UVR, and the latter usually arise in sun-exposed areas and may be found in conjunction with other keratinocytic tumours, mostly squamous cell carcinomas. Two viral oncoproteins, large T antigen (LT; coded by MCPyV_gp3) and small T antigen (sT; coded by MCPyV_gp4), promote different carcinogenic pathways. OBJECTIVES: To determine which genes are differentially expressed in MCPyV-positive and MCPyV-negative MCC; to describe the mutational burden and the most frequently mutated genes in both MCC subtypes; and to identify the clinical and molecular factors that may be related to patient survival. METHODS: Ninety-two patients with a diagnosis of MCC were identified from the medical databases of participating centres. To study gene expression, a customized panel of 172 genes was developed. Gene expression profiling was performed with nCounter technology. For mutational studies, a customized panel of 26 genes was designed. Somatic single nucleotide variants (SNVs) were identified following the GATK Best Practices workflow for somatic mutations. RESULTS: The expression of LT enabled the series to be divided into two groups (LT positive, n = 55; LT negative, n = 37). Genes differentially expressed in LT-negative patients were related to epithelial differentiation, especially SOX9, or proliferation and the cell cycle (MYC, CDK6), among others. Congruently, LT displayed lower expression in SOX9-positive patients, and differentially expressed genes in SOX9-positive patients were related to epithelial/squamous differentiation. In LT-positive patients, the mean SNV frequency was 4.3; in LT-negative patients it was 10 (P = 0.03). On multivariate survival analysis, the expression of SNAI1 [hazard ratio (HR) 1.046, 95% confidence interval (CI) 1.007-1.086; P = 0.02] and CDK6 (HR 1.049, 95% CI 1.020-1.080; P = 0.001) were identified as risk factors. CONCLUSIONS: Tumours with weak LT expression tend to co-express genes related to squamous differentiation and the cell cycle, and to have a higher mutational burden. These findings are congruent with those of earlier studies.

Merkel cell carcinoma (MCC) is an aggressive form of skin tumour. There are two subtypes of MCC: one of them is related to a virus called Merkel cell polyomavirus (MCPyV); the other one is related to persistent exposure to sunlight. The aim of this research was to find differences between these subtypes in their molecular behaviour (the genes that are expressed and the mutations that may be found). To do this, we carried out two studies, one to investigate gene expression (the process cells use to convert the instructions in our DNA into a functional product such as a protein) and one to look at gene mutations (changes in the DNA sequence). We found that the tumours that were not related to MCPyV expressed genes related to epithelial differentiation (the process by which unspecialized cells gain features characteristics of epithelial cells, which, among other things, make up the outer surface of the body), which means that the origin of both MCC subtypes may be different. We also found that MCPyV-related tumours had fewer mutations. Our findings are important because they help us to understand the biology of the MCC subtypes and could help with the development of new treatments for people diagnosed with skin tumours.

Membrane-bound Merkel cell polyomavirus middle T protein constitutively activates PLCγ1 signaling through Src-family kinases.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMTY114), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

Phosphorylation of Human Polyomavirus Large and Small T Antigens: An Ignored Research Field.

Protein phosphorylation and dephosphorylation are the most common post-translational modifications mediated by protein kinases and protein phosphatases, respectively. These reversible processes can modulate the function of the target protein, such as its activity, subcellular localization, stability, and interaction with other proteins. Phosphorylation of viral proteins plays an important role in the life cycle of a virus. In this review, we highlight biological implications of the phosphorylation of the monkey polyomavirus SV40 large T and small t antigens, summarize our current knowledge of the phosphorylation of these proteins of human polyomaviruses, and conclude with gaps in the knowledge and a proposal for future research directions.

Characterization of a Human Gastrointestinal Stromal Tumor Cell Line Established by SV40LT-Mediated Immortalization.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the digestive tract and originate from the interstitial cells of Cajal (ICC), which is the pacemaker for peristaltic movement in the gastrointestinal tract. Existing GIST cell lines are widely used as cell models for in vitro experimental studies because the mutation sites are known. However, the immortalization methods of these cell lines are unknown, and no Chinese patient-derived GIST cell lines have been documented. Here, we transfected simian virus 40 large T antigen (SV40LT) into primary GIST cells to establish an immortalized human GIST cell line (ImGIST) for the first time. The ImGIST cells had neuronal cell-like irregular radioactive growth and retained the fusion growth characteristics of GIST cells. They stably expressed signature proteins, maintained the biological and genomic characteristics of normal primary GIST cells, and responded well to imatinib, suggesting that ImGIST could be a potential in vitro model for research in GIST to explore the molecular pathogenesis, drug resistance mechanisms, and the development of new adjuvant therapeutic options.

Establishment of SV40 Large T-Antigen-Immortalized Yak Rumen Fibroblast Cell Line and the Fibroblast Responses to Lipopolysaccharide.

The yak lives in harsh alpine environments and the rumen plays a crucial role in the digestive system. Rumen-associated cells have unique adaptations and functions. The yak rumen fibroblast cell line (SV40T-YFB) was immortalized by introducing simian virus 40 large T antigen (SV40T) by lentivirus-mediated transfection. Further, we have reported the effects of lipopolysaccharide (LPS) of different concentrations on cell proliferation, extracellular matrix (ECM), and proinflammatory mediators in SV40T-YFB. The results showed that the immortalized yak rumen fibroblast cell lines were identified as fibroblasts that presented oval nuclei, a fusiform shape, and positive vimentin and SV40T staining after stable passage. Chromosome karyotype analysis showed diploid characteristics of yak (n = 60). LPS at different concentrations inhibited cell viability in a dose-dependent manner. SV40T-YFB treated with LPS increased mRNA expression levels of matrix metalloproteinases (MMP-2 and MMP-9), inflammatory cytokines (TNF-α, IL-1ß, IL-6), and urokinase-type plasminogen activator system components (uPA, uPAR). LPS inhibits the expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2), plasminogen activator inhibitor-2 (PAI-2), fibronectin (FN), anti-inflammatory factor IL-10, and collagen I (COL I) in SV40T-YFB. Overall, these results suggest that LPS inhibits cell proliferation and induces ECM degradation and inflammatory response in SV40T-YFB.

Animal Models for Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects mammals and birds throughout the world. This protocol describes murine models of acute T. gondii infection, toxoplasmic encephalitis and toxoplasma retinochoroiditis. T. gondii infection in severe combined immunodeficient (SCID) mice, deficient in T and B cells, has allowed for the study of T cell-independent mechanisms of defense against intracellular organisms, as described here. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii induce protection against challenge with virulent strains of the parasite. They have allowed studies of immunization and adoptive-transfer experiments. A protocol is provided for infection with these mutant strains. The EGS strain of T. gondii has the unique feature of spontaneously forming tissue cysts in cell culture. Dual fluorescent reporter stains of this strain have allowed the study of tachyzoite to bradyzoite transitions in vitro and in vivo. A protocol for in vitro and in vivo growth of this strain and tissue cyst isolation is provided. Genetic manipulation of T. gondii and mice has led to the development of parasites that express fluorescent proteins as well as mice with fluorescently labeled leukocytes. This together with the use of T. gondii that express model antigens and transgenic mice that express the appropriate T cell receptor have facilitated the in vivo study of parasite host-interaction. In addition, parasites that express bioluminescent markers have made it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite that includes these newer methodologies. In addition, support protocols address the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of acute T. gondii infection in mice Basic Protocol 2: Model of toxoplasmic encephalitis and toxoplasma retinochoroiditis in chronically infected mice Basic Protocol 3: Assessment of T. gondii invasion into neural tissue Basic Protocol 4: T. gondii infection in scid/scid (SCID) mice Basic Protocol 5: Infection with the uracil auxotroph strain CPS1-1 or the temperature-sensitive TS-4 strain of T. gondii Basic Protocol 6: In vivo and in vitro maintenance of the EGS strain of T. gondii Support Protocol 1: Assessment of progression of infection and immune response to T. gondii Support Protocol 2: Maintenance of a bank of T. gondii cysts of the ME49 strain Support Protocol 3: Maintenance of T. gondii tachyzoites using human foreskin fibroblasts Support Protocol 4: Maintenance of T. gondii tachyzoites in mice Support Protocol 5: Preparation of T. gondii lysate antigens Support Protocol 6: Isolation of T. gondii tissue cysts from brain.

LAMP1 targeting of the large T antigen of Merkel cell polyomavirus results in potent CD4 T cell responses and tumor inhibition.

Introduction: Most cases of Merkel cell carcinoma (MCC), a rare and highly aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of oncoproteins including a truncated form of the viral large T antigen (LT) in infected cells. These oncoproteins are an attractive target for a therapeutic cancer vaccine. Methods: We designed a cancer vaccine that promotes potent, antigen-specific CD4 T cell responses to MCPyV-LT. To activate antigen-specific CD4 T cells in vivo, we utilized our nucleic acid platform, UNITE™ (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and potent antigen-specific T cell responses. LTS220A, encoding a mutated form of MCPyV-LT that diminishes its pro-oncogenic properties, was introduced into the UNITE™ platform. Results: Vaccination with LTS220A-UNITE™ DNA vaccine (ITI-3000) induced antigen-specific CD4 T cell responses and a strong humoral response that were sufficient to delay tumor growth of a B16F10 melanoma line expressing LTS220A. This effect was dependent on the CD4 T cells' ability to produce IFNγ. Moreover, ITI-3000 induced a favorable tumor microenvironment (TME), including Th1-type cytokines and significantly enhanced numbers of CD4 and CD8 T cells as well as NK and NKT cells. Additionally, ITI-3000 synergized with an α-PD-1 immune checkpoint inhibitor to further slow tumor growth and enhance survival. Conclusions: These findings strongly suggest that in pre-clinical studies, DNA vaccination with ITI-3000, using the UNITE™ platform, enhances CD4 T cell responses to MCPyV-LT that result in significant anti-tumor immune responses. These data support the initiation of a first-in-human (FIH) Phase 1 open-label study to evaluate the safety, tolerability, and immunogenicity of ITI-3000 in patients with polyomavirus-positive MCC (NCT05422781).

Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells.

BACKGROUND: Patients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous T-cell products and fratricide of CAR-T cells targeting T-lineage antigens. Recently, allogeneic double-negative T cells (DNTs) have been shown to be safe as an off-the-shelf adoptive cell therapy and to be amendable for CAR transduction. Here, we explore the antitumor activity of allogeneic DNTs against T-cell malignancies and the potential of using anti-CD4-CAR (CAR4)-DNTs as adoptive cell therapy for T-cell malignancies. METHODS: Healthy donor-derived allogeneic DNTs were ex vivo expanded with or without CAR4 transduction. The antitumor activity of DNTs and CAR4-DNTs against T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) were examined using flow cytometry-based cytotoxicity assays and xenograft models. Mechanisms of action were investigated using transwell assays and blocking assays. RESULTS: Allogeneic DNTs induced endogenous antitumor cytotoxicity against T-ALL and PTCL in vitro, but high doses of DNTs were required to attain therapeutic effects in vivo. The potency of DNTs against T-cell malignancies was significantly enhanced by transducing DNTs with a third-generation CAR4. CAR4-DNTs were manufactured without fratricide and showed superior cytotoxicity against CD4+ T-ALL and PTCL in vitro and in vivo relative to empty-vector transduced-DNTs. CAR4-DNTs eliminated T-ALL and PTCL cell lines and primary T-ALL blasts in vitro. CAR4-DNTs effectively infiltrated tumors, delayed tumor progression, and prolonged the survival of T-ALL and PTCL xenografts. Further, pretreatment of CAR4-DNTs with PI3Kδ inhibitor idelalisib promoted memory phenotype of CAR4-DNTs and enhanced their persistence and antileukemic efficacy in vivo. Mechanistically, LFA-1, NKG2D, and perforin/granzyme B degranulation pathways were involved in the DNT-mediated and CAR4-DNT-mediated killing of T-ALL and PTCL. CONCLUSIONS: These results demonstrate that CAR4-DNTs can effectively target T-ALL and PTCL and support allogeneic CAR4-DNTs as adoptive cell therapy for T-cell malignancies.

BK Polyomavirus-Associated Urothelial Carcinoma of the Bladder with a Background of BK Polyomavirus Nephropathy in a Kidney Transplant Recipient.

Renal transplant recipients are at increased risk for the development of a malignant neoplasm. Polyomavirus-associated urothelial carcinoma is a rare tumor that occurs in renal transplant recipients, with approximately 41 cases reported since 2002. It accounts for 27-31% of all post-transplant urothelial carcinomas and develops at an average of 8.5 years after transplantation. Histologically, it shows high-grade urothelial carcinoma (95.1%) with a high frequency of glandular differentiation and micropapillary structures (58.5%) and positive immunohistochemistry for polyomavirus large T antigen, p53 (92.9%), and p16 (100%). We encountered a case of BK polyomavirus (BKPyV)-associated urothelial carcinoma of the bladder diagnosed 54 months after kidney transplantation. Histologically, it was a high-grade urothelial carcinoma with micropapillary features, and immunohistochemically, it was diffusely positive for polyomavirus large T antigen, p16, and p53. BKPyV DNA and mRNA for BKPyV large T antigen have been identified in tissues using real-time polymerase chain reaction. The same sequence of the BKPyV VP1 genome hypervariable region was detected in both transplanted kidney tissue with polyomavirus nephropathy and urothelial carcinoma tissue, suggesting that polyomavirus-associated urothelial carcinoma developed in a background of persistent polyomavirus nephropathy. This case showed typical histological features and was detected and treated at an earlier stage than has been reported. It is important to keep in mind that polyomavirus-associated urothelial carcinoma can develop early after transplantation and might be associated with polyomavirus nephropathy. Because of its rapidly progressive nature, careful follow-up with urine cytology and cystoscopy is necessary. We report this case with a literature review.

SND1, a novel co-activator of HIF1α, promotes tumor initiation in PyMT-induced breast tumor.

The multifunctional protein staphylococcal nuclease domain-containing protein 1 (SND1) is conserved and has been implicated in several aspects of tumor development, such as proliferation, epithelial-mesenchymal transition, and immune evasion. Despite this, the precise role of SND1 in the initiation and metastasis of mammary gland tumors remains largely unexplored. In this study, we utilized a mouse model of breast tumors induced by polyomavirus middle T antigen (PyMT) to demonstrate that the knockout of SND1 significantly delayed the onset of primary mammary tumor formation induced by PyMT. Histological staining and cytometric analysis were conducted to confirm the reduction of tumor-initiating cells and lung metastasis following depletion of SND1. Additionally, our findings demonstrate that enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a crucial epigenetic modifier implicated in PyMT-induced breast tumors, serves as an essential mediator of SND1-promoted primary mammary tumor formation. Mechanistic investigations revealed that SND1 functions as a transcriptional co-activator of hypoxia-inducible factor 1 subunit alpha (HIF1α), thereby regulating the downstream target gene EZH2 and promoting tumorigenesis. Overall, this study provides novel insights into the role of SND1 as a co-activator of HIF1α in the acceleration of PyMT-induced spontaneous breast tumor formation through the promotion of EZH2 transcription. The findings provide novel insights into the relationship between SND1 and the formation of tumor-initiating cells.

Reduction in disialyl-T antigen levels in mice deficient for both St6galnac3 and St6galnac4 results in blood filling of lymph nodes.

Sialic acid (SA) is present at the terminal ends of carbohydrate chains in glycoproteins and glycolipids and is involved in various biological phenomena. The biological function of the disialyl-T (SAα2-3Galß1-3(SAα2-6)GalNAcα1-O-Ser/Thr) structure is largely unknown. To elucidate the role of disialyl-T structure and determine the key enzyme from the N-acetylgalactosaminide α2,6-sialyltransferase (St6galnac) family involved in its in vivo synthesis, we generated St6galnac3- and St6galnac4-deficient mice. Both single-knockout mice developed normally without any prominent phenotypic abnormalities. However, the St6galnac3::St6galnact4 double knockout (DKO) mice showed spontaneous hemorrhage of the lymph nodes (LN). To identify the cause of bleeding in the LN, we examined podoplanin, which modifies the disialyl-T structures. The protein expression of podoplanin in the LN of DKO mice was similar to that in wild-type mice. However, the reactivity of MALII lectin, which recognizes disialyl-T, in podoplanin immunoprecipitated from DKO LN was completely abolished. Moreover, the expression of vascular endothelial cadherin was reduced on the cell surface of high endothelial venule (HEV) in the LN, suggesting that hemorrhage was caused by the structural disruption of HEV. These results suggest that podoplanin possesses disialyl-T structure in mice LN and that both St6galnac3 and St6galnac4 are required for disialyl-T synthesis.

ST6GALNAC4 promotes hepatocellular carcinogenesis by inducing abnormal glycosylation.

Hepatocellular carcinoma (HCC) is one of the most lethal tumor types worldwide. Glycosylation has shown promise in the study of tumor mechanisms and treatment. The glycosylation status of HCC and the underlying molecular mechanisms are still not fully elucidated. Using bioinformatic analysis we obtained a more comprehensive characterization of glycosylation of HCC. Our analysis presented that high glycosylation levels might correlate with tumor progression and poor prognosis. Subsequent Experiments identified key molecular mechanisms for ST6GALNAC4 promoting malignant progression by inducing abnormal glycosylation. We confirmed the contribution of ST6GALNAC4 to proliferation, migration, and invasion in vitro and in vivo. Mechanistic studies revealed that ST6GALNAC4 may be induced abnormal TGFBR2 glycosylation, resulting in the higher protein levels of TGFBR2 and TGF[Formula: see text] pathway increased activation. Our study also provided a further understand of immunosuppressive function of ST6GALNAC4 through T antigen-galectin3+ TAMs axis. This study has provided one such possibility that galectin3 inhibitors might be an acceptable treatment choice for HCC patients with high T antigen expression.