Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.

The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells.

The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.

Dual-inhibitory domain iCARs improve the efficiency of the AND-NOT gate CAR T strategy.

CAR (chimeric antigen receptor) T cell therapy has shown clinical success in treating hematological malignancies, but its treatment of solid tumors has been limited. One major challenge is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected nonmalignant tissue sites. However, the strategy seems inefficient, requiring high levels of iCAR and its target antigen for inhibition. Using a TROP2-targeting iCAR with a single PD1 inhibitory domain to inhibit a CEACAM5-targeting CAR (CEACAR), we observed that the inefficiency was due to a kinetic delay in iCAR inhibition of cytotoxicity. To improve iCAR efficiency, we modified three features of the iCAR-the avidity, the affinity, and the intracellular signaling domains. Increasing the avidity but not the affinity of the iCAR led to significant reductions in the delay. iCARs containing twelve different inhibitory signaling domains were screened for improved inhibition, and three domains (BTLA, LAIR-1, and SIGLEC-9) each suppressed CAR T function but did not enhance inhibitory kinetics. When inhibitory domains of LAIR-1 or SIGLEC-9 were combined with PD-1 into a single dual-inhibitory domain iCAR (DiCARs) and tested with the CEACAR, inhibition efficiency improved as evidenced by a significant reduction in the inhibitory delay. These data indicate that a delicate balance between CAR and iCAR signaling strength and kinetics must be achieved to regulate AND-NOT gate CAR T cell selectivity.

IRIS: Discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing.

Alternative splicing (AS) is prevalent in cancer, generating an extensive but largely unexplored repertoire of novel immunotherapy targets. We describe Isoform peptides from RNA splicing for Immunotherapy target Screening (IRIS), a computational platform capable of discovering AS-derived tumor antigens (TAs) for T cell receptor (TCR) and chimeric antigen receptor T cell (CAR-T) therapies. IRIS leverages large-scale tumor and normal transcriptome data and incorporates multiple screening approaches to discover AS-derived TAs with tumor-associated or tumor-specific expression. In a proof-of-concept analysis integrating transcriptomics and immunopeptidomics data, we showed that hundreds of IRIS-predicted TCR targets are presented by human leukocyte antigen (HLA) molecules. We applied IRIS to RNA-seq data of neuroendocrine prostate cancer (NEPC). From 2,939 NEPC-associated AS events, IRIS predicted 1,651 epitopes from 808 events as potential TCR targets for two common HLA types (A*02:01 and A*03:01). A more stringent screening test prioritized 48 epitopes from 20 events with "neoantigen-like" NEPC-specific expression. Predicted epitopes are often encoded by microexons of ≤30 nucleotides. To validate the immunogenicity and T cell recognition of IRIS-predicted TCR epitopes, we performed in vitro T cell priming in combination with single-cell TCR sequencing. Seven TCRs transduced into human peripheral blood mononuclear cells (PBMCs) showed high activity against individual IRIS-predicted epitopes, providing strong evidence of isolated TCRs reactive to AS-derived peptides. One selected TCR showed efficient cytotoxicity against target cells expressing the target peptide. Our study illustrates the contribution of AS to the TA repertoire of cancer cells and demonstrates the utility of IRIS for discovering AS-derived TAs and expanding cancer immunotherapies.

Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros.

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Liver resection with two-step vascular exclusion, in situ hypothermic portal perfusion for the treatment of end-stage hepatic alveolar echinococcosis.

PURPOSE: To evaluate the safety and efficacy of two-step vascular exclusion and in situ hypothermic portal perfusion in patients with end-stage hepatic hydatidosis. METHODS: This study involved patients with advanced hepatic hydatid disease undergoing surgical treatment between 2022 and 2023, which included resection and reconstruction of the hepatic veins, inferior vena cava (IVC), and portal vein (PV). We described the technical details of liver resection and vascular reconstruction, as well as the use of two-step vascular exclusion and in situ hypothermic portal perfusion techniques during the vascular reconstruction process. RESULT: We included 7 patients with advanced hepatic hydatid disease who underwent surgical resection using two-step vascular exclusion and in situ hypothermic portal perfusion. The mean duration of surgery was 12.5 h (range, 7.5-15.0 h). The average hepatic ischemia time was 45 min (range, 25-77 min), while the occlusion time of the IVC was 87 min (range, 72-105 min). The total blood loss was 1000 milliliters (range, 500-1250 milliliters). Postoperatively, patients exhibited good recovery of liver and renal function. The mean ICU stay was 2 days (range, 1-3 days), and the mean postoperative hospital stay was 13 days (range, 9-16 days), with no Grade III or above complications observed during a mean follow-up period of 15 months (range, 9-24 months), CONCLUSION: two-step vascular exclusion and in situ hypothermic portal perfusion for surgical resection of end-stage hepatic hydatid disease is safe and effective. This significantly reduces the anhepatic time.

Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning.

T cell receptors (TCRs) are generated by somatic recombination of V/D/J segments to produce up to 1015 unique sequences. Highly sensitive and specific techniques are required to isolate and identify the rare TCR sequences that respond to antigens of interest. Here, we describe the use of mRNA sequencing via cross-linker regulated intracellular phenotype (CLInt-Seq) for efficient recovery of antigen-specific TCRs in cells stained for combinations of intracellular proteins such as cytokines or transcription factors. This method enables high-throughput identification and isolation of low-frequency TCRs specific for any antigen. As a proof of principle, intracellular staining for TNFα and IFNγ identified cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-reactive TCRs with efficiencies similar to state-of-the-art peptide-MHC multimer methodology. In a separate experiment, regulatory T cells were profiled based on intracellular FOXP3 staining, demonstrating the ability to examine phenotypes based on transcription factors. We further optimized the intracellular staining conditions to use a chemically cleavable primary amine cross-linker compatible with current single-cell sequencing technology. CLInt-Seq for TNFα and IFNγ performed similarly to isolation with multimer staining for EBV-reactive TCRs. We anticipate CLInt-Seq will enable droplet-based single-cell mRNA analysis from any tissue where minor populations need to be isolated by intracellular markers.

Real-time indocyanine green fluorescence imaging and navigation for cone unit laparoscopic hepatic resection of intrahepatic duct stone: a case series study.

BACKGROUND: Intrahepatic bile duct stones, although common and benign, require varying therapeutic strategies due to their recurrent nature. Inadequate management can escalate to liver cirrhosis or cholangiocarcinoma. A surgical method merging indocyanine green fluorescence imaging (ICG-FI) with liver cone unit resection is optimal, ensuring complete lesion removal and healthy liver tissue conservation. METHOD: A retrospective descriptive study was conducted on 15 patients with intrahepatic bile duct stones who were admitted to Sichuan Provincial People's Hospital from January 2021 to December 2023. All patients underwent laparoscopic anatomical liver resection guided by ICG-FI. RESULTS: Among the 15 patients included in the study, ten were male and five were female, with an average age of 52 years. All patients were free from underlying medical conditions. Intraoperatively, ICG-FI was good, with clear boundaries, and all patients successfully underwent surgery without any conversions to open surgery. The mean operative time was 236 ± 56 min, and the estimated blood loss was 320 ± 75 ml. Patients had a postoperative hospital stay of 5.5 ± 1.5 days. No severe complications occurred. CONCLUSIONS: Real-time ICG-FI with anatomical liver resection is a safe and effective approach for managing intrahepatic bile duct stones.

Pathway-guided analysis identifies Myc-dependent alternative pre-mRNA splicing in aggressive prostate cancers.

We sought to define the landscape of alternative pre-mRNA splicing in prostate cancers and the relationship of exon choice to known cancer driver alterations. To do so, we compiled a metadataset composed of 876 RNA-sequencing (RNA-Seq) samples from five publicly available sources representing a range of prostate phenotypes from normal tissue to drug-resistant metastases. We subjected these samples to exon-level analysis with rMATS-turbo, purpose-built software designed for large-scale analyses of splicing, and identified 13,149 high-confidence cassette exon events with variable incorporation across samples. We then developed a computational framework, pathway enrichment-guided activity study of alternative splicing (PEGASAS), to correlate transcriptional signatures of 50 different cancer driver pathways with these alternative splicing events. We discovered that Myc signaling was correlated with incorporation of a set of 1,039 cassette exons enriched in genes encoding RNA binding proteins. Using a human prostate epithelial transformation assay, we confirmed the Myc regulation of 147 of these exons, many of which introduced frameshifts or encoded premature stop codons. Our results connect changes in alternative pre-mRNA splicing to oncogenic alterations common in prostate and many other cancers. We also establish a role for Myc in regulating RNA splicing by controlling the incorporation of nonsense-mediated decay-determinant exons in genes encoding RNA binding proteins.

T cell antigen discovery via trogocytosis.

T cell receptor (TCR) ligand discovery is essential for understanding and manipulating immune responses to tumors. We developed a cell-based selection platform for TCR ligand discovery that exploits a membrane transfer phenomenon called trogocytosis. We discovered that T cell membrane proteins are transferred specifically to target cells that present cognate peptide-major histocompatibility complex (MHC) molecules. Co-incubation of T cells expressing an orphan TCR with target cells collectively presenting a library of peptide-MHCs led to specific labeling of cognate target cells, enabling isolation of these target cells and sequencing of the cognate TCR ligand. We validated this method for two clinically employed TCRs and further used the platform to identify the cognate neoepitope for a subject-derived neoantigen-specific TCR. Thus, target cell trogocytosis is a robust tool for TCR ligand discovery that will be useful for studying basic tumor immunology and identifying new targets for immunotherapy.

Ras Homolog Family Member F, Filopodia Associated Promotes Hepatocellular Carcinoma Metastasis by Altering the Metabolic Status of Cancer Cells Through RAB3D.

BACKGROUND AND AIMS: The mechanism by which tumor cells resist metabolic stress remains unclear, but many oncogenes are known to regulate this process. Accordingly, metabolic stress is closely associated with tumor metastasis. In this study, gene chip technology showed that Ras homolog family member F, filopodia associated (RHOF), a member of the Rho guanosine triphosphatase family, is an oncogene that is significantly related to hepatocellular carcinoma (HCC) metastasis; however, it has rarely been reported in tumors. Our aim was to determine the clinicopathological significance and role of RHOF in HCC progression and investigate the associated mechanisms. APPROACH AND RESULTS: The results showed that compared to expression in adjacent noncancerous tissues, RHOF was frequently up-regulated in HCC tumor samples and elevated under conditions of glucose deprivation. RHOF expression was associated with tumor-node-metastasis stage, T grade, metastasis status, recurrence, and survival in HCC. RHOF also affected cell morphology and promoted migration, invasion, and epithelial-mesenchymal transition (EMT) of HCC cell lines. Analysis of the underlying mechanism showed that RHOF promoted the Warburg effect by up-regulating the expression and function of several glycolytic enzymes in HCC cells. This metabolic shift enhanced HCC cell migration and invasion. Specifically, RHOF exerted a tumor-promoting effect by directly interacting with AMP-activated protein kinase (AMPK) and increasing the phosphorylation of AMPK. This subsequently affected RAB3D mRNA stability and led to elevated RAB3D expression, thereby amplifying the Warburg effect and malignant biological behaviors of HCC cells. CONCLUSIONS: RHOF helps tumor cells resist metabolic stress through modulating the Warburg effect and plays a critical role in promoting HCC cell migration, invasion, and EMT, highlighting its important role in remodeling the metastatic microenvironment and regulating tumor metastasis. RHOF shows potential as a therapeutic target and prognostic biomarker for HCC.

Isolation and characterization of NY-ESO-1-specific T cell receptors restricted on various MHC molecules.

Tumor-specific T cell receptor (TCR) gene transfer enables specific and potent immune targeting of tumor antigens. Due to the prevalence of the HLA-A2 MHC class I supertype in most human populations, the majority of TCR gene therapy trials targeting public antigens have employed HLA-A2-restricted TCRs, limiting this approach to those patients expressing this allele. For these patients, TCR gene therapy trials have resulted in both tantalizing successes and lethal adverse events, underscoring the need for careful selection of antigenic targets. Broad and safe application of public antigen-targeted TCR gene therapies will require (i) selecting public antigens that are highly tumor-specific and (ii) targeting multiple epitopes derived from these antigens by obtaining an assortment of TCRs restricted by multiple common MHC alleles. The canonical cancer-testis antigen, NY-ESO-1, is not expressed in normal tissues but is aberrantly expressed across a broad array of cancer types. It has also been targeted with A2-restricted TCR gene therapy without adverse events or notable side effects. To enable the targeting of NY-ESO-1 in a broader array of HLA haplotypes, we isolated TCRs specific for NY-ESO-1 epitopes presented by four MHC molecules: HLA-A2, -B07, -B18, and -C03. Using these TCRs, we pilot an approach to extend TCR gene therapies targeting NY-ESO-1 to patient populations beyond those expressing HLA-A2.

Comparison of flexural properties and cytotoxicity of interim materials printed from mono-LCD and DLP 3D printers.

STATEMENT OF PROBLEM: Because few 3D-printing resins have been specifically developed for liquid crystal display (LCD) 3D printers, mono-LCD users may use digital light processing (DLP) 3D-printing resins. However, the suitability of these resins requires evaluation. PURPOSE: The purpose of this in vitro study was to evaluate whether 3D-printing resins designed for DLP 3D printers can be used successfully in a mono-LCD 3D printer. MATERIAL AND METHODS: 3D printers based on photopolymerization and 3D-printing resin for interim restorations were used in this study. Enlighten AA temp and NextDent C&B MFH were printed from both the MiiCraft Ultra 125 and Phrozen Sonic printers, followed by postpolymerization by using the FormCure and PhrozenCure units for different times. The flexural strength and cytotoxicity of the specimens were evaluated. RESULTS: After postpolymerization treatment, the flexural strength of Enlighten AA temp and NextDent C&B MFH from both 3D printers was over the 50-MPa minimal requirement for the flexural strength of interim resins specified in the International Standards Organization (ISO) 10477 standard. With 15 minutes of FormCure and 1 minute of PhrozenCure postpolymerization, 4 material-printer combinations reached nearly 100% in cell viability. CONCLUSIONS: Interim resins designed for DLP 3D printers can be successfully used in mono-LCD 3D printers if the printed specimens are postpolymerized in a more powerful postpolymerization unit or in a less powerful postpolymerization unit for a longer time.

Bond strength of three chairside crown reline materials to milled polymethyl methacrylate resin.

STATEMENT OF PROBLEM: Information on the bond strength of milled polymethyl methacrylate interim restorations when relined with chairside reline materials is lacking. PURPOSE: The purpose of this in vitro study was to measure the shear bond strength of various combinations of 3 different chairside reline materials bonded to milled polymethyl methacrylate blocks with 3 different types of surface treatments. MATERIALS AND METHODS: Uniform blocks (10×10×22 mm) were milled from tooth-colored polymethyl methacrylate disks (Vivid PMMA; Pearson Dental Supply Co). The surface treatments tested were airborne-particle abrasion with 50-µm particle size aluminosilicate, application of acrylic resin monomer (Jet Liquid; Lang Dental Manufacturing Co) for 180 seconds, and airborne-particle abrasion with monomer application. The control groups were blocks with no surface treatment. The chairside reline materials tested were Jet acrylic resin (Jet Powder; Lang Dental Manufacturing Co), bis-acryl resin (Integrity; Dentsply Sirona), and flowable composite resin (Reveal; Bisco). All materials were applied through a Ø1.5×3-mm bonding ring. Ten specimens for each of the 12 groups were tested in a universal testing machine. Load was applied at a crosshead speed of 1 mm/min. Fracture surfaces were then analyzed for cohesive versus adhesive or mixed failure. Data were analyzed using 2-way ANOVA and Tukey-Kramer post hoc analysis (α=.05). RESULTS: The mean shear bond strength values ranged from 1.77 ±0.79 MPa to 28.49 ±5.75 MPa. ANOVA revealed that reline material (P<.05), surface treatment (P<.05), and their interactions (P<.05) significantly affected the shear bond strength among the experimental groups. The strongest combination was Jet acrylic resin applied on specimens treated with airborne-particle abrasion and monomer. All 3 failure modalities (adhesive, cohesive, and mixed modes) were observed. CONCLUSIONS: Of the materials tested, the most reliable material to bond to milled polymethyl methacrylate was Jet acrylic resin, and the bond strength values were increased substantially when the milled polymethyl methacrylate surface was airborne-particle abraded and monomer was applied.

Prostate epithelial cell of origin determines cancer differentiation state in an organoid transformation assay.

The cell of origin for prostate cancer remains a subject of debate. Genetically engineered mouse models have demonstrated that both basal and luminal cells can serve as cells of origin for prostate cancer. Using a human prostate regeneration and transformation assay, our group previously demonstrated that basal cells can serve as efficient targets for transformation. Recently, a subpopulation of multipotent human luminal cells defined by CD26 expression that retains progenitor activity in a defined organoid culture was identified. We transduced primary human prostate basal and luminal cells with lentiviruses expressing c-Myc and activated AKT1 (myristoylated AKT1 or myrAKT1) to mimic theMYCamplification andPTENloss commonly detected in human prostate cancer. These cells were propagated in organoid culture before being transplanted into immunodeficient mice. We found that c-Myc/myrAKT1-transduced luminal xenografts exhibited histological features of well-differentiated acinar adenocarcinoma, with strong androgen receptor (AR) and prostate-specific antigen (PSA) expression. In contrast, c-Myc/myrAKT1-transduced basal xenografts were histologically more aggressive, with a loss of acinar structures and low/absent AR and PSA expression. Our findings imply that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types subjected to the same oncogenic insults. This study provides a platform for the functional evaluation of oncogenes in basal and luminal epithelial populations of the human prostate. Tumors derived in this fashion with defined genetics can be used in the preclinical development of targeted therapeutics.

Activation of Notch1 synergizes with multiple pathways in promoting castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.