Cross-sensitivity immune SPR sensor based on fan-shaped microstructured optical fiber for temperature and refractive index sensing.

To avoid coating and filling into the fiber holes, facilitate the phase-matching and eliminate cross-sensitivity problems, we propose a surface plasmon resonance sensor based on a fan-shaped microstructured optical fiber (MOF) for the simultaneous sensing of temperature and refractive index (RI). The fan-shaped structure is fabricated by polishing two sides of MOF with an angle of 120°. One side is coated with the gold film and polydimethylsiloxane layer for temperature sensing, and the other side is only coated with the gold film for RI sensing. The two sensing sides can support resonance peaks with two polarized directions at the angle of 120°, which are independent without cross-sensitivity. By monitoring the shifts of the two polarized peaks, our numerical results show that the temperature sensitivity is 2.932 nm/°C in the range of 30 °C to 40 °C, and RI sensitivity is 4235 nm/RIU in the range of 1.38 to 1.39, respectively.

ZNF683 marks a CD8+ T cell population associated with anti-tumor immunity following anti-PD-1 therapy for Richter syndrome.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.

Isolation of Limosilactobacillus reuteri Strain with Anti-porcine Epidemic Diarrhea Virus from Swine Feces.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea diseases in piglets, which has brought huge economic losses to the pig industry. As the dominant Lactobacillus species in the piglet intestine, the antiviral effect of Limosilactobacillus reuteri (L. reuteri) has been reported. Nine L. reuteri strains were isolated and identified from swine feces in this study. The CCK-8 assay examined the anti-PEDV potential of their cell-free supernatant (CFS). Among the nine L. reuteri isolates examined, LRC8 had a higher inhibition rate to PEDV than the other strains. Thus, the biological properties of the LRC8 strain, such as growth ability, acid production ability, acid and bile salt tolerance, and adhesion to IPEC-J2 cells, were evaluated. Besides, the anti-PEDV activity of LRC8-CFS (LRC8 metabolites, LRM) was assessed using plaque reduction assays, indirect immunofluorescence assays, RT-qPCR, and western blotting. The mRNA relative expression levels of inflammatory factors including IL-1ß, IL-6, IL-8, MCP1, and TNF-α were determined by RT-qPCR. The results showed that the LRC8 strain grew well, was resistant to acid, tolerated bile salts, and adhered strongly to IPEC-J2 cells. In addition, treatment with its CFS (LRM) dramatically downregulated the mRNA expression levels of inflammatory cytokines, and in the Vero cell culture, prophylactic, therapeutic, competitive, and direct-inhibitory actions were seen against PEDV. Finally, we explored the anti-PEDV effects of the LRC8 strain in piglets and found that the LRC8 strain effectively relieved the clinical symptoms and intestinal damage of piglets infected by PEDV. To sum up, we found a L. reuteri strain with an anti-PEDV effect.

Characterization and pathogenicity evaluation of recombinant novel duck reovirus isolated from Southeast China.

The novel duck reovirus (NDRV) emerged in southeast China in 2005. The virus causes severe liver and spleen hemorrhage and necrosis in various duck species, bringing serious harm to waterfowl farming. In this study, three strains of NDRV designated as NDRV-ZSS-FJ20, NDRV-LRS-GD20, and NDRV-FJ19 were isolated from diseased Muscovy ducks in Guangdong and Fujian provinces. Pairwise sequence comparisons revealed that the three strains were closely related to NDRV, with nucleotide sequence identities for 10 genomic fragments ranging between 84.8 and 99.8%. In contrast, the nucleotide sequences of the three strains were only 38.9-80.9% similar to the chicken-origin reovirus and only 37.6-98.9% similar to the classical waterfowl-origin reovirus. Similarly, phylogenetic analysis revealed that the three strains clustered together with NDRV and were significantly different from classical waterfowl-origin reovirus and chicken-origin reovirus. In addition, the analyses showed that the L1 segment of the NDRV-FJ19 strain was a recombinant of 03G and J18 strains. Experimental reproduction of the disease showed that the NDRV-FJ19 strain was pathogenic to both ducks and chickens and could lead to symptoms of hemorrhage and necrosis in the liver and spleen. This was somewhat different from previous reports that NDRV is less pathogenic to chickens. In conclusion, we speculated that the NDRV-FJ19 causing duck liver and spleen necrosis is a new variant of a duck orthoreovirus that is significantly different in pathogenicity from any previously reported waterfowl-origin orthoreovirus.

An ultrasensitive electrochemical sensor for detecting porcine epidemic diarrhea virus based on a Prussian blue-reduced graphene oxide modified glassy carbon electrode.

This study developed a novel, ultrasensitive sandwich-type electrochemical immunosensor for detecting the porcine epidemic diarrhea virus (PEDV). By electrochemical co-deposition of graphene and Prussian blue, a Prussian blue-reduced graphene oxide-modified glassy carbon electrode was made, further modified with PEDV-monoclonal antibodies (mAbs) to create a new PEDV immunosensor using the double antibody sandwich technique. The electrochemical characteristics of several modified electrodes were investigated using cyclic voltammetry (CV). We optimized the pH levels and scan rate. Additionally, we examined specificity, reproducibility, repeatability, accuracy, and stability. The study indicates that the immunosensor has good performance in the concentration range of 1 × 101.88 to 1 × 105.38 TCID50/mL of PEDV, with a detection limit of 1 × 101.93 TCID50/mL at a signal-to-noise ratio of 3σ. The composite membranes produced via co-deposition of graphene and Prussian blue effectively increased electron transport to the glassy carbon electrode, boosted response signals, and increased the sensitivity, specificity, and stability of the immunosensor. The immunosensor could accurately detect PEDV, with results comparable to real-time quantitative PCR. This technique was applied to PEDV detection and served as a model for developing additional immunosensors for detecting hazardous chemicals and pathogenic microbes.

Difference analysis of intestinal microbiota and metabolites in piglets of different breeds exposed to porcine epidemic diarrhea virus infection.

The gut microbial composition of the Luchuan (LC) piglet, one of China's native breeds, has rarely been studied, especially when compared to other breeds. This study developed a porcine epidemic diarrhea virus (PEDV) infection model in LC and Largewhite (LW) piglets, and analyzed the patterns and differences of intestinal microbial communities and metabolites in piglets of these two breeds after infection. The diarrhea score, survival time, and distribution of viral antigens in the intestine of piglets infected with PEDV differed among breeds, with the jejunal immunohistochemistry score of LW piglets being significantly higher than that of LC piglets (P < 0.001). The results of 16S rRNA sequencing showed differences in microbial diversity and community composition in the intestine of piglets with different breeds between PEDV infection piglets and the healthy controls. There were differences in the species and number of dominant phyla and dominant genera in the same intestinal segment. The relative abundance of Shigella in the jejunum of LC piglets after PEDV infection was significantly lower than that of LW piglets (P < 0.05). The key microorganisms differed in the microbiota were Streptococcus alactolyticus, Roseburia faecis, Lactobacillus iners, Streptococcus equi, and Lactobacillus mucosae (P < 0.05). The non-targeted metabolite analysis revealed that intestinal metabolites showed great differences among the different breeds related to infection. Spearman correlation analysis was conducted to examine any links between the microbiota and metabolites. The metabolites in the intestine of different breeds related to infection were mainly involved in arginine biosynthesis, synaptic vesicle cycle, nicotinic acid and nicotinamide metabolism and mTOR signaling pathway, with significantly positive or negative correlations (P < 0.05) between the various microorganisms. This study provides a theoretical foundation for investigating the application of core microorganisms in the gut of piglets of different breeds in the digestive tracts of those infected with PEDV, and helps to tackle the antimicrobial resistance problem further.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Landscape of helper and regulatory antitumour CD4+ T cells in melanoma.

Within the tumour microenvironment, CD4+ T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules1,2, but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4+ T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4+ T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4+ T regulatory (TReg) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4+ TReg clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4+ T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4+ TReg cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

Genetic variation analysis of the sigma B protein gene of novel duck reovirus in southeastern China from 2011 to 2020.

The novel duck reovirus (NDRV) disease first appeared in China in 2011. Infected ducks may be of various ages and breeds. Sigma B protein, which is the key component of NDRV's outer capsid, may trigger group-specific neutralizing antibodies linked to NDRV infection, pathogenicity, and immune defense. The sigma B protein gene of fourteen NDRV field strains was amplified by RT-PCR and cloned into the pMD-18 T vector for sequencing to examine the genetic variation in sigma B proteins of NDRVs in southeastern China between 2011 and 2020. The sigma B protein gene of the fourteen NDRV southeastern strains included in this analysis had 96.3 %-99.8 % nucleotide, and 96.2 %-99.7 % deduced amino acid sequence homology. Phylogenetic analysis revealed that the fourteen southeastern strains belonged to a well-supported lineage that included NDRV and Muscovy duck reovirus (MDRV) strains. However, Avian reovirus (ARV) formed a distinct genetic lineage in the gene tree. The sigma B protein gene sequences of NDRV strains found in southeastern China are substantially conserved, according to these findings. There is no significant geographical difference between NDRV southeastern strains and DRV strains in other regions of China. Our findings will add to the molecular epidemiological picture of NDRV strain spread in southeastern China between 2011 and 2020, laying the groundwork for potential in-depth research on vaccine collection and comprehensive prevention.

Unannotated proteins expand the MHC-I-restricted immunopeptidome in cancer.

Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets.

Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment.

Lineage-tracing methods have enabled characterization of clonal dynamics in complex populations, but generally lack the ability to integrate genomic, epigenomic and transcriptomic measurements with live-cell manipulation of specific clones of interest. We developed a functionalized lineage-tracing system, ClonMapper, which integrates DNA barcoding with single-cell RNA sequencing and clonal isolation to comprehensively characterize thousands of clones within heterogeneous populations. Using ClonMapper, we identified subpopulations of a chronic lymphocytic leukemia cell line with distinct clonal compositions, transcriptional signatures and chemotherapy survivorship trajectories; patterns that were also observed in primary human chronic lymphocytic leukemia. The ability to retrieve specific clones before, during and after treatment enabled direct measurements of clonal diversification and durable subpopulation transcriptional signatures. ClonMapper is a powerful multifunctional approach to dissect the complex clonal dynamics of tumor progression and therapeutic response.

Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma.

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.

Longitudinal Single-Cell Dynamics of Chromatin Accessibility and Mitochondrial Mutations in Chronic Lymphocytic Leukemia Mirror Disease History.

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics. SIGNIFICANCE: Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution. See related commentary by Hilton and Scott, p. 2965. This article is highlighted in the In This Issue feature, p. 2945.

Molecular and cellular features of CTLA-4 blockade for relapsed myeloid malignancies after transplantation.

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma.

Personal neoantigen vaccines have been envisioned as an effective approach to induce, amplify and diversify antitumor T cell responses. To define the long-term effects of such a vaccine, we evaluated the clinical outcome and circulating immune responses of eight patients with surgically resected stage IIIB/C or IVM1a/b melanoma, at a median of almost 4 years after treatment with NeoVax, a long-peptide vaccine targeting up to 20 personal neoantigens per patient ( NCT01970358 ). All patients were alive and six were without evidence of active disease. We observed long-term persistence of neoantigen-specific T cell responses following vaccination, with ex vivo detection of neoantigen-specific T cells exhibiting a memory phenotype. We also found diversification of neoantigen-specific T cell clones over time, with emergence of multiple T cell receptor clonotypes exhibiting distinct functional avidities. Furthermore, we detected evidence of tumor infiltration by neoantigen-specific T cell clones after vaccination and epitope spreading, suggesting on-target vaccine-induced tumor cell killing. Personal neoantigen peptide vaccines thus induce T cell responses that persist over years and broaden the spectrum of tumor-specific cytotoxicity in patients with melanoma.

A large peptidome dataset improves HLA class I epitope prediction across most of the human population.

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies.

Mitochondrial apoptosis can be effectively targeted in lymphoid malignancies with the FDA-approved B cell lymphoma 2 (BCL-2) inhibitor venetoclax, but resistance to this agent is emerging. We show that venetoclax resistance in chronic lymphocytic leukemia is associated with complex clonal shifts. To identify determinants of resistance, we conducted parallel genome-scale screens of the BCL-2-driven OCI-Ly1 lymphoma cell line after venetoclax exposure along with integrated expression profiling and functional characterization of drug-resistant and engineered cell lines. We identified regulators of lymphoid transcription and cellular energy metabolism as drivers of venetoclax resistance in addition to the known involvement by BCL-2 family members, which were confirmed in patient samples. Our data support the implementation of combinatorial therapy with metabolic modulators to address venetoclax resistance.

Growth dynamics in naturally progressing chronic lymphocytic leukaemia.

How the genomic features of a patient's cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to analyse the growth rates and corresponding genomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo.

Conversion of Aldehydes to Branched or Linear Ketones via Regiodivergent Rhodium-Catalyzed Vinyl Bromide Reductive Coupling-Redox Isomerization Mediated by Formate.

A regiodivergent catalytic method for direct conversion of aldehydes to branched or linear alkyl ketones is described. Rhodium complexes modified by P tBu2Me catalyze formate-mediated aldehyde-vinyl bromide reductive coupling-redox isomerization to form branched ketones. Use of the less strongly coordinating ligand, PPh3, promotes vinyl- to allylrhodium isomerization en route to linear ketones. This method bypasses the 3-step sequence often used to convert aldehydes to ketones involving the addition of pre-metalated reagents to Weinreb or morpholine amides.