bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel, non-feeder-cell approach to generate large numbers of Cytokine-Induced Memory-Like NK cells for adoptive cells therapies
Authors: Shrestha Niraj , Dee Michael , Chaturvedi Pallavi , Leclerc Gilles , Zhu Xiaoyun , George Varghese , Prendes Caitlin , You Lijing , Spanoudis Catherine , Rhode Peter , Wong Hing C 
Published: January 2023

Abstract
We have constructed two heterodimeric multi-cytokine fusions, HCW9201 and HCW9206, to support a “Kick and Expand” approach to activate and induce proliferation of purified NK cells for adoptive cell therapy (ACT). HCW9201 is a heterodimeric fusion protein complex comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-18 and IL-12. HCW9206 is heterodimeric fusion protein comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-7, and IL-21. When purified human NK cells from peripheral blood were activated with HCW9201 for three hours and then incubated with HCW9206 in combination with a capture antibody, the HCW9201-activated NK cells expanded approximately 100–300 folds within 14 days. The expanded NK cells exhibit a cytokine-induced memory-like (CIML) phenotype with a high metabolic rate and respiratory capacity, remarkable anti-tumor activity, and persistence when they were adoptively transferred into NSG mice. They also retained their heightened responsiveness when re-stimulated with tumor targets. Cytokine dependent epigenetic demethylation imprints of the Ifng promoter region were also observed for at least 10 days after the NK cells were adoptively transferred to NSG mice. In conclusion, a simple, scalable, non-feeder-cell-based “Kick and Expand” process was developed to support the generation of large numbers of CIML NK cells for multiple rounds of ACT using peripheral blood NK cell. We also provide data to show that these CIML NK cells are an excellent source for generation of CAR-NK cells.
Satb1 deficiency licenses TFH-differentiation and Tertiary Lymphoid Structure formation in cancer
Authors: Gonzalez Ricardo A Chaurio , Payne Kyle K , Galindo Carmen Maria Anadon , Costich Tara Lee , Harro Carly , Birwas Subir , Rigolizzo Kristen , Mine Jessica A , Mandal Gunjan , Powell Chase , Martin Alexandra L , Wang Zhitao , Kroeger Jodi , Robinson John , Melendez Johana , Rodriguez Paulo C , Conejo-Garcia Jose R 
Published: January 2023

Abstract
Tertiary Lymphoid Structures (TLS) are commonly identified in human tumors with improved outcome, but how they are orchestrated remains elusive. Here we show that silencing of the master genomic organizer Satb1 results in enhanced antigen-specific T Follicular Helper (TFH) differentiation. Increased TFH thereby promoted antigen-specific intra-tumoral CD19+B220+ B cell responses and spontaneous TLS assembly upon ovarian tumor challenge. Mechanistically, Satb1 deficiency drives increased TFH formation through de-repression of ICOS and PD-1. Accordingly, TGF-β1-driven downregulation of Satb1 licenses activated human CD4+ T-cells for enhanced antigen-specific T Follicular Helper (TFH) differentiation. Furthermore, Satb1 deficiency abrogates the generation of PD-1highCXCR5+Foxp3+ T Follicular Regulatory (TFR) cells during the TFH differentiation process. Importantly, functional TFH cell accumulation, in the absence of Satb1 specifically in CD4+ T cells, resulted in corresponding isotype-switched B cell responses and spontaneous formation of TLS, while B cell depletion accelerated malignant progression. Our results indicate that the formation of TLS in cancer depends on enhanced B cell responses driven by TFH cells generated through Satb1 down-regulation.
Analysis of stability CRISPR/Cas9 engineered B cells using flow cytometry
Authors: Inwood Sarah L , Tian Linhua , Maragh Samantha , Wang Lili 
Published: January 2023

Abstract

Objective
CRISPR/Cas9, a widely used genome editing system, has huge potential and is being used to improve protein, cell and gene therapies. Though an exciting tool, there is not a lot of long-term data about the genomic and phenotypic stability and off-target effects that may arise during the editing process. Since the cells are used in the patient with cell and gene therapies, cell characterization is essential for safety.
In support of production of safe and effective CRISPR/Cas9 engineered therapies, the objective of this work is to study both on- and off-target effects of CRISPR/Cas9 using flow cytometry on a B-lymphoblast cell line, GM24385, whose genome sequence has been well characterized.

Methods
The cultured B cells were characterized with a B cell panel using flow cytometry. The CD19 marker was knocked out using CRISPR/Cas9 RNPs with a nucleofection method using four gRNAs. The resulting pools were then evaluated with sequencing and a flow cytometry B cell panel and sorted using a FACSAria II flow sorter.

Results
The CD19− B cells were created from nucleofection of cultured GM24385 cells with CRISPR/Cas9 RNPs. The characteristics obtained from a B cell subtyping panel showed different efficiencies of CD19 knockout and different B cell subtypes. The growth rate of CD19− B cells was unpredictable over time post transfection. Additionally, different gRNAs against CD19 resulted in different editing efficiencies evaluated at genomic and proteomic levels.

Conclusions
Using CRISPR/Cas9 with different gRNAs to knock out CD19 in B lymphoblasts, GM24385, influences the B cell subtype profile. The work with well sequenced GM24385 enables the identification of off-target effects of the editing method, the next step of the study.
characterization of newly isolated porcine epidemic diarrhea virus (PEDV) and its resistance to interferon and neutralizing antibody
Authors: Shin Hyun-Jin 
Published: January 2023

Abstract
Porcine epidemic diarrhea virus (PEDV) infects pigs and causes enteric disease characterized by watery diarrhea and dehydration. Despite the extensive vaccination, PEDV strains with variations in the spike gene have been reported in several countries, including Korea, China, and the United States. Variant PEDV belonging to genogroup G2 has higher pathogenicity and morbidity than conventional PEDV belonging to genogroup G1. To understand the pathogenesis of the variant PEDV, we examined the susceptibility of the variant PEDV to interferon and PEDV specific neutralizing antibodies, which are known to important for the control of PEDV infection. We found that variant PEDV (WG strain) showed higher resistance to interferon than conventional PEDV (KPEDV-9 strain). Similarly, KPEDV-9 strain grew well in interferon-deficient Vero cells but did not grow in interferon-releasing porcine alveolar macrophages, whereas WG strain showed similar infectivity in both cells. To elucidate the possible mechanism of resistance to interferon and neutralizing antibody in variant PEDV, sequencing analysis was performed. We found huge variations in spike and ORF3 proteins. To test the impacts of these mutations on the acquisition of interferon and neutralizing antibody resistance, we are currently generating pseudotyped PEDV and recombinant PEDVs with mutations. Our results indicate that the recently introduced variant PEDV exhibits higher resistance to the interferon than the existing PEDV and avoids the neutralizing antibody against the existing PEDV. These results also provide understanding of the occurrence of variant PEDV and its pathogenesis.
Inflammatory reactions and melanogenesis in salmon during virus infection
Authors: Bjoergen Haavard , Malik Muhammad Salman , Rimstad Espen , Koppang Erling Olaf 
Published: January 2023

Abstract
Melanised skeletal muscle changes (black spots) are a serious problem for the salmon industry world-wide, causing extensive production losses. Piscine orthoreovirus (PRV) is associated with the condition. The initial cause for the focal changes remains obscure. Early changes start as muscle bleedings and extensive muscle necrosis, and so far, these changes cannot be explained, However, from this acute phase, the changes may progress into melanised focal changes appearing as chronic granulomatous reactions. Granulomatous melanised changes have been consistently observed in PRV infected fish, and in serious changes, the tissue persistence of virus dominate the changes despite the inflammatory changes. Here, we address the pathogenesis of PRV infection and phases of melanised focal changes development. Fluorescent in situ hybridisation (FISH) assay confirmed the colocalisation of iNOS2 transcripts (M1 activated macrophages) with PRV in red spot whereas both M1 and M2 (Arg2 transcripts) macrophages localized at PRV infected clusters in black spots. Melano-macrophages were found positive with Arg2 transcripts demonstrating their role in tissue repair mechanism. In PRV negative fish low expression of M1 and M2 specific transcripts suggest PRV act as an accelerator for the melanisation in these focal changes. Transcripts specific for melanin production were observed in non-melanised cells in early stages of the condition and in melano-macrophages in late stages. The nature of the melano-macrophages is disputed and the rationale for their melanin production is not understood. Our investigations reveal new characteristics both of the pathogenesis of focal melanised changes as well as of the melano-macrophages involved.
Hypothetical evolutionary models of the variable lymphocyte receptor genes in jawless vertebrates
Authors: Das Sabyasachi , Hirano Masayuki , Rast Jonathan , Cooper Max D 
Published: January 2023

Abstract
Both humoral and cellular arms of an adaptive immune system are common to jawed and jawless vertebrates. However, whereas antigen recognition in the jawed vertebrates is mediated by immunoglobulin domain-based B cell receptors and T cell receptors, antigen recognition in jawless vertebrates is mediated by leucine-rich repeat (LRR) sequences in variable lymphocyte receptors (VLRs). Three types of VLR genes (VLRA, VLRB and VLRC) have been characterized in the extant jawless vertebrates, lampreys and hagfishes. VLRA- and VLRC-producing cells are T cell-like, while VLRB-producing cells are B cell-like. The VLRs are assembled by copying neighboring LRR sequences into the incomplete germline genes. In searching for the evolutionary origin of the VLRs we identified hypothetical VLR-like progenitors in amphioxus and tunicates (VLRPs). Molecular cladistics markers and phylogenetic analysis suggest the VLRPs are structurally more similar than other ancestral candidates to assembled VLRs. Based on our further analysis we propose alternative models for the origin of VLRs: (A) Among the VLRs, VLRB is most similar to the ancestral state and two subsequent duplication events gave rise to VLRC and VLRA-like genes; each of the three ancestral VLR genes in a common jawless vertebrate ancestor was then modified by cut and paste transposition of the internal DNA region encoding multiple LRR modules. (B) A cut and paste transposition event in an ancestral VLRB-like gene was followed by sequential duplication events which gave rise to VLRC and VLRA. In both models, subsequent duplications and fragmentations have generated a large array of genomic donor cassettes. Thus, after the emergence of the three VLR loci, each has undergone independent evolution.
Genetic variation in the RSV fusion protein controls RSV strain-dependent inflammasome activation and the development of a pathogenic Th17 response
Authors: Waldstein Kody , Weiss Kayla A , Hartwig Stacey M , Varga Steven M 
Published: January 2023

Abstract
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide. RSV-induced disease ranges from mild to severe, with severe disease correlated with elevated levels of pro-inflammatory cytokines, including the Th17-associated cytokine, IL-17. To better understand the viral factors that influence the induction of a Th17 response, we compared the inflammatory responses elicited by different RSV strains. In contrast to the commonly used RSV A2 strain, RSV 2-20 infection resulted in increased protein levels of IL-6 and IL-1β, which were required to induce a pathogenic Th17 response. RSV 2–20 infection increased mucus production and airway hyperreactivity indicating strain-specific disease induction. RSV 2–20 and A2 induced similar protein levels of caspase-1, however, 2–20 infection resulted in increased protein levels of pro-IL-1β suggesting differential activation of inflammasome signal 1. Increased production of IL-1β following RSV 2–20 infection was dependent on TLR4 expression and signaling, a canonical inflammasome signal 1 activator. A recombinant A2 virus expressing the 2–20 fusion (F) protein induced IL-1β to similar levels as wild-type 2–20 indicating the 2–20 F protein is sufficient to increase pro-inflammatory cytokine production. Reversion of three-point mutations in a region likely to interact with host cells in the RSV A2/2–20 F recombinant virus to the A2 sequence significantly reduced IL-6 and IL-1β protein production. Our results demonstrate that variation in the RSV F protein plays a crucial role in the induction of pro-inflammatory cytokines and modulating disease severity in the animal model and may contribute to differential RSV pathogenesis.
Generating protective immunity to severe influenza disease in Indigenous Australians
Authors: Hensen Luca , Illing Patricia , Rowntree Louise , Habel Jennifer , Mifsud Nicole , Koutsakos Marios , Loh Liyen , van de Sandt Carolien , Nguyen Andrea , Gras Stephanie , Rockman Steve , Miller Adrian , Nguyen Oanh , Clemens Bridie , Tong Steven , Purcell Anthony , Kedzierska Katherine 
Published: January 2023

Abstract
The 2009 influenza pandemic caused generally mild infections due, at least partially, to broadly cross-reactive pre-existing CD8+ T cell immunity. In contrast, severe disease was observed in Indigenous populations worldwide, as shown by disproportionate hospitalisation rates. Human leukocyte antigen-I (HLA-I) molecules present viral peptides to CD8+ T cells, eliciting anti-viral responses that contribute to accelerated viral clearance. We identified several HLA-I alleles which are highly prevalent in Indigenous Australians (HLA-A*11:01, A*24:02, A*34:01, B*13:01 and B*15:21) but largely understudied. Using specific HLA-allomorph expressing C1R cell lines, we identified influenza A and B immunopeptidomes by mass spectrometry. A total of 653 influenza peptides derived from various viral proteins were presented during infection by different HLA-Is. Using virus-specific PBMC expansions, we screened these peptides for their potential to reactivate influenza-specific memory CD8+ T cells. We identified on average 4.6 (2–8) immunogenic epitopes per HLA-I for influenza A and B. We defined epitope-specific CD8+ T cells ex vivo across different human tissues and determined their memory subsets, phenotype and activation in healthy donors and influenza-infected patients. Overall, our approach is effective in detection of immunogenic epitopes and understanding CD8+ T cell pools in Indigenous populations, thus providing potential vaccine targets to protect Indigenous populations globally, from severe influenza disease. This is the first study to identify hallmarks of immunogenic responses and reveal key targets of an effective CD8+ T cell vaccine in Indigenous people.
Chronic ethanol consumption causes lesions in CD8 T cell memory and is associated with increased susceptibility to influenza A virus infections
Authors: Zacharias Zeb Raplh , Legge Kevin L 
Published: January 2023

Abstract
Chronic alcohol consumption is associated with increased risk of severe disease during both bacterial and viral infections. We have previously shown that this increased disease severity during a primary influenza A virus (IAV) infection is due to deficits in the developing IAV-specific CD8 T cell response. Recent studies have highlighted that human alcoholics have increased susceptibility to IAV infections, even though they likely possess pre-existing immune memory to IAV. However, the effects of chronic alcohol consumption on pre-existing immune memory responses have not been explored. Here, our results show that mice previously exposed to IAV infection that then consumed alcohol (IAV-immune➞EtOH) exhibited increased morbidity and mortality to secondary heterologous IAV challenge compared to IAV-immune mice that had consumed water (IAV-immune➞H2O). This increased susceptibility in IAV-immune➞EtOH mice was associated with reduced IAV-specific killing of target cells. Furthermore, we observed a universal numerical reduction of all memory CD8 T cell subsets within the lungs of IAV-immune➞EtOH mice compared to IAV-immune➞H2O mice. Finally, our results show that IAV-specific CD8 T cell recall responses upon secondary IAV challenge are altered in IAV-immune➞EtOH mice, particularly within the lung tissue, and were associated with reduced expression of CXCL10 and CXCL11, suggesting defects in cell trafficking. Overall, these data demonstrate that the negative impacts of chronic alcohol consumption occur across the full CD8 T cell compartment, including both the naïve and memory response, contributing to lesions in the development of de novo CD8 T cell and pre-existing memory CD8 T cell responses.
Lung-resident memory CD8+ T cells prevent transmission of respiratory viruses
Authors: Uddback Ida Elin Maria , Laccette Kristiana H , Li Zheng-Rong Tiger , Kohlmeier Jacob E 
Published: January 2023

Abstract
Lung tissue-resident memory CD8+ T cells (TRM) reduce viral replication and limit pathology following respiratory virus infections. In particular, TRM are crucial for protection against infections with heterosubtypic influenza viruses, where pre-existing antibody does not provide sterilizing immunity. In addition to providing individual host immunity, vaccination programs are designed to limit pathogen spread at population level. Despite the interest in lung TRM for intra-host protective immunity, their effect on the transmission respiratory viruses has been understudied due to the lack of animal models where influenza infection can be tracked longitudinally, and where the immunological parameters are easily identified and manipulated. To circumvent these issues, we used a luciferase-expressing Sendai virus, a natural mouse parainfluenza virus that readily transmits via the aerosol and contact routes, to evaluate the efficacy of lung TRM against transmission of respiratory viruses. Using a recombinant influenza virus expressing the immunodominant SenNP324–332Kb epitope to generate SenNP+ memory CD8+ T cells and IVIS imaging to non-invasively measure Sendai virus infection over time, we found that mice with pre-existing SenNP+ lung TRM don’t transmit Sendai virus to naïve mice when co-housed. In contrast, immunized mice that had pre-existing circulating effector memory CD8+ T cells (TEM), but no lung TRM, failed to prevent transmission of Sendai virus to naïve mice when co-housed. These results show the ability of lung TRM to contribute to herd immunity by preventing respiratory virus transmission and underscore the potential of a TRM-inducing vaccine for protection against respiratory viruses.
A BAFF-ling new role for NK cells in promoting marginal zone antibacterial resistance during chronic virus infection
Authors: Ali Ayad , Cranert Stacey A , Moran Michael T , Rajaram Sanjeeth , Waggoner Stephen N 
Published: January 2023

Abstract
Natural killer (NK) cells are critical for both direct control of virus replication and regulation of potentially pathogenic antiviral responses. Yet, whether NK cells also contribute to immune control of bacterial superinfections during viral infection, a significant clinical cause of morbidity and mortality, remains unknown. We find that NK cells are vital to prevent a near complete loss of B cells and macrophages associated with the marginal zone (MZ) during chronic virus infection in mice. Establishment of chronic infection in the absence of NK cells results in hyper-susceptibility to Listeria monocytogenes infection that cannot be recapitulated by NK-cell depletion prior to bacterial challenge of chronically infected mice. Therefore, we posit that preservation of MZ leukocytes by NK cells, and not direct NK cell activity, curtails post-viral susceptibility toward bacterial superinfection. Mechanistically, we show that NK cells begin expressing the crucial MZ survival factor, B-cell activating factor (BAFF), during chronic virus infection, gradually becoming a prominent source of BAFF in the spleen. In vivo blockade of BAFF in NK-sufficient mice recapitulated the infection-associated loss of MZ cells seen in NK-cell deficient mice. These results reveal a new mechanism whereby NK cells promote bacterial resistance during chronic infection by maintaining BAFF-dependent immune structures. Disruption of this activity of NK cells during certain virus infections in humans may provoke post-viral pneumonia or invasive pneumococcal disease.
DNA Methylation Tightly Regulates TIGIT Expression in CD8+ T cells During Chronic HIV Infection
Authors: Corley Michael J , Chew Glen , Pang Alina PS , Ndhlovu Lishomwa 
Published: January 2023

Abstract
The T cell Ig and ITIM domain (TIGIT) receptor is a next generation negative immune checkpoint that is unregulated on exhausted CD8+ T cells in cancer and in chronic HIV-1 infection. TIGIT is currently being pursued in Phase 1 human clinical studies for immune restoration using targeted monoclonal antibodies. However, the regulation of TIGIT expression on T cells still remain unclear. We characterized and compared genome-wide epigenetic and transcriptional profiles in sorted TIGIT+ and TIGIT− CD4 and CD8 T cells in peripheral blood obtained from three virally-suppressed chronic HIV participants on antiretroviral therapy. Unsupervised hierarchical clustering analysis of differentially methylated loci indicated TIGIT+ T cells stratified from TIGIT− T cells based on cellular DNA methylation profiles. Compared to cell sorted purified TIGIT− T cells, 86.5% of CpG loci exhibiting hypomethylated states in TIGIT+ T cells. Specifically, the levels of DNA methylation at the nine CpG loci on the methylation array related to the TIGIT gene showed significant demethylation in TIGIT+ compared to TIGIT− T cells. We found 6/7 sites located at the TIGIT promotor were significantly decreased in methylation in TIGIT + compared to TIGIT− cells (cg19959917: TIGIT+ = 0.605, TIGIT− = 0.715, P = 0.002). Moreover, the CpG located intergenic (cg19959917: TIGIT+ = 0.605, TIGIT− = 0.715, P = 0.002 and at the 3′ UTR (cg19959917: TIGIT+ = 0.605, TIGIT− = 0.715, P = 0.002) were significantly demethylated in TIGIT+ compared to TIGIT− T cells. DNA methylation changes correlated significantly with mRNA expression and surface protein expression. These reference methylomes expose a distinct DNA methylation state of TIGIT+ T cells and reference for epigenetic targeting.
Anaphylaxis
Authors: Stoland O. O. , Sherwood N. P. , Woodbury R. A. 
Published: January 2023

Summary and Conclusions
The chronaxie of the vagus nerves in dogs sensitized to horse serum is lower than for normal animals indicating an increased irritability associated with the development of the hypersensitive state. The decrease in chronaxie may reach such a state that nerve block or apparent decreased irritability occurs. The vagus nerve irritability is not appreciably affected by anaphylactic shock. Anaphylactic shock as shown by fall of blood pressure and prolonged coagulation time does not occur in all dogs supposedly sensitized to horse serum. Vagus nerve stimulation before injection of shocking dose of antigen may or may not modify the anaphylactic shock. The increased irritability and permeability of tissue in sensitized animals is not the primary factor in anaphylactic shock.