bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oligo-conjugated antibodies (Ab-seq) and massively parallel single cell sequencing reveal the high parameter correlation of protein and mRNA expression in individual immune cells.
Authors: Martin Jody , Fan H. Christina , Shum Eleen 
Published: January 2023

Abstract
Single-Cell mRNA sequencing has enhanced our knowledge of the cell populations found in individual tissues or disease states. However, low correlation between mRNA and protein expression levels hinders our understanding of biology and disease. Traditional single cell protein assays are limited in the number of parameters that can be achieved within one experiment.
Here we use oligo-conjugated antibodies (Ab-seq) in combination with massively parallel single cell mRNA sequencing on the BD Rhapsody™ platform to simultaneously measure the protein and mRNA content of individual human blood cells (PBMCs). To accomplish this, a 30+ parameter Ab-seq panel against immune relevant cell surface markers was paired with the BD Rhapsody™ Immune Response panel (a targeted gene expression panel consisting of 399 targets). The Multi-omics data from this single workflow experiment provides a measurement of both gene expression and protein expression. The digital measurements are rendered free of PCR bias through the use of the unique molecular indices (UMIs).
Our results show that protein expression detected with Ab-seq is highly sensitive and specific. Protein expression patterns correlate well with results from flow cytometry data on the same samples. Ab-seq allowed the robust detection of expressed genes even when their cognate mRNA transcripts have low abundance. Certain markers cannot be easily distinguished at the mRNA level (i.e. isoforms CD45RA and CD45RO) but are easily distinguished at the protein level. Our study shows successful analysis of protein and mRNA expression data within a single workflow, and should enable the further elucidation single cells within different tissues, developmental time points, and disease states.
Multicolor Immunophenotyping using Flow Cytometry: Evaluation of Multiple Methods for Instrument Optimization
Authors: Bradford Jolene A. , Shallice Matthew , Diz Ramiro , Oxford Carol , Punj Manik , Alford Kate , Donaldson Phil , Seredick Barbara , Buller Gayle , Sardina Patricia 
Published: January 2023

Abstract
A basic tool of the Immunologist, multicolor flow cytometry is a technology that uses multiple fluorescent markers to identify and characterize populations of cells. To obtain high quality fluorescent flow cytometry data, a well-optimized instrument is required. The most common type of detector is the photo-multiplier tube (PMT), which amplifies signal from emitted light photons by applying a voltage to the PMT. As the voltage is increased the fluorescent signal is increasingly separated from background, providing greater resolution of the positive signal. However, at a certain voltage, the increasing separation of fluorescent signal from background will plateau and the separation of fluorescent signal from background will remain constant. This is called the minimum voltage required; an ideal minimal voltage will amplify dim signal above background, but is not so high that the fluorescent signal exceeds the upper range of PMT linearity. In most cases, adjusting voltages above the minimal voltage does not increase the separation, while voltages below the minimal voltage requirement compromise detection of dim fluorescent signal. A variety of methods have been proposed to optimize the PMT voltage, most using a technique called the voltage walk, or voltration. This study compares a variety of techniques and calculations, using different types of hard-dyed beads that are detected in all channels but do not include specific fluorophores used for typical experimentation; both cells and antibody-capture beads labeled with fluorophores specific to a detector; Electronic Noise (EN) of the PMT, and methods combining these approaches. For Research Use Only. Not for use in diagnostic procedures.
The utilization of ctDNA technology as an early diagnostic tool for Cancer detection in Tonga
Authors: Sika-Paotonu Dianne , Vaea Kolinisiupeli , ‘Ulufonua Lisiate Kilipeni , ‘Akau’ola Siale , Aho George , Tuipulotu Amelia Afuha’amango , Toumoua Seventeen , Lilo Filimone , Fotu Latu , Guilford Parry 
Published: January 2023

Abstract
Circulating tumour DNA (ctDNA) refers to small fragments of tumour DNA found in the blood circulation. These DNA fragments can be isolated and quantified to obtain data about a cancer’s size and progression. The possibility of utilizing a simple blood sample to allow early detection and monitoring of cancer growth is a highly desirable application for this ground breaking technology. Significant health inequalities exist for Pacific peoples with cancer living in New Zealand and in the Pacific region, where the burden of cancer remains a public health concern. The application of this simple ctDNA method of detecting and monitoring cancer to enhance the precision of early cancer diagnostics and surveillance to improve cancer health outcomes for Pacific peoples in Tonga was investigated. Consultation was sought with senior Government officials, Medical, Nursing, Health and Community research staff concerning the development and implementation of ctDNA as a diagnostic tool within the health care setting throughout Tonga. Preliminary assessment of the on-the-ground laboratory requirements needed for the implementation of the ctDNA technology was also undertaken. Strong support for the development of ctDNA as an early diagnostic tool within the clinical setting was expressed. A key feature of the ctDNA technology within this resource constrained environment was the anticipated detection of cancers at an earlier stage with a greater chance of being treatable given that most cancer presentations in Tonga are late. This work sought to explore and progress the implementation of ctDNA as an early cancer diagnostic tool within the Pacific setting, and remains a highly feasible early cancer detection tool within Tonga.
Magnetic Delivery of Antibody-Coated Beads to Live Lymph Node Tissue Slices
Authors: Belanger Maura C. , Pompano Rebecca R. 
Published: January 2023

Abstract
Lymph nodes have a high degree of spatial organization, characterized by dynamic zones of cells secreting changing sets of cytokines. Experimental measurement of the local distribution of secreted cytokines in live tissue would provide new insights into the dynamics of cell-cell communication, and may help inform spatially targeted therapies. As a step towards bead-based immunoassays for cytokine detection in situ, here we report our findings for delivering beads uniformly to live tissue, a significant technical challenge due to a dense tissue matrix and potential internalization by endocytic cells. We utilized live slices of lymph node tissue ex vivo to allow access to the interior of the node while maintaining structural integrity and dynamics. Lymph node tissue viability post slicing was confirmed by flow cytometry (80% viable by PI exclusion). We conjugated capture antibodies to magnetic beads using carboxylic acid-amine linker chemistry; linkage was confirmed by a secondary antibody. To test penetrance into a dense matrix, beads with various diameters were delivered to 2 % agarose gel by magnetic force. Large (4 and 6 μm) beads were excluded, while smaller beads (0.22, 0.56 and 1.0 μm) were delivered successfully. Small antibody-conjugated beads also penetrated into live, 300-μm thick lymph node tissue slices, but penetrated only 40 μm deep and were localized to the sinuses. Covalent conjugation of polyethylene glycol (PEG) alongside antibody allowed beads to penetrate up to 70 μm into the tissue and provided a more uniform distribution. Having delivered beads successfully, future work will implement a complete multi-step immunoassay in the tissue.
Early Treatment of Experimental Autoimmune Encephalomyelitis with Cannabidiol Suppresses Tc1 Cells and Reduces Neuroinflammation
Authors: Nichols James Matthew , Kummari Evangel , Sherman Jessica , Szafran Brittany , Kaplan Barbara 
Published: January 2023

Abstract
Multiple sclerosis (MS) is a neurodegenerative, T cell-mediated, autoimmune disease. In this study, we examined use of the phytocannabinoid, cannabidiol (CBD), in the experimental autoimmune encephalomyelitis (EAE) model to determine CBD’s potential as an oral therapy for MS. We hypothesized that 75mg/kg of oral CBD for 5 days after initiation of disease would reduce EAE severity through suppression of either the early peripheral immune or late neuroimmune response. EAE was induced in C57BL/6 mice at two different magnitudes, and peripheral inflammatory and neuroinflammatory responses were measured at days 3, 10, and 18. Th1, Th17, Tc1, Tc17, Tregs, and myeloid derived suppressor cells (MDSC) were identified in lymph node and spleen by flow cytometry to determine if CBD altered these cell populations in the secondary lymphoid tissues. Additionally, neuroinflammation was identified in brain and spinal cord using hematoxylin and eosin (H&E) and CD3 staining to detect cellular infiltrates and T cells, respectively. Our results show a clear correlation between reduction in the percentage of MOG-35–55 specific Tc1 cells in the spleen at day 10, and reduced neuroinflammation and clinical scores at day 18 in EAE mice treated with CBD; however, this correlation was not seen in the milder form of disease. These results suggest CBD’s ability to reduce neuroinflammation in EAE might be due to early suppression of the Tc1 phenotype in the peripheral inflammatory response, and that CBD’s ability to suppress disease is dependent on the magnitude of the inflammatory response. Furthermore, this study highlights the effectiveness of oral CBD as a treatment for EAE and suggests that CBD might be effective for treatment of other T cell-mediated diseases.
Delta-Like Ligand 4 (DLL4)/Notch axis regulates EAE development by modulating T cell trafficking across the blood brain barrier
Authors: Walter Fruzsina R. , Hsu Martin , Gilpin Trey E. , Sandor Matyas , Fabry Zsuzsanna , Karpus William J. 
Published: January 2023

Abstract
The Notch receptor system has been suggested to play a critical role in the regulation of chemokine receptor expression on encephalitogenic T cells and subsequent T cell trafficking to the central nervous system (CNS). Here we tested whether Delta-Like Ligand (DLL) 4 (DLL4)/Notch axis regulates EAE development by modulating T effector cell trafficking mechanisms across the blood-brain barrier (BBB). Primary cultures of brain microvessel endothelial cells were established and migration of splenocytes was tested in the presence of DLL4 blocking antibody. Our data show that anti DLL4 blocks transendothelial migration of non-activated T cells, but does not influence the migration of anti CD28 and CD3-activated splenocytes. Additionally, activation of brain microvessel endothelial cells with TNFα abrogated the blocking effect of anti DLL4 in transendothelial migration of T cells. We also demonstrate that blockade of DLL4 function inhibits the development of a progressive model of clinical experimental autoimmune encephalomyelitis (EAE), in C57Bl6 mice. Based on these results, we propose that endothelial DLL4 engagement with Notch expressing T cells is critical in strengthening the neuroinflammation and blockage of DLL4 could provide novel therapies in early phases of CNS neuroinflammatory diseases.
A non-redundant role for RORα in TH17 cell development and TH17-driven inflammatory disorders
Authors: Solt Laura A. , Wang Ran , Amir Mohammed , Chaudhari Sweena M. , Campbell Sean , Bassette Molly A. , Kamenecka Theodore M. 
Published: January 2023

Abstract
Dysregulated TH17 immune responses have been associated with the pathogenesis of several autoimmune diseases. While full development of TH17 cells requires both nuclear receptors RORα and RORγt, most of the work surrounding TH17 cells has focused on the lineage defining transcription factor RORγt. However, TH17 cells are not absent in RORγ−/− mice, suggesting that RORα plays an important role. Despite this, RORα is considered redundant to RORγt and little is known about its function in TH17 cells. The aim of this study was to determine whether RORα played a significant role in TH17 cell development and TH17-driven inflammatory disorders.
Using molecular, genetic, and pharmacological approaches our data suggests that RORα has non-redundant functions to RORγt driving TH17 cell development. Loss or overexpression of RORα in CD4+ T cells significantly decreased or increased IL-17A, respectively, despite no change in RORγt expression. Using two mouse models of autoimmunity known to be TH17 driven [experimental autoimmune encephalomyelitis (EAE) and adoptive transfer model of colitis], we found that loss of RORα significantly inhibited the development of EAE and colitis, largely as a consequence of decreased frequencies of pro-inflammatory TH17 cells and increased frequencies of anti-inflammatory Foxp3+ T regulatory cells. Finally, using a RORα-selective small molecule that we developed, we found that modulation of RORα activity significantly inhibited the development of autoimmunity. Our data suggests that RORα plays a non-redundant role to RORγt in TH17 cell development. Studies focusing on assessing RORα activity alone or along with RORγt may lend novel insight into future therapeutic design for TH17-mediated autoimmunity.
Interferon-β-driven inflammatory B cells promote TH17-mediated neuro-inflammation
Authors: Axtell Robert C. , Agasing Agnieshka , Quinn James L. , Kumar Gaurav , Ko Rose M. 
Published: January 2023

Abstract
Interferon (IFN)-β is a popular therapy for multiple sclerosis (MS). However, neuro-autoimmune diseases that have strong T Helper (TH) 17 signatures are exacerbated with IFN-β; these includes MS patients who are non-responders to IFN-β and patients with neuromyelitis optica (NMO). Previous studies using experimental autoimmune encephalomyelitis (EAE) demonstrated that IFN-β treatment has exacerbated TH17-induced disease. These results are confounding because one proposed mechanism of the efficacy of IFN-β treatment is that it inhibits IL-17 expression in T-cells. Our objective is to elucidate the conflicting theories on how IFN-β affects TH17 function. Here, we demonstrate that IFN-β enhances the effects of IL-23 on TH17 cells and promotes their pathogenicity by elevating the expression of TH1 and TH17 genes as well as GM-CSF. We next determined the precise cellular mechanism by which IFN-β drives an inflammatory TH17 response. We found that the IFN-β exerts its inflammatory effect by directly acting on B cells which in turn effected the function of T helper cells. Specifically, we found that IFN-β elevates the expression of the co-stimulatory receptors CD80 and CD86 as well as the inflammatory cytokine IL-6 in B-cells. Furthermore, we found that B cells previously stimulated with IFN-β drive the differentiation of myelin-specific TH17 cells, which secrete the inflammatory cytokines GM-CSF, IL-17A, IFNγ and IL-6. Taken together, these findings resolve a mechanistic paradox surrounding type 1 interferon and TH17-induced disease and illuminates possible cellular pathways responsible for the pathophysiology of NMO and of MS patients who are IFN-β non-responders.
PD-1 blockade markedly enhances immunity to prostate tumors by adoptively transferred human Vγ2Vδ2 T cells in an NSG mouse model
Authors: Morita Craig T. , Wang Hong , Nada Mohanad H. 
Published: January 2023

Abstract
Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Bisphosphonate treatment of tumors results in increases in isopentenyl pyrophosphate that is sensed by butyrophilin 3A1. This allows Vγ2Vδ2 T cells to recognize and kill all tumor types independent of MHC expression or the number of tumor mutations. In clinical trials, adoptive immunotherapy with Vγ2Vδ2 cells has few side effects but has only resulted in a few partial and complete remissions with the most common response being stable disease. To improve effectiveness, we have now tested the combination of PD-1 blockade with Vγ2Vδ2 T cells and the bisphosphonate, pamidronate. Vγ2Vδ2 T cells were found to express PD-1, CTLA-4, LAG-3, and TIM-3 inhibitory receptors during the 14 day ex vivo expansion. Moreover, tumor expression of PD-L1 was increased by co-culture with activated Vγ2Vδ2 cells and by exogenous IFN-γ. To assess the in vivo effect of PD-1 blockade, immunodeficient NSG mice were inoculated with human PC-3 prostate cancer cells that naturally express PD-L1. After 13 days, the mice were treated with pamidronate followed 1 day later by purified Vγ2Vδ2 cells combined with either an anti-PD-1 or a control IgG1 mAb. This regimen was repeated weekly for 5 weeks. Anti-PD-1 mAb treatment markedly enhanced Vγ2Vδ2 immunity to PC-3 tumors, reducing tumor volume nearly to zero after 5 weeks. These results demonstrate that PD-1 checkpoint blockade can enhance the effectiveness of adoptive immunotherapy with γδ T cells in treating prostate tumors in a preclinical model and suggests that this combination should be tested in patients.
NKT cell ligand-induced cytokines enhance antitumor effects with PD-1 blockade by reinvigorating exhausted CD8 T cells.
Authors: Bae Eun-ah , Seo Hyungseok , Choi Jeongwon , Lee Jeongmi , Kang Chang-Yuil 
Published: January 2023

Abstract
Various immune checkpoint blockades have shown great promise for cancer patients. Anti-PD-1 therapies are known to reinvigorate exhausted immune cells, which progressively have lost effector function after chronic exposure to antigen. Although anti-PD-1 blockades were tested in various clinical trials on a wide range of tumors, many have observed clinical efficacy in only a portion of the patients tested. As such, these anti-PD-1 resistance or non-responsiveness needs to be overcome by combination therapeutic approaches that can synergize with existing immune checkpoint blockades. In this study, we investigated the effects of cytokines induced by invariant Natural Killer T (iNKT) cell ligand stimulation, more specifically α-galactosylceramide (αGC), on exhausted CD8 T cells. We used a mouse tumor model expressing ovalbumin antigen (OVA) and tracked OVA-specific CD8 T cells to show the effects on exhausted CD8 T cells in the tumor. αGC-induced cytokines not only enhanced the effector functions, but also diversified the functionality of exhausted CD8 T cells in vitro. Also, administration of αGC-loaded antigen presenting cells (APCs) restored exhausted CD8 T cell functions and led to higher cytotoxic effects in vivo. Among αGC-induced cytokines, we found IL-2 and IL-12 to be crucial in enhancing exhausted CD8 T cell function in mouse tumors and patient tumors. Furthermore, we showed that αGC-loaded APCs bring synergistic effects with PD-1 blockade in a mouse therapeutic tumor model. Our studies suggest a new insight for applying αGC as an adjuvant in cancer immunotherapy.
Targeting type I interferon tumor entry by anti-PD-L1 creates feedforward antitumor responses to overcome innate and adaptive resistance
Authors: Tang Haidong , Liang Yong , Guo Jingya , Qiu Xiangyan , Peng Hua , Fu Yang-Xin 
Published: January 2023

Abstract
Most patients remain unresponsive to intensive PD-1/PD-L1 blockade therapy despite the presence of tumor-infiltrating lymphocytes. We propose that impaired innate sensing might limit the complete activation of tumor-specific T cells after PD-1 blockade. Local delivery of type I interferon (IFN) restored antigen presentation but also upregulated PD-L1, dampening subsequent T-cell activation. Therefore, we armed anti-PD-L1 antibody with IFN (IFN-anti-PD-L1) to create feedforward loops targeting both PD-L1 blockade and IFN-alpha receptor (IFNAR) activation within tumor microenvironment. A synergistic effect of anti-PD-L1 and IFN was achieved to overcome both PD therapy and IFN resistance with least side effects in advanced tumors. Intriguingly, we observed that PD-L1 in either tumor cells or tumor-associated host cells was sufficient for fusion protein targeting and antitumor effects. IFN-anti-PD-L1 activated IFN receptor signaling in host cells but not tumor cells to initiate T-cell reactivation. Clinically, patients with higher type I IFN levels in tumor tissues show better prognosis after PD-1 blockade therapy. Our data suggest that a next-generation PD-L1 antibody armed with IFN improves tumor targeting and antigen presentation while countering innate or T-cell-driven PD-L1 upregulation and overcoming innate and adaptive tumor resistance to checkpoint blockade therapy.
Single Cell RNA Sequencing of Lung Adenocarcinoma Reveals Heterogeneity of Immune Response-related Genes
Authors: Ma Keyue , Schonnesen Alexandra A. , Brock Amy , Van Den Berg Carla , Eckhardt S. Gail , Liu Zhihua , Jiang Ning 
Published: January 2023

Abstract
Immunotherapies have emerged to be promising approaches to treating cancers, while the partial responses in multiple clinical trials suggest the significance of characterizing intertumoral and intratumoral heterogeneity for better prognosis and decision-making in treatment. The lack of intratumoral characterization of immune response-related genes in cancer cells, however, hinders the further development of metrics to select and predict immunotherapies. Therefore, we analyzed single cell RNA-seq data from lung adenocarcinoma patients to identify intratumoral heterogeneity of immune response-related genes and demonstrated their potential effect on immunotherapy efficacy. We found IFNγ signaling pathway genes are heterogeneously expressed and co-regulated in cancer single cells, including MHC class II molecules (MHCII), which form a set of favorable prognosis markers. The upregulation of MHCII is also mutually exclusive with upregulation of cell cycle pathways that are associated with unfavorable prognosis. Moreover, analysis of two groups of tumor restricted antigens, neoantigens and cancer testis antigens, revealed heterogeneity in their expression in single cells. These analyses provide the rationale of applying combinatorial therapies to prevent tumor escape and basis for future development of prognosis metrics based on intratumoral heterogeneity.
Linkage between TIM3 genotype and human HER2 vaccine response in Diversity Outbred (DO) HER2 transgenic (Tg) mice
Authors: Gibson Heather , Jones Richard , Dyson Greg , Reyes Joyce , Morris John , Wei Wei-Zen 
Published: January 2023

Abstract
Regulatory T cell (Treg) depletion boosts HER2 vaccine response in human HER2 transgenic (Tg) mice. To test whether polymorphisms of Treg-associated genes impact vaccine response, individually distinct F1(DOxB6 HER2 Tg) mice were vaccinated 2× with Adv/HER2 to generate highly variable responses. Single nucleotide polymorphisms (SNP) of 16 candidate genes were analyzed by MonsterPlex sequencing or TaqMan assay, with TIM3 showing an association with vaccine response. There are14 SNPs in the 2kb proximal promoter region and 10 SNPs in a 1kb region containing TIM3 exon 7. Four distinct TIM3 genotypes were identified for either region with one genotype consistently matching between the promoter and exon 7 loci, i.e. the CAST/EiJ strain genotype. Analysis of 84 vaccinated DOxB6 HER2 Tg mice using a tag SNP rs28230613 showed that mice with homozygous CC produce HER2 Ab at 26.9±28.9 μg/mL, and those with heterozygous CA (CAST/Eij genotype) 12.8±19.4 μg/mL (p=0.0075). T cell response was absent in most CA heterozygotes. Relative transcript levels of splenocyte TIM3 over CD4 showed heterozygotes at 2.06±2.27 and homozygotes 0.54±0.51 (p=0.0073, n=24), indicating higher TIM3 transcript in CA heterozygotes. Thus, CAST/EiJ TIM3 genotype is associated with higher TIM3 transcript level and lower vaccine response to self HER2. This correlation is absent in mice depleted of CD25+ Treg, implicating Treg and their TIM3 in regulating HER2 vaccine response. The segregation of TIM3 genotype with vaccine response is not absolute, suggesting additional molecular regulation in HER2 vaccine response. CA76340