bluuebunny/crossref_metadata_2025 huggingface dataset:
E3 ligase FBXW7 is critical for RIG-I stabilization during antiviral responses
Authors: Wang Qingqing , Song Yinjing , Xue Yue , Yu Hai
Published: January 2023

Viruses can escape from the host recognition by degradation of RIG-I or interference of the RIG-I signaling to establish persistent infections. However, the mechanisms how host cells stabilize RIG-I protein for avoiding its degradation are largely unknown. We report here that, upon virus infection, the E3 ubiquitin ligase FBXW7 translocates from the nucleus into the cytoplasm and stabilizes RIG-I. FBXW7 interacts with SHP2 and mediates the degradation and ubiquitination of SHP2, thus disrupting the SHP2/c-Cbl complex which mediates RIG-I degradation. When infected with VSV or influenza A virus, FBXW7 conditional knockout mice (Lysm+FBXW7f/f) show impaired antiviral immunity. FBXW7 deficient macrophages have decreased RIG-I protein levels and type I interferon signaling. Furthermore, PBMCs from RSV infected children have reduced FBXW7 mRNA levels. Our results identify FBXW7 as an important interacting partner for RIG-I. These findings provide insights into the function of FBXW7 in antiviral immunity and its related clinical significance.
Cytokines and miRNAs levels in nasal lavage correlate with symptoms in patients infected with respiratory viruses
Authors: Rosas-Taraco Adrian Geovanni , Arredondo-Loza Erick , Martinez-Castilla Azalia , Mata-Tijerina Viviana , Salinas-Carmona Mario C
Published: January 2023

Respiratory viruses are responsible of acute respiratory infections that affect worldwide population causing morbi-mortality. Cytokines and microRNAs can modulate the immune response but little is known in nasal mucosa and less if these would correlate with symptoms severity. We included hundred patients diagnosed with possible viral acute respiratory infections. Patients were sequentially enrolled previous written informed consent was obtained. Virus was detected in pharyngeal exudate by Luminex xTAG RVP kit and qRT-PCR. Nasal lavage were performed in each patients to evaluate microRNAs and cytokines by RT-qPCR, multiplex immunoassay and ELISA, respectively. The symptoms severity was daily monitored throughout seven days by surveys. The respiratory viruses found in these patients were influenza A and B, human rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza, and coronavirus. Viral loads showed no difference between mono-infected patients compared to co-infected; however nasal cytokines and microRNAs profiles differ between these two groups in some cases. Regardless of the viral agent, several nasal microRNAs correlated with symptoms severity. On the other hand, pro-inflammatory and Th2 cytokines correlated positively and negatively with respiratory infection symptoms, respectively. Moreover, correlation between miRNAs and cytokine were found in these patients. Our findings suggest that microRNAs and cytokines in nasal lavage may regulate local immune response against respiratory virus and specific profile may be possible candidates for severity biomarkers.
Three distinct pneumotypes characterize the microbiome of the lung in BALB/cJ mice.
Authors: Scheiermann Julia , Klinman Dennis M
Published: January 2023

Bacteria can rarely be isolated from normal healthy lungs using conventional culture techniques, supporting the traditional belief that the lungs are sterile. Yet recent studies using next generation sequencing report that bacterial DNA commonly found in the upper respiratory tract (URT) is present at lower levels in the lungs. Interpretation of that finding is complicated by the technical limitations and potential for contamination introduced when dealing with low biomass samples. The current work sought to overcome those limitations to clarify the number, type and source of bacteria present in the lungs of normal mice. Results showed that the oral microbiome is large, less diverse and highly conserved whereas murine lung samples fall into three distinct patterns. Most pulmonary samples were sterile, as they lacked culturable bacteria and their bacterial DNA content did not differ from background. 11% of samples contained comparatively higher amounts of bacterial DNA whose composition mimicked that detected in the URT, consistent with (micro)aspiration. A final group (26%) contained smaller amounts of microbial DNA whose composition was similar to that of rodent chow, perhaps acquired by inspiration of food fragments. This work eliminated the bias introduced by aspiration-contaminated samples to establish that three distinct microbiome pneumotypes are present in normal murine lungs of BALB/cJ mice. The presented findings contribute to the knowledge in the growing field of microbiome research in the respiratory tract and are of relevance to researchers evaluating respiratory diseases in murine models.
NLRP3 Inflammasome controls adipose tissue macrophage activation during aging
Authors: Camell Christina , Sander Jil , Spadaro Olga , Lee Aileen , Nguyen Kim Y. , Wing Allison , Goldberg Emily L. , Youm Yun-Hee , Rodeheffer Matthew S. , Schultze Joachim L. , Dixit Vishwa Deep
Published: January 2023

Aging is associated with chronic inflammation and increased risk for degenerative disorders and diseases. In the elderly there is a development of insulin resistance, increased visceral adiposity and dysfunctional adipose tissue (AT) homeostasis; however the mechanisms of age-induced immune defects in AT is unclear. AT contains numerous immune populations, which play well-known roles in regulating both inflammatory responses and metabolic homeostasis. Our prior studies have identified that the canonical inflammasome, NLRP3, is required for diet-induced AT inflammation and age-related inflammation in multiple organs. We hypothesized that NLRP3 is required for age-induced changes in AT immune cells and that deficiency in Nlrp3 will improve AT function during aging. Here we show that unlike obesity, aging is associated with reduction in specific AT macrophages subsets. Whole transcriptome sequencing of AT macrophages from aged mice revealed increased caspase-1 activation and distinct signatures that were regulated in part by Nlrp3. Linear support vector regression and bioinformatics analyses revealed that AT macrophages do not display classical M1–M2 like transcriptome and display AT-specific signatures that are distinct from other tissue macrophages in gut, brain, liver and spleen. We identified that as compared to 24-month old WT, the aged Nlrp3−/− animals were protected from alterations in AT macrophage activation. Furthermore, Nlrp3-mediated reduction in AT inflammation and macrophage activation led to protection from age-related defects in AT function. Overall our findings suggest that NLRP3-dependent immune-metabolic interactions within AT contribute to age-related inflammation and metabolic dysfunction.
Nanoparticle based enrichment and expansion of self and neo-epitope specific CD8+ T cells in murine melanoma.
Authors: Bessell Catherine Ami , Bieler Joan G , Oelke Mathias , Schneck Jonathan P
Published: January 2023

Neo-antigen T cell responses against tumors has created a new avenue to target and kill patients’ tumors while possibly decreasing autoimmune side effects. CD8+ Neo-antigen T cells have the potential benefit to be a source of T cells that are not limited by peripheral tolerance or deleted from the T cell repertoire by central tolerance. To understand the differences between self and neo-antigens T cell responses, the two types of T cell populations must be compared side-by-side by anti-tumor responses and phenotypic differences. Following another group (Castle; 2012) that identified mutated proteins from long sequences in murine melanoma our current work has identified the short peptide epitopes that can stimulate neo-antigen T cell responses. The candidate neo-epitopes were validated by peptide stabilization assays and in vitro T cell stimulations with nanoparticle artificial antigen presenting cells. 11 naïve neo-antigen T cells responses were identified out of a total of 19 possible candidate neo-antigens. Artificial antigen presenting cells were used to assess neo and self-antigen tumor specific responses in the naïve T cell repertoire and after tumor exposure to B16 murine melanoma. After exposure to tumor, neo-epitope T cell responses showed larger fold expansion as compared to a self-antigens expressed by the tumor cells. From this work, we deliver a proof of concept of a streamlined way to screen candidate neo-epitopes and identify rare endogenous T cell populations; and evaluate the benefit of T cell directed immunotherapies on different T cell populations.
Plasmid electrotransfer activates DNA pattern recognition receptors and induces production of proinflammatory molecules in both B16.F10 and TS/A cells in vitro
Authors: Semenova Nina P. , Znidar Katarina , Bosnjak Masa , Cemazar Maja , Heller Loree
Published: January 2023

Electroporation or electrotransfer effectively delivers plasmid DNA (pDNA) into cells and tissues. pDNA primarily enters the cell via endocytosis, although some pDNA may reach the cytosol to be detected by endosomal, cytosolic, and nuclear DNA-specific pattern recognition receptors (PRRs) which have been described in many cell types. Activated PRRs induce the production of proinflammatory molecules and programmed cell death.

Our previous studies on B16.F10 mouse melanomas showed that electrotransfer of backbone pDNA induced the production of several proinflammatory molecules including IFNβ, implicating PRR binding and activation. Histologically, caspase-3 independent tumor necrosis was observed. In B16. F10 cells, IFNβ mRNA and protein levels were also significantly upregulated. The mRNAs for several PRRs were present in these cells and DAI, DDX60, and p204 mRNAs were significantly upregulated after pDNA electrotransfer. DDX60 protein levels were coordinately upregulated. The goal of our study was to find out whether the effects observed in B16.F10 cells are widespread among tumor types.

The electrotransfer of empty backbone pDNA into TS/A murine mammary adenocarcinoma cells induced the production IFNβ and TNFα mRNA and protein. The mRNA levels for several PRRs (including DAI, DDX60, DDX36, p202 and p204) were significantly upregulated with DAI level upregulation being the most prominent. This upregulation in DAI mRNA translated to the protein level, which was confirmed with Western blotting analysis. This increased IFNβ and DNA sensor expression indicate that pDNA electrotransfer activates intracellular PRRs in multiple tumor cell types, producing in vitro effects similar to what was observed in B16.F10 cells.
Ensemble computational analysis and systems immune monitoring reveal a double negative T cell subset enriched in melanoma tumors following dabrafenib and trametinib
Authors: Greenplate Allison , Oberholtzer Brian K. , Doxie Deon B. , Roe Caroline , Kelley Mark C. , Sosman Jeffrey A , Johnson Douglas B , Irish Jonathan Michael
Published: January 2023

Advances in single cell biology have made it possible to measure > 40 features on millions of cells within clinical samples, but data analysis quickly becomes logistically complex and time-consuming for immune monitoring studies collecting many samples per patient. Here we created an ensemble analysis strategy for longitudinal immune monitoring using single cell platforms like mass cytometry. Multiple tools were employed to give key immunology information in an automated “first pass” analysis and highlight cell subsets for in-depth review. Cells identified by immunologists or computational tools were assessed for four complementary types of cellular information: 1) relative abundance, 2) phenotypic plasticity, 3) novelty of phenotype, and 4) signature features. To challenge this analysis approach, we selected three systems immune monitoring studies focusing on blood from melanoma patients, blood and bone marrow from acute myeloid leukemia (AML) patients, and new mass cytometry data characterizing tumor infiltrating immune cells from melanoma patients. Phenotypic plasticity was revealed in T cells within tumors over the course of MEK/BRAF inhibitor therapy. Changes in the overall T cell landscape were driven by expansion of double negative (DN) T cells (12.38% ± 23.52% pre-therapy; 37.67% ± 17.97% post-therapy, p<0.01). Unlike healthy DN T cells, intratumoral DN T cells were CD45RO+. The ensemble analysis strategy tracked the immune systems of patients over time and quantified key differences in the system as well as cell subsets. The features revealed by this method can now be tested as markers for cell isolation, targets for drug development, receptors for immune engineering, and potential mechanisms of treatment response.
Non-pathogenic CD8+ T cells in lupus-prone mice regulate neurological and peripheral disease.
Authors: Morawski Peter A , Qi Chen-Feng , Bolland Silvia
Published: January 2023

Lupus is a systemic autoimmune disorder affecting multiple organ systems including the skin, kidneys, spleen, lungs, and brain. Severe SLE often manifests with multiple psychiatric and neurological sequelae, and may be accompanied by CNS vasculitis. However, the cellular contributors to CNS disease in lupus patients remain poorly defined. We have uncovered a unique bias for CD8+ T cell infiltration in the CNS of mice expressing multiple copies of Toll-like receptor 7 (TLR7[Tg]), which present with a lupus-like disease. These animals exhibit blood brain barrier damage and neuropathology in the presence of infiltrating T cells. However, genetic ablation of CD8+ T cells in lupus-prone mice by removal of the major histocompatibility complex (MHC) adaptor protein b2m results in aggravation of disease, suggesting the removal of a protective population. Relative to the peripheral CD8+ T cells in lupus-prone mice, those in the CNS represent the most activated lymphocytes in the animal, display a tissue resident-memory phenotype, and can home to the brain. We have also identified a population of CD8+ T cells in the cerebrospinal fluid (CSF) of several lupus patients. Similar to our animal model findings the human CD8+ T cells in the CSF have a large increase in antigen experience by surface phenotyping relative to lymphocytes from peripheral blood. Our ongoing studies aim to elucidate specifically how these CD8+ T cells enter the CNS and further clarify their role in neuropathology. Our findings suggest a possible mechanism of CNS pathology regulation that could have applicability to human neuropsychiatric manifestations of SLE.
System control of auto-reactive T cells
Authors: Wong Harikesh S. , Gola Anita , Angermann Bastian , Germain Ronald N.
Published: January 2023

Potentially auto-reactive T cells routinely escape thymic negative selection, but rarely elicit peripheral autoimmunity. We investigated the fate of these cells in murine secondary lymphoid organs using quantitative imaging approaches. During steady state, a small number of seemingly auto-reactive CD4+ T cells were detected along the T cell zone border. These cells displayed clear signs of antigenic stimulation, specifically plasmalemma expression of PD-1, in the absence of foreign bacteria or host commensals. Remarkably, high densities of regulatory T cells routinely converged on the auto-reactive CD4+ T cells, which in turn, exhibited compromised plasma membrane integrity consistent with cell death. Acute depletion of regulatory T cells resulted in uncontrolled expansion of the auto-reactive cells and correlated with reduced death. Collectively, these results suggest that regulatory T cells may not only suppress, but also prune the repertoire of dangerous clones, thereby minimizing the risk of autoimmunity.
Selective PRMT5 inhibition suppresses inflammatory T cell responses and autoimmunity.
Authors: Webb Lindsay M , Savardekar Himanshu , Jablonski Kyle A , Amici Stephanie A , Panfil Amanda , Li Linsen , Green Patrick L , Li Chenglong , Baiocchi Robert A , Guerau-De-Arellano Mireia
Published: January 2023

In the autoimmune disease Multiple Sclerosis (MS), pro-inflammatory, pathogenic T helper (Th) 1 and Th17 responses promote demyelination and disability. MS treatments that preferentially target inflammatory T cell responses may be beneficial to the field. Epigenetic modifications regulate cellular functions and phenotype and inhibition of all methylation reactions suppresses T cell proliferation and experimental autoimmune encephalomyelitis (EAE). However, the specific enzyme(s) responsible for these effects remain to be determined. Protein arginine methyltransferase 5 (PRMT5), the major Type II methyltransferase enzyme, mediates symmetric dimethylation of arginine residues of histones. Although PRMT5 is known to be up-regulated in many lymphoid malignancies, currently there is no known role for PRMT5 in T cells. In this study, we use memory Th cells and animal models to study the role of PRMT5 in T cell responses and autoimmunity. PRMT5 was up-regulated in memory Th cells upon activation in a process. We found that PRMT5 drove T cell proliferation, and pathogenic Th1 cells were more susceptible to PRMT5-selective inhibitor treatment than benign Th2 cells. PRMT5-selective inhibitors ameliorated disease severity in EAE, the mouse model of MS. This disease suppression correlated with suppression of myelin-specific T cell proliferation as well as IFNg and IL-17 cytokine production. Additionally, PRMT5 inhibitors suppressed pathogenic Th17 cell populations ex vivo. These results indicate that PRMT5 may be a promising therapeutic target in T cell-mediated autoimmune diseases such as Multiple Sclerosis.
Inhibition of Th17 by targeted degradation of HIF1-α
Authors: Lai Ming-Zong , Chou Ting-Fang , Chuang Ya-Ting , Hsieh Wan-Chen , Chang Pei-Yun , Hsu Tzu-Sheng , Miaw Shi-Chuen , Chen Ruey-Hwa , Kimchi Adi
Published: January 2023

Death-associated protein kinase (DAPK) is a tumor suppressor, while hypoxia-inducible factor 1α (HIF-1α) promotes tumorigenesis in various type of cancers. Here we report a targeted degradation of HIF-1α mediated by DAPK in T helper 17 (Th17) cells. HIF-1α is a transcription factor critical for Th17 development. We found that DAPK inhibits Th17 differentiation and prevents Th17-mediated autoimmune diseases. DAPK suppresses the expression of RORγt and IL-17, which could be attributed to a specific suppression of HIF-1α. In contrast, Th1 cell differentiation and T-bet expression is not affected by DAPK. We demonstrate that DAPK specifically downregulates HIF-1α in a unique process. While HIF-1α is predominant nuclear localized in many cancer cells, HIF-1α is located in both cytosol and nucleus in T cells. This leads to an interaction between HIF-1α and DAPK in T cell cytoplasm. The simultaneous binding of DAPK to prolyl hydroxylase domain protein 2 (PHD2) increases the association between HIF-1α and PHD2. Consequently, DAPK enhances the proline hydroxylation and proteasome degradation of HIF-1α in Th17 cells. DAPK-deficiency thus results in excess HIF-1α accumulation, enhanced Th17 generation, and exacerbated experimental autoimmune encephalomyelitis (EAE). Transgenic DAPK expression, in contrast, suppresses Th17 differentiation and prevents EAE generation. The overproduction of Th17 cells and exacerbated EAE development in Dapk−/− mice is fully reversed by knockout of HIF-1α. Our results demonstrate a new process involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that a potential therapy for Th17-associated inflammatory diseases by targeted elevation of DAPK.
Micromanaging lupus nephritis: miR17-92 modulates TFH development and regulatory T cell activity
Authors: Yang Huang-Yu , Wu Chao-Yi , Pan Fan , Pardoll Drew , Yang Chih-Wei
Published: January 2023

T follicular helper (TFH) cell provide crucial growth signals to germinal center (GC) B cells supporting antibody production. Tight control of TFH numbers maintains self-tolerance. Regulatory T (Treg) cells play a critical role in maintaining self-tolerance and controlling the magnitude of physiologic immune response. The Treg transcription factor forkhead box P3 (Foxp3) works in concert with other co-regulator molecules to determine suppressive phenotype of Treg. Compiling evidence show that aberrant TFH, GC responses and deficiencies of Treg are associated with systemic lupus erythematous and autoantibody production. We induce pristane nephropathy on T cell specific miR-17–92 knockout (miR-17–92−/−) mice. Mir17-92 T cell specific deficiency mitigates pristane induced-lupus nephropathy in mice. The mice showed less TFH cells, less GC B cells and lower autoantibody formation. Consistent with the reduction in autoantibody production, histological analysis revealed a lower mean renal histopathology score and less compliment deposition. We demonstrate that the miR17-92 cluster regulates TFH development by targeting Akt pathway. Moreover, miR17-92 mitigate the suppression function of Tregs by targeting Foxp3 co-regulators. Ectopic expression of miR17-92 downmodulates the suppression functions of Tregs and provides Tregs with partial effector activity via de-repression of cytokine genes. Our studies suggest that miR17-92 modulates lupus activity through critical regulation in TFH and Treg, unveiling the future therapeutic potential of microRNA manipulation in lupus nephritis.
Validation of murine lymph node slices as a viable and functional experimental platform
Authors: Belanger Maura C , Ross Ashley E , Kinman Andrew W L , Groff Benjamin D , Pompano Rebecca R
Published: January 2023

Lymph nodes are highly organized secondary lymphoid organs that undergo dynamic spatial rearrangement during an immune response. Unlike either in vitro cultures or in vivo animal experiments, ex vivo tissues slices both maintain the cellular arrangement and dynamics of the original organ and also make it experimentally accessible. In this study we perform the first systematic approach to validating live lymph node slices as an experimental platform by assessing their viability and functionality. A method was developed for fluorescent immunostaining of 300-μm thick live tissue slices for cellular and structural markers, e.g. B220 (B cells), Lyve-1 (lymphatics), and CD69 (T cell activation), minimizing background and maintaining viability. Slicing and culture conditions were optimized to maximize viability and minimize inflammatory consequences of slicing. Flow cytometry and fluorescent microscopy data were collected from individual slices, providing detailed information about the state of the tissue. In preliminary experiments, tissue slices were maintained for 24 hours with 71.9 ± 5.49% viable cells (propidium iodide negative); this number is expected to increase after optimization experiments are completed. Cell motility and cytokine secretion were intact. Average cell motility of adoptively transferred splenocytes was measured to be 9 ± 2 μm/min in immunostained slices at 37 °C, consistent with reported values. Treatment with phytohemagglutinin-L produced 3-fold higher IFN-γ in treated vs. untreated slices after 48 hr, and 15-fold higher than treated cell cultures at a matching density. This study sets up live tissue slices as an experimental platform to study dynamic cellular and molecular events within the lymph node.
Transcriptomic dissection of whole lymphoid organs using high throughput single cell sequencing.
Authors: Chang Christina , Bansal Nidhanjali , Fan H. Christina
Published: January 2023

Secondary lymphoid organs contain heterogeneous immune cell populations that play central roles in the defense against pathogens. Proper immune response relies on dynamic and complex changes in the composition and function of these immune cells. Until recently, high throughput single cell analysis was primarily achieved through flow cytometry, which is limited by the number of cell surface markers that can be examined simultaneously. But advances in single cell sequencing technology has made transcriptional profiling of single cells possible. In this study, we demonstrate the power of using the BDTM Resolve single cell sequencing platform to delineate immune cell types in mouse spleen and lymph node tissues.

Using BDTM Resolve’s micro-well array and molecular indexing technology, we captured thousands of cells from disassociated mouse spleen and lymph nodes. Around 1000 cells were subsampled for single cell analysis using two approaches: whole transcriptome amplification(WTA) and targeted highly multiplexed PCR, which surveys over 400 immune relevant genes. Based on transcriptional profiles, we were able to reconstruct all the major immune cell compartments as well as rarer subsets using both assays. While WTA analysis offers an unbiased approach for identifying novel genes and pathways, our study shows that highly multiplexed PCR is a cost effective alternative for profiling immune population in tissues, requiring significantly fewer reads and achieving higher sensitivity for low abundance transcripts. The BDTM Resolve single cell system streamlines whole tissue immune profiling from thousands of cells, and can easily be applied to other tissues to understand immunological changes in cell composition and function.
High throughput single cell analysis of human T cell differentiation dynamics by multiplexed PCR.
Authors: Chang Christina , Shum Eleen Y , Bansal Nidhanjali , Dreux Joanna , Lam Gretchen , Fan Jue , Fan H. Christina
Published: January 2023

CD4 T cells are critical components of the adaptive immune system and can differentiate into specialized effector subsets to mediate immune response against a wide host of pathogens. Studying the transcriptional profile of distinct T helper subsets has illuminated important pathways and mechanisms for effector cell function and lineage. Recently, single cell whole transcriptome analysis (WTA) provides a high resolution view of the complex and highly heterogeneous transcriptional dynamics of immune cell populations. However, such methods are often labor and cost prohibiting for high throughput analysis. Moreover, a significant portion of sequencing reads consist of high expressing housekeeping genes that make meaningful cluster analysis difficult.

In this study we explored a targeted approach to single cell sequencing of in vitro differentiated human Th1 and Th17 cells, by way of highly multiplexed PCR, and compared the results to WTA of the same samples. Using the BD Resolve Targeted T cell assay, we surveyed more than 300 genes in thousands of cells. We were able to distinguish between T cells that were cultured in different polarizing conditions using cluster analysis, and identify cells in different activation and differentiation states. In addition, the targeted assay produced better sensitivity for T cell critical genes with less than one tenth of the sequencing reads needed in WTA to achieve similar sequencing depths, and allowed us to detect low abundance T cell markers that are difficult to capture in WTA. Our results show that single cell analysis by multiplexed PCR is a powerful and cost effective way to study the functional states of T cells.
Exploring the potential application of ctDNA technology within a resource constrained environment
Authors: Sika-Paotonu Dianne , Viali Satu Urima , Lameko Viali , Bunch Shelley , Best Kilistina , Fiu Fa’avaivai , Iaapo Fo’a , Ilaoa-Auva’a Leveti , Polatavao Tasi , Savea Lagaau , Guilford Parry
Published: January 2023

A novel cancer detection method known as circulating tumour DNA (ctDNA) monitors small fragments of tumour DNA found in the blood circulation. These fragments can be isolated and quantified to obtain data related to cancer progression and treatment response. Since gaining access to and obtaining biopsy samples from solid cancers in people is not always possible or straightforward, the possibility of utilizing a simple blood sample to allow detection and monitoring of cancer growth and behaviour is highly desirable. It is anticipated this simple method of detecting and monitoring cancer will enhance the precision of cancer care for patients, and will eventuate into a particularly useful tool within resource constrained environments such as the Pacific. The purpose of this work was to explore the potential utility of ctDNA to support the management, treatment and diagnosis of cancer patients in Samoa and identify the diagnostic methods, treatment and pharmacologic options currently available for cancer patients. Strong interest was expressed by medical, nursing, health and community research staff and lay persons in the development of ctDNA particularly as a potential diagnostic tool within the health setting in Samoa. A key feature of interest concerning the ctDNA technology was the potential to monitor patients in remote locations by visiting them in their homes rather than having them travel to the main hospitals often with their families. ctDNA remains an attractive tool with potential to support cancer patients in Samoa.
Characterization of B- and T-cell immune repertoires using Anchored Multiplex PCR and Next-Generation Sequencing.
Authors: Eberlein Jens , Harrison Thomas , Sims Jennifer , McKittrick Ian , Wemmer Megan , Griffin Laura M , Culver Brady P , Johnson Laura , Kudlow Brian A
Published: January 2023

Introduction

NGS-based analysis of the immune repertoire (IR) is a powerful tool to monitor disease, adaptive immune responses to disease, vaccination and therapeutic interventions. IR characterization by NGS usually requires large primer panels, due to the extensive combinatorial diversity, and a complex system of synthetic controls to account for differential amplification efficiency across segment combinations. Anchored Multiplex PCR (AMP™) uses molecular barcoded (MBC) adapters and gene-specific primers (GSPs), enabling NGS-based immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Here, we describe AMP-based NGS assays for IR characterization, Immunoverse™ IGH and TCR, which utilize a minimal set of unidirectional GSPs and MBC adapters that reduce amplification bias.

Methods

The quantitative reproducibility and sensitivity of our assays was validated using mRNA isolated from PBMCs of healthy donors, B-cell chronic lymphocytic leukemia donors and formalin-fixed paraffin-embedded (FFPE) tissue.

Results

Both assays demonstrated high reproducibility between replicates with quantitative clone tracking down to 0.01%. The ability to determine isotype, clonotype and IGHV mutational status in a single assay was demonstrated. Preliminary TCR assay data indicates that CDR3 sequence capture is possible from FFPE tissue with clonotype calling being driven by input quantity, T-cell content, and, to a lesser degree, mRNA quality.

Conclusions

AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire.
High-throughput single cell gene expression profiling of peripheral blood mononuclear cells from healthy donor and individuals with rheumatoid arthritis
Authors: Fan H. Christina , Cordes Metzler Kimberly R. , Bansal Nidhanjali , Fan Jue , Mao Rong , Elliott Serra , Robinson William H
Published: January 2023

Peripheral blood has been extensively studied using gene expression microarrays in the past. These studies have revealed numerous genes that are differentially expressed between diseased and healthy populations. However, almost all these studies utilized bulk RNA isolated from blood cells for analysis, therefore masking the contribution from different types of cells in the sample. Here, we demonstrated the use of high throughput single cell gene expression analysis to profile peripheral blood mononuclear cells. Using BDTM Resolve and next-gen sequencing, we studied the single cell profiles of a total of ~27,000 cells obtained from two individuals with rheumatoid arthritis (RA) and a healthy donor. By studying either the profiles of the entire transcriptome or a representative set of ~500 gene markers via targeted amplification, we were able to observe distinct expression patterns in the monocyte population in RA patients as compared to controls, as well as difference in overall population architecture. At similar or even lower sequencing cost per cell, targeted amplification of representative gene markers offers much higher resolution of gene expression analysis of highly similar subtypes of cells (such as T cells) as compared to the whole transcriptome approach. These results demonstrated that high-resolution single-cell gene expression analysis using targeted amplification can potentially be used to routinely examine peripheral blood cells in various conditions, and may serve as new screening or diagnostic approach.
Optimization of a Multi-Parametric Activation-Induced Marker (AIM) Assay for Identifying Antigen-Specific CD4+ and CD8+ T-cells
Authors: Victor Kaitlin , Friberg Heather , Jarman Richard G , Currier Jeffrey R
Published: January 2023

Interest in methods to identify and isolate reactive, antigen-specific CD4+ and CD8+T-cells has increased due to their potential for use as immunotherapeutics in cancer and infectious disease. Current methods use one or two markers of T-cell activation to identify the cells of interest after stimulating and culturing PBMCs for up to two weeks in vitro. Here we describe an activation-induced marker (AIM) assay that uses five surface markers to reliably identify all subsets of activated CD4+ and CD8+T-cell populations after overnight stimulation. Conditions have been optimized to either capture activation marker expression during the stimulation or surface stain for expression post-stimulation in the absence of protein transport inhibitors. We successfully used this approach to identify subsets of T cells expressing CD134(OX40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS) and CD69, capturing the total antigen-specific population and enabling characterization of typically hard-to-identify T helper subsets including Th2 and Tfh cells. As there is nointracellular staining component, this method allows for sorting of the populations of interest. This assay is also compatible with downstream applications such as single-cell transcriptional profiling and in vitro expansion – both of which require intact and viable antigen-specific T cells.
Seq-Well: A portable, low-cost platform for massively-parallel single-cell RNA-Seq profiling of sparse clinical samples
Authors: Gierahn Todd M , Wadsworth Marc H , Hughes Travis K , Love J. Christopher , Shalek Alex K
Published: January 2023

Many disease-relevant immune processes occur within tissues. Comprehensive phenotyping of the scarce cells acquired from tissue samples is challenging using traditional analytical approaches. Single-cell RNA-Seq (scRNA-Seq) has recently emerged as a potent new tool for deep profiling of cells, but most approaches suffer from low cell throughput, high costs and significant equipment requirements. To overcome these barriers, we have developed Seq-Well, a technique that enables massively parallel scRNA-Seq analysis of sparse clinical specimens. Seq-Well utilizes nanowell arrays to capture the transcripts from up to 80% of loaded cells on DNA-barcoded poly(dT) beads. After capture, all transcripts are processed and sequenced in bulk. The bead-derived barcode associated with each sequencing read is then used to deconvolute single cell transcriptomes in silico. We validated that the transcriptomes acquired through Seq-Well are derived from single cells through species mixing experiments and analysis of PBMC subtypes. To date, Seq-Well has been utilized to profile cells present in cytobrushes, tumor aspirates, biopsies, cerebral spinal fluid and sputum. Seq-Well requires no specialized equipment beyond a simple manual clamp and costs less than $200 to achieve a sequencing library for thousands of single cells, making the technique deployable in nearly any laboratory at scale. Overall, Seq-Well provides an effective and accessible means to acquire deep phenotypic profiles of leukocytes from nearly any clinical sample.
NLRX1 promotes HIV-1 and DNA viruses replication by blocking STING-TBK1 innate immune signaling
Authors: Deng Meng , Guo Haitao , König Renate , Riess Maximilian , Mo Jinyao , Zhang Lu , Petrucelli Alex , Yoh Sunnie M. , Zhang Aiping , Colberg-Poley Anamaris M. , Feng Hui , Lemon Stanley M. , Zhang Zhigang , Damania Blossom , Tsao Li-Chung , Wang Qi , Su Lishan , Duncan Joseph A. , Chanda Sumit K. , Ting Jenny P
Published: January 2023

Microbial infections are the most common cause of death in humans. Type I interferon (IFN-I) is a key element bridging the host innate and adaptive immune response against infections. Delineating the molecular regulation network of IFN-I signaling is critical for developing novel antiviral strategy and benefiting rational therapy. Using an unbiased siRNA screen, we find NLRX1, one nucleotide-binding leucine-rich-repeat-containing protein, is a host factor that promotes an early step of HIV-1 infection. NLRX1 suppresses type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA and enhances the nuclear import of HIV-1 DNA. In addition to HIV, NLRX1 also reduces STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, and DNA virus. Mechanistically, NLRX1 associates with STING in mitochondria-associated ER membranes, and prevents STING recruiting TBK1 and activating downstream interferon signaling. By using purified recombinant proteins, we found NLRX1 interacts directly with STING. Furthermore, DNA virus infected Nlrx1−/− mice exhibited enhanced innate immunity and reduced morbidity and viral load. In summary, these findings reveal that NLRX1 is a checkpoint protein for DNA sensing adaptor STING and may represent a novel precision target for anti-viral therapy.
Identification of CMPK2 as an Interferon Stimulated Gene that Restricts HIV Infection
Authors: Soliman Mary , El-Diwany Ramy , Wheelan Sarah , Thomas David L , Balagopal Ashwin
Published: January 2023

Type 1 interferons upregulate many interferon-stimulated genes (ISGs) with diverse antiviral effects. ISGs that restrict HIV in humans have been poorly characterized. Exogenous interferon-α decreases HIV viremia: we hypothesized that interferon-α induces expression of ISGs with anti-HIV activity in activated CD4+ T cells, the cells in which HIV chiefly replicates.

Activated CD4+ T cells (CD3+/CD4+/CD38+/HLA-DR+) were sorted from PBMCs taken from 19 HIV infected patients with uncontrolled viremia before and after peginterferon alpha 2b (IFN) administration. RNAseq was performed and ISGs were identified if they were up-regulated in 11/19 people. Viral kinetics were measured pre- and post-interferon and correlated with each ISG to identify putative HIV restriction factors. In vivo findings were tested in THP-1 cells. mRNA (measured by qPCR) and protein (measured by Western blot) were quantified pre- and post-IFNα treatment (1000 IU/mL). RNA interference was used to test whether the anti-HIV effect of IFNα could be attributable to CMPK2.

Among the 19 persons, 99 ISGs were defined in activated CD4+ T cells: the magnitude of upregulation of 13 ISGs correlated strongly with plasma HIV RNA decline (r>−0.8 for all, p<0.001 for all). Cytidine/Uridine Monophosphate Kinase 2 (CMPK2) was selected for examination. CMPK2 mRNA was upregulated by IFNα 40-fold, and protein upregulation was confirmed. IFNα restricted HIV replication in vitro 1000-fold, and siRNA knockdown of CMPK2 restored HIV replication to 72.1% of untreated cells (p=0.019).

CMPK2, a gene involved in maintenance of intracellular UTP/CTP, is an ISG with potential to restrict HIV replication. Future work will focus on the mechanism underlying the antiviral effects of CMPK2.
Cellular immunity patterns of rDEN2Δ30 (Tonga/74) support its suitability as a human DENV challenge strain.
Authors: Grifoni Alba , Angelo Michael , Sidney John , Paul Sinu , Peters Bjoern , de Silva Aruna D , Phillips Elizabeth J , Mallal Simon Alexander , Diehl Sean A , Botten Jason , Boyson Jonathan E , Kirkpatrick Beth D , Whitehead Stephen S , Durbin Anna , Sette Alessandro , Weiskopf Daniela
Published: January 2023

The need for human DENV challenge model to better evaluate DENV candidate vaccines has become of extreme importance in the past years after neutralizing antibody failure to be a good correlate of protection and the lack of valid alternatives. rDEN2Δ30, a variant of DENV2 Tonga/74 strain lacking 30 nucleotides from its 3′ untranslated region, has been established as DENV human challenge model. In this study, DENV cellular immune responses of rDEN2Δ30 strain have been compared with natural infection to derive correlates of protection for DENV vaccines based on cellular immunity. For this purpose, HLA class I and class II restricted peptides have been predicted and tested in an IFN-gamma ELISPOT assay, to assess CD8+ and CD4+ T cell responses in healthy donors infected with rDEN2Δ30. CD8 and CD4 responses were detected in approximately 80% and 90% of donors respectively. Strong CD8 responses are detected in rDEN2Δ30 recipients and when compared with natural infections similarity in terms of magnitude and numbers of recognized epitopes is observed. In particular, the immunodominance hierarchy of the DENV nonstructural proteins NS3, NS5 is maintained in both analyzed cohorts. On the contrary, CD4 responses were mainly focused on nonstructural proteins and less strong respect to natural infection. Large overlap is observed in the epitopes recognized by rDEN2Δ30 infection and natural infection both for CD8 (100%) and CD4 (85%) responses. Finally, when rDEN2Δ30 CD8 response is compared with the one induced by other attenuated DENV isolates, a stronger response is observed. In conclusion, T cell response of rDENV2D30 has very similar specificity to natural infection supporting its suitability as an appropriate human DENV challenge model.
In vitro expansion and characterization of virus-specific memory B cells
Authors: Murtaugh Michael P , Rahe Michael C , Gustafson Kevin
Published: January 2023

In the pig, the memory B cell is largely uncharacterized and as a result, our ability to determine long-term vaccine efficacy has been hindered. To address this unmet need, CD21+ porcine B cells were activated in vitro with CD40L and stimulated with various cytokines to test for cellular activation and expansion into antibody secreting cells (ASC). IL-21 was a potent inducer of proliferation and differentiation, inducing a 3-fold expansion in total cell numbers with about 15% of all B cells differentiating to IgM or IgG ASCs. But even with robust proliferation the viability of CD21+ B cells deteriorated rapidly following initial expansion. Thus, APRIL and BAFF were tested as cell survival and maintenance factors. BAFF enhanced mature B cell and ASC viability, but APRIL was only effective for ASCs. They induced a 2-fold increase in secreted IgM and IgG. CD21+ B cells from porcine reproductive and respiratory virus (PRRSV) immune and naïve pigs were expanded and tested for reactivity to nonstructural protein 7 (nsp7), a highly immunogenic PRRSV protein. Immune, but not naïve, pigs displayed positive reactivity on both ELISPOT and ELISA. These results highlight the expansion and differentiation potential of memory B cells in vitro without the stimulation of surface Ig, as well as the establishment of a culture system to interrogate the porcine memory B cell. A PRRSV nsp7-B-cell tetramer was produced for identification and FACS sorting of antigen-specific B cells. Combining B cell tetramers with the in vitro model of memory B cell expansion is expected to facilitate the evaluation of vaccination efficacy through analysis of memory B cell response quality and magnitude without requiring live animal challenge.
Bordetella pertussis vaccine composition drives hematopoietic stem and progenitor cell expansion and immune response during vaccination and subsequent challenge.
Authors: Varney Melinda , Boehm Dylan , DeRoos Katherine , Wong Ting , Kilic Emel Sen , Bradford Shelby , Elkins Cody , Epperly Matthew , Witt William , Barbier Mariette , Damron Fredrick H
Published: January 2023

Pertussis is a re-emerging infectious disease world-wide. It is hypothesized that current acellular vaccines (ACVs) wane in efficacy each year after immunization. We hypothesized that whole cell vaccines (WCVs) induce hematopoietic stem and progenitor cell (HSPC) expansion and differentiation processes necessary for life-long protection. While HSPC innate immune signaling and expansion play a critical role in directing immune response to infection, their role in vaccine efficacy is unclear. To test our hypothesis, we assessed bone marrow (BM) HSPC frequency, peripheral blood composition, and immune cell proportions in the spleen and thymus upon vaccination. We find that the BM Lineage-Sca1+cKit+ (LSK) cell population undergoes progressive expansion in WCV mice when compared to ACV and PBS-injected control mice. Additionally, peripheral white blood cells and spleen size increase in WCV mice, suggesting that BM cells mobilize to the blood and spleen, where extramedullary hematopoiesis can occur. Upon infection, myeloid cells have been demonstrated to leave the spleen, enter infected tissues, and produce cytokines that favor the generation of Th1 cells. Occurring more rapidly in WCV mice, LSK cell frequency increases and spleen size decreases upon B. pertussis challenge in both WCV and PBS-injected mice when compared to ACV mice. WCV mice exhibit increased peripheral blood monocytes, neutrophils, and lymphocytes when compared to other groups. Taken together, our data suggest that HSPC expansion, differentiation, and mobilization upon WCV immunization may prime the host to better respond to pathogen, and that ACVs formulated with adjuvants that stimulate HSPC expansion, such as TLR4 agonists, may enhance vaccine efficacy.
Characterization of pathways involved in the suppression of T follicular helper cells in newborns
Authors: Yang Ji Yeon , Siddiqui Shafiuddin , Uslu Kadriye , Lee Robert C , Ireland Derek DC , Allman Windy R , Verthelyi Daniela , Akkoyunlu Mustafa
Published: January 2023

The immature immune system of newborns renders poor vaccine response. However, mechanisms of limited T follicular helper (Tfh)-mediated antibody production in newborns are largely unknown.

We found significantly more expression of the Tfh inhibitory genes associated with regulatory T cell (Treg) cells and IL-2 signaling in immunized newborn mice CD4+CXCR5+PD-1+Tfh cells than in adult mice. The IL-2-STAT5 signaling in CD4+CXCR5+PD-1+Foxp3+T follicular regulatory cells (Tfr) was hyperactive in newborns as compared to adults. Also, in newborns IL-6-STAT3 signaling was lower in Tfh cells but higher in Tfr cells compared to adult mice. Immunization did not elicit IL-21 expressing Tfh cells but when provided sufficient IL-21, in vitro cultured newborn T cells differentiated into Tfh-like cells. These data indicate that in newborns, dendritic cell-derived IL-6 signaling preferentially induces Treg cells to differentiate into Tfr cells, which in turn suppresses Tfh generation. Supporting this hypothesis, co-injection of newborn mice with conjugate type 14 pneumococcal polysaccharide vaccine together with IL-21 or anti-CD25 blocking antibody reduced Tfr/Tfh ratio and enhanced Tfh formation. While IL-21 elicited both IgG and IgA antibodies against vaccine, anti-CD25 antibody only improved IgA production, due in part to the elevated APRIL and TGF-β expression in T cells and fibroblast reticular cells. Since IL-21 acts both on T cells and B cells, improved IgG response in IL-21 injected mice is likely due to its direct effect on B cells.

These findings highlight the differences in the programming of IL-2 and IL-6 signaling in newborn and adult Tfh cells. These differences may be exploited in devising improved vaccines for infants.