bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract

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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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&gt;−0.8 for all, p&lt;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

Abstract
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

Abstract
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.