bluuebunny/crossref_metadata_2025_split huggingface dataset:
HIVE Single-Cell TCR Sequencing: Assay Principle and Applications
Authors: Whitney Irene E , Chen Li , Gierahn Todd , Flanigon Jim 
Published: January 2023

Abstract
Biological resolution at the level of individual cells is powering the next phase of precision health. Honeycomb Biotechnologies currently offers an assay for single-cell RNA sequencing with the HIVE™ scRNAseq Solution. The HIVE is a portable, handheld, single-use device that enables gentle capture, robust storage, and easy processing for the analysis of single-cell samples. The HIVE scRNAseq assay integrates sample storage and single-cell profiling into a single workflow, facilitating decentralized collection and centralized processing of samples. The design of the HIVE is ideal for the recovery of fragile cells, loading of large sample volumes, and efficient processing of numerous samples in parallel, particularly valuable for clinical applications.
Here, we extend the application of the HIVE by offering a single-cell T cell receptor enrichment and sequencing (scTCRseq) assay. Using the stable whole transcriptome amplification (WTA) product obtained from the scRNAseq assay as the sample input, the HIVE scTCRseq assay enriches TCR in a streamlined target-specific amplification workflow. The assay is compatible with all human tissues, and can accommodate a wide range of cell numbers. We typically achieve over 75% TCR molecule recovery from the WTA, and over 60% sequencing reads are mapped to TCR. Here we also present TCR profiling of several specimens, demonstrating its value in clinical applications.
Honeycomb aims to expand single-cell opportunities across basic, translational, and clinical research throughout the world.
Single-cell multi-omic analysis identify heterogeneity and distinct features in fate-mapped tissue-resident alternatively activated macrophages
Authors: Lin Jian-Da , Chou Tzu-Yin , Yang Chen-Hsuan , Chao Pei-An , Chen Yen-Ting , Loke P’ng 
Published: January 2023

Abstract
Peritoneal cavity cells play pivotal roles in inflammation, repair, and maintaining homeostasis in response to pathogenic infections and tissue injury. Two major tissue-resident macrophages (TRMs) are large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs). These are the major macrophage subsets in the peritoneal cavity and originate from embryogenic (LPMs) or bone-marrow-derived myeloid precursors (SPMs). CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) provides simultaneous information for single cells in both cell-surface protein and gene expression levels. Here, we used CITE-seq to profile peritoneal cells by an oligonucleotide-labeled antibody panel designed to react with 189 unique mouse cell surface antigens. We identify 14 markers exclusively expressed on TRMs but not other immune cell types. These markers can classify phenotype differences between LPMs and SPMs during IL-4 stimulation. We further profile fate-mapped TRMs by scRNA-seq and identified more heterogenous phenotypes of TRMs that originated from embryogenic rather than bone-marrow-derived myeloid precursors. Notably, serum amyloid A-3 (Saa3) and platelet factor 4 (Pf4), can distinguish TRM clusters from alternative activation to IL-4 stimulation and Heligmosomoides polygyrus infection. Hence, we identify distinct markers that can be used to distinguish the different origins and heterogenous TRM phenotypes under steady state and type 2 immune responses.
Cell type-specific differential expression in Alzheimer’s disease and cancer using spatial transcriptomics
Authors: Cable Dylan , Irizarry Rafael , Chen Fei 
Published: January 2023

Abstract
Spatial transcriptomics enables spatially resolved gene expression measurements at near single-cell resolution. The detection of genes that are differentially expressed across tissue context for cell types of interest is an essential challenge for dissecting pathological mechanisms. However, changes in cell type composition across space and measurement contamination from other cell types introduce complex statistical challenges. Here, we introduce a statistical method, Generalized Linear Admixture Models for Differential Expression (GLAMDE), that estimates cell type-specific patterns of differential gene expression while accounting for localization of other cell types. By modeling spatial transcriptomics gene expression as an additive mixture, across cell types, of generalized log-linear functions, we provide a unified framework for identifying gene expression changes for a wide-range of relevant contexts: changes due to pathology, anatomical regions, physical proximity to specific cell types, and cellular microenvironment. Our approach enables statistical inference across multiple samples and replicates. We demonstrate, through simulations and validation experiments on Slide-seq and MERFISH data, that our approach accurately identifies cell type-specific differential gene expression with valid uncertainty quantification. Lastly, we apply GLAMDE to characterize spatially-localized tissue changes in the context of disease. In an Alzheimer’s mouse model Slide-seq dataset, we identify plaque-dependent patterns of cellular immune activity. We also find a putative interaction between tumor cells and myeloid immune cells in a Slide-seq tumor dataset. GLAMDE is available within the spacexr R package.
D.C. was supported by a Fannie and John Hertz Foundation Fellowship and an NSF Graduate Research Fellowship. This work was supported by an NIH Early Independence Award (DP5, 1DP5OD024583 to F.C.), the NHGRI (R01, R01HG010647 to F.C.), as well as the Burroughs Wellcome Fund, the Searle Scholars Award, and the Merkin Institute to F.C.. R.A.I. was supported by NIH grants R35GM131802 and R01HG005220.
Analysis of human BCR-IgH from genomic DNA with UMI incorporation
Authors: Brown Brittany E , Pan Wenjing , Berngruber Chelsea , Byrne-Steele Miranda , Han Jian 
Published: January 2023

Abstract
Immune repertoire analysis can be performed from practically any biological sample as long as viable nucleic acid can be extracted. While using RNA as a template has its benefits, gDNA is often preferred as gDNA is often easier to obtain, especially from difficult samples. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may better reflect the quantity of cells. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement.
We developed a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted since the relationship of one V(D)J per cell will be maintained and will have a unique barcode added during the processing. The inclusion of UMI also assists with the elimination of PCR and sequencing error, which will lead to more confident analysis of low frequency clones and hypermutations. The primers were first tested on a BCR-IgH synthetic library. The sequencing result demonstrated &gt;85% correlation with the known synthetic library gene expression. These results showed that the new gDNA BCR-IgH panel was sufficient at amplifying all known J gene targets.
Presently, we have also applied this method to gDNA extracted from B cell enriched PBMC samples including the spike-in of a known BCR clone at various concentrations. To our knowledge, this is the first assay of its kind to incorporate UMI into BCR-IgH gDNA to capture both the BCR repertoire and true cell frequency.
Microfluidic cell sorting for gentle isolation of immune cells.
Authors: Jagnandan Nicole , Morachis Jose 
Published: January 2023

Abstract
Isolation of specific immune cells is critical to biomedical research, however, techniques such as droplet-based fluorescence-activated cell sorting can mechanically irritate cells resulting in cell death or unwanted activation of immune cells that affect experimental analysis. Here we show that T cell subsets and neutrophils can be isolated using a low pressure (&lt;2 psi) microfluidic cell sorter (WOLF G2®, NanoCellect®). These experiments show that this 11-parameter cell sorter can isolate complex cell types at high purity and high viability. In addition, due to the low-pressure microfluidic sorting mechanism, laminar flow reduces shear stress relative to traditional droplet sorters and cells remain functional and unaltered for subsequent experiments. Furthermore, we demonstrate high quality single-cell genomics data when combined downstream with single-cell partitioning and plate-based workflows. These findings demonstrate a novel workflow for isolating immune cells using the next generation of cell sorters.
Lumit: a novel bioluminescent, homogenous immunoassay platform for analyte detection
Authors: Bach Maggie L , O’Brien Martha A , Nath Nidhi , Zegzouti Hicham , Lazar Dan , Cali James 
Published: January 2023

Abstract
The detection of a specific analyte in a complex mixture by an immunoassay is a common procedure in many bioanalytical workflows. Immunoassays rely on the interaction between labeled antibodies and the analyte of interest. We have developed Lumit, a novel bioluminescent labeling approach allowing no-wash immunoassays amenable for high-throughput applications.
Lumit immunoassay technology is based on an adaptation of NanoBiT® luciferase, a structural complementation system that consists of the Large BiT (LgBiT; 18 kDa) subunit and a small, complementary peptide, Small BiT (SmBiT; 11 amino acids), that has low affinity for LgBiT. Antibodies labeled with LgBiT or SmBiT subunits are brought into proximity due to binding to a target analyte. This facilitated complementation reconstitutes an active luciferase and generates light proportional to the amount of analyte present. This detection chemistry has several advantages over traditional ELISA workflows, including a no-transfer and no-wash protocol suitable for automation, fast detection time (30 – 90 minutes), and a broad dynamic range mitigating the need for sample dilutions.
Here, we present a variety of applications of Lumit technology to detect analytes in immunology research. Examples include detection of cytokines from activated cultured immune cells using labeled primary antibodies, the detection of antibody-Fc receptor binding, the detection of protein phosphorylation using labeled secondary antibodies, and detection of the PD1: PDL1 interaction using labeled anti-tag antibodies.
Buoyancy Activated Cell Separation (BACS™) Microbubbles for Lysis and Ficoll Free Processing of Leukopaks
Authors: Grimley Edward C , Roussey Jon , Ku Chia , Snow Tiffany , Wegner Casey , McNaughton Brandon H 
Published: January 2023

Abstract
Leukopaks are highly-enriched apheresis product containing large numbers of mononuclear cells. In applications where large numbers of primary immune cells are required, leukopaks offer an ideal starting material. For this reason, leukopaks are a vital source material for immunophenotyping studies, cell therapy manufacturing, and pharmaceutical development.
The enrichment of leukocytes from leukopaks have traditionally required an upfront density gradient separation to remove red blood cells (RBCs) which has made leukopak processing time consuming, inefficient, and inconsistent. To overcome these obstacles, Akadeum has developed a gentle and effective approach for density gradient and lysis-free processing of leukopaks that is based on our proprietary Buoyancy Activated Cell Sorting (BACS™) Microbubbles.
Here we report on the isolation of untouched T cell and CD4+ T cell populations directly from leukopak material. Untouched T cells and CD4+ T cells were isolated in roughly 30 minutes using our quick and easy protocol. T cells were isolated with a final purity of 91.8% while CD4+ T cells were isolated with a final purity of 93.9%. In addition to high purity, we achieved greater than 90% recovery of both cell populations. These data clearly demonstrate the value of Akadeum’s easy, gentle, and effective sample preparation platform.
The immune dysregulations in inflammatory arthritis immune related adverse events
Authors: Zeng Hu , Zhu Xingxing , Li Yanfeng , Jaquith Jane , McCarthy-Fruin Kathleen , Thanarajasingam Uma 
Published: January 2023

Abstract
The immune checkpoint inhibition therapy induces various immune related adverse events (irAEs). The immunological basis of irAEs remains unclear. We recruited a cohort of patients with inflammatory arthritis irAE (IA-irAE), the most common form of rheumatic irAEs, together with matched patients with conventional rheumatoid arthritis (RA) and cancer patients without irAEs. Comprehensive clinical and immunological investigations reveal an imbalance of T cell exhaustion and senescence in IA-irAE patients, and immunological similarities between IA-irAE patients and seronegative RA patients. These data suggest that IA-irAE and seronegative RA may have shared immunological mechanisms. Thus, our study identifies potential immunological underpinnings of IA-irAE and the relationship between IA-irAE and conventional RA. The results may have implications for managing irAE and seronegative RA patients.
Supported byR01AR0077518 Arthritis National Research Foundation
ST8Sia6 expression in beta cells mitigates onset of autoimmune diabetes in the murine NOD model
Authors: Choe Justin , Belmonte Paul , Rajcula Matthew , Theodore Keith , Lee Hyun Se Kim , Shapiro Michael J , Shapiro Virginia 
Published: January 2023

Abstract
In this study, we harnessed immunomodulatory Siglec-sialic acid interactions to mitigate autoimmune rejection of beta cells in Type 1 Diabetes (T1D). Mice engineered to express the sialyltransferase ST8Sia6 in beta cells (βST mice) were backcrossed to the non-obese diabetic mouse model of T1D and followed for disease; 60% of female littermates spontaneously developed diabetes by 300 days, compared to minimal occurrence (5%) in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration at 4 and 8 weeks of age, but the severity of insulitis was drastically reduced in βST mice beyond 20 weeks of age as compared to littermates. IHC for various immune populations suggested no difference in composition of insulitis at 8 weeks between βST and littermate mice. ELISA for serum anti-insulin antibodies demonstrated no difference in concentration between female βST and littermate mice. After female euglycemic βST and littermate mice at 16 weeks of age were challenged with anti-PD-L1 monoclonal antibodies, both groups resulted in similarly rapid induction of disease, suggesting that ST8Sia6 dependent protection occurs peripherally. Shut-off of ST8Sia6 expression in euglycemic βST mice at 20 weeks of age did not abrogate protection from disease, supporting persistent immune tolerance beyond this time. Flow analysis of islet infiltrating immune cells at 300 days confirmed reduced number of infiltrating immune cells in βST mice. Thus, induced expression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD mouse and presents a novel path forward for improving the success of curative islet transplant into T1D patients.
Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564).
Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564).
Induction of tolerance to a CD4 T cell hybrid insulin peptide epitope prolongs islet graft survival and suppresses autoreactive CD8 T cells
Authors: DiLisio James E , Jamison Braxton L , Neef Tobias , Miller Stephen D , Baker Rocky , Haskins Kathryn 
Published: January 2023

Abstract
Autoreactive T cells are thought to drive autoimmune diabetes by recognizing beta cell-derived peptide antigens. We previously discovered that hybrid insulin peptides (HIPs) are potent neoantigen peptide ligands for a subset of autoreactive CD4 T cells in both the NOD mouse model and human type 1 diabetes patients. Inducing tolerance to prominent T cell autoantigens through antigen-specific immunotherapy could prevent disease onset or recurrence after islet transplantation without the need for broad immunosuppression. Here we show that tolerogenic nanoparticle (NP) delivery of the 2.5HIP, a dominant CD4 T cell HIP epitope in the NOD mouse, can prolong islet graft survival in transplanted diabetic NOD mice. Tolerance induction to the 2.5HIP not only suppressed 2.5HIP tetramer+ CD4 T cells but also autoreactive CD4 and CD8 T cells specific for different islet antigens. 2.5HIP NP treatment induced a dysfunctional state in graft-infiltrating T cells characterized by increased expression of anergic markers on CD4 T cells and reduced inflammatory cytokine production in 2.5HIP tetramer+ CD4 T cells as well as IGRP (islet specific glucose-6-phosphatase catalytic subunit-related protein) tetramer+ CD8 T cells. In conclusion, we demonstrate that antigen-specific immunotherapy aimed at inducing tolerance to a single CD4 T cell HIP epitope can prolong islet graft survival and suppress autoreactive CD8 T cells in the NOD mouse model of autoimmune diabetes.
Supported by grants from NIH (R01 DK122566, R01 DK081166, T32 DK120520) and JDRF (2-SRA-2018-566-S-B)
Tolerogenic Immune Modifying Nanoparticles (TIMP) Encapsulating Multiple Diabetogenic Epitopes Prevent Onset of Type 1 Diabetes in NOD Mice
Authors: Genardi Samantha , Podojil Joseph R , Kakade Sandeep , Boyne Michael , Elhofy Adam , Miller Stephen D 
Published: January 2023

Abstract
Type 1 diabetes (T1D) is an autoimmune disease characterized by inflammatory lesions within islets (insulitis) and β cell loss which is associated T cell and B cell responses specific for pancreatic islet β cell proteins. Data show that treatment with poly(lactide-co-glycolide) (PLGA) nanoparticles (i.e. TIMP/COUR’s CNP) containing a single diabetogenic peptide blocks T1D induced by transfer of CD4+ BDC2.5 or CD8+ NY8.3 TCR transgenic T cells to NOD-scid mice. In contrast, Ag-specific tolerance approaches targeting single β-islet cell protein epitopes, e.g. GAD65 or Insulin, have failed to achieve efficacy in both wildtype NOD mice and clinical trials. The possibility exists that T1D initiation and progression is due to activation of T cell populations specific for multiple diabetogenic epitopes. To test this hypothesis, recombinant Chormogranin A, GAD65, and Insulin proteins were encapsulated within CNPs to assess Treg/Tr1 cell induction, inhibition of Ag-specific T cell responses, and blockade of T1D in NOD mice. While treatment of NOD mice with CNPs containing a single protein inhibited the corresponding Ag-specific T cell response, the inhibition of T1D development only occurred if all three diabetogenic proteins were included within the CNPs (CNP-T1D). CNP-T1D-induced blockade of T1D was characterized by Treg/Tr1 cell induction and a significant decrease in both peri-insulitis and immune cell infiltration into pancreatic islets. We have recently published that CNP treatment is both safe and induces Ag-specific tolerance in Phase I/IIa celiac disease clinical trials. Therefore, utilization of this technology including multiple diabetogenic proteins is a promising Ag-specific tolerance approach to T1D treatment.
An IL-2 mutein promotes Foxp3+ Treg-mediated suppression of dendritic cell activation in response to inflammatory stimuli
Authors: Jamison Braxton L , Campbell Daniel J 
Published: January 2023

Abstract
Foxp3+ regulatory T (Treg) cells are essential for maintaining immune tolerance and the development of strategies that boost Tregs is a promising approach for the treatment of autoimmune disorders. We previously developed a murine Fc-fused IL-2 mutein (Fc.Mut24) with decreased affinity for IL-2Rβ resulting in increased CD25 dependency and potent Treg selectivity. High-dose Fc.Mut24 administration prevents autoimmunity in the non-obese diabetic (NOD) mouse; however, the mechanisms by which Fc.Mut24 affects Treg suppressive function remain unclear. In this study, we have performed a phenotypic analysis of Treg and classical dendritic cells (DC) from IL-2 mutein-treated mice. We have found that Fc.Mut24 treatment increases CTLA-4 recycling/expression on Treg with a concurrent decrease in PD-1 expression specifically on effector regulatory T (eTreg) cells. Both CD8+ cDC1 and DCIR2+ cDC2 subsets from Fc.Mut24-treated mice have reduced expression of the co-stimulatory molecules CD80/CD86 following lipopolysaccharide (LPS) injection. Furthermore, RNA-sequencing reveals that Tregs regulate the transcriptional response of DCs to LPS and may preferentially suppress cDC1 activation in vivo. These results suggest that a major mechanism of action by IL-2 mutein may be promoting CTLA-4-mediated transendocytosis of CD80/CD86 while simultaneously reducing PD-1: PD-L1 co-inhibitory signaling between eTreg and DCs. Our findings address unanswered questions about the role of IL-2 in modulating Treg suppressive function and have important implications for the use of Treg-selective IL-2 therapeutics in the clinic.
B.L.J. is supported by a postdoctoral fellowship from the Washington Research Foundation.
Chromatin profiling of renal allograft-infiltrating cells reveals epigenetic regulation of CD8+ T cell exhaustion in chronically rejected human kidney allografts
Authors: Yi Stephanie Grace , Zou Dawei , Dai Yulin , Zhao Zhongming , Gaber Osama , Chen Wenhao 
Published: January 2023

Abstract

Purpose
Our single cell RNA sequencing (scRNA-seq) analysis found that T cells in chronically rejected kidney allografts and peripheral blood mononuclear cells (PBMCs) of the same kidney transplant recipients (KTR) exhibited a spectrum of dynamic cell states; herein, we investigate how T cell states are regulated at the epigenetic level during chronic rejection.

Methods
H3K27me3 and H3K27ac CUT&Tag was performed in live T cells isolated from PBMCs and allografts of 3 KTR with failed transplants. All transplants had chronic cellular and antibody mediated rejection on pathology.

Results
With CD8+ T cell infiltration into allografts, H3K27me3 was deposited at the gene loci associated with T cell quiescence and stemness (TCF7, BACH2, FOXP1, IL7R, etc.), whereas H3K27ac was mostly deposited at the gene loci involved in T cell effector (GZMB, ID2, ZEB2, etc) and exhaustion (TOX, TOX2, TIGIT, PDCD1, HAVCR2, etc) programs.

Conclusion
Our prior scRNA-seq analysis revealed that most CD8+ T cells in PBMCs maintain TCF7+ stem-like cell phenotypes. However, most CD8+ T cells infiltrated in chronically rejected allografts lose stemness, becoming either TCF7− GZMBhi effector or TCF7− PDCD1hi HAVCR2hi exhausted T cells. Herein our CUT&Tag analysis reveals that chromatin remodeling mediates such dynamic changes of T cell states. Of note, although T-cell exhaustion occurs in chronically rejected allografts, it only represents dynamic changes of T cell states. We propose that TCF7+ allogenic T cells in the periphery maintain stem-like property. Without proper epigenetic silencing of their function, TCF7+ allogenic T cells produce not only exhausted progeny but also effector cell progeny to mediate allograft rejection.
Supported by internal