bluuebunny/crossref_metadata_2025_split huggingface dataset:
An automated approach to high-plex cytometric immunophenotyping with CyTOF XT
Authors: Li Stephen K H , Zabinyakov Nick , Bouzekri Alexandre , Straus Rita , Jong Raymond , Sullivan Michael , Loboda Alexander , Majonis Daniel , Loh Christina 
Published: January 2023

Abstract
CyTOF® mass cytometry is a single-cell analysis platform that uses isotope-tagged antibodies to resolve 50-plus markers in a single tube without signal compensation, making CyTOF ideal for routine immunophenotyping. CyTOF XT™, the latest CyTOF system, features automated sample acquisition. Stained samples were acquired in parallel using the automated CyTOF XT system and manually, using the Helios™ system, to assess performance of the automated system.
Multiple suspension mass cytometry staining workflows were evaluated. Population frequencies and resolution indices for markers were assessed by manual gating. There was no significant difference between population frequencies analyzed between the two CyTOF systems. On average, samples acquired on CyTOF XT resulted in greater resolution between positive and negative populations compared to Helios.
The Maxpar® Direct™ Immune Profiling System, which comprises the Maxpar® Direct™ Immune Profiling Assay™ and Maxpar Pathsetter™ software, was also compared on the CyTOF XT and Helios systems. The Maxpar Direct Immune Profiling Assay includes a 30-marker panel in a dry, single-tube format for staining human whole blood or PBMC. Maxpar Pathsetter automates reporting of population statistics and stain assessments for the panel. Maxpar Pathsetter showed comparable population frequencies between the two CyTOF systems and improved staining assessment on CyTOF XT.
Overall, these studies find that the CyTOF XT system generates better signal resolution than the Helios system. Automated acquisition by CyTOF XT enables researchers to accurately and reproducibly streamline human immunophenotyping.
For Research Use Only. Not for use in diagnostic procedures.
An integrated single-cell multiomics approach to characterize mRNA, intracellular, and surface proteins using intracellular AbSeq and BD® AbSeq Immune Discovery Panel
Authors: Song Hye-Won , Hatami Elham , Vadrevu Samatha , Martin Jody , Snowden Eileen , Zhu Timothy Zheng , Wright Adam , Nakamoto Margaret , Dillmore Shannon , Ayer Aruna 
Published: January 2023

Abstract
DNA-barcoded antibodies enable single-cell multiomics approaches that examine protein alongside mRNA in a single cell. Although it is a powerful approach, leading methods are currently limited to surface proteins, which limits understanding of signal-transduction and transcriptional pathways that are often governed by expression changes and posttranslational modifications of intracellular proteins. The ability to analyze intracellular proteins alongside the transcriptome is complicated by the fix and perm processes necessary to access intracellular epitopes, which can negatively impact RNA assay sensitivity. Furthermore, nonspecific binding of antibody-oligos to nucleic acids can generate high background signals.
To address these issues, we generated a protocol that can recover high-quality RNA alongside specific intracellular and surface protein information. Using this protocol in conjunction with the BD Rhapsody™ Single-Cell Analysis System, we simultaneously profiled over 50 protein markers including 14 intracellular proteins with the whole transcriptome. We identified predicted patterns, including increased phosphorylation of AKT protein correlated with the proliferation marker Ki67 upon immune stimulation, and transcription factor (T-bet) expression in a specific cell subset. Together these preliminary results suggest that analysis of intracellular proteins together with the transcriptome may be possible using single-cell sequencing technologies and can enable a deeper understanding of cellular states.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Quick and Easy Isolation of Immune Cells From Large-Volume Samples
Authors: Posarac Vesna , MacDonald G Neil , Buck Chris A , Toombs Eric , Gellner Savannah D , de Jong Susan , Williamson Mark E , Egeler Oliver , Dalton Bob , Kokaji Andy I , Eaves Allen C , Louis Sharon A , Antignano Frann 
Published: January 2023

Abstract
Large-scale cell isolation is commonly performed in labs and core facilities for cell banking and drug discovery, and as a critical step in cell therapy manufacturing. Current methods can be a significant bottleneck in a lab’s workflow, often requiring a full day for sample processing and cell isolation. To address this need, we have developed two methods: manual column-free isolation using the Easy 250 EasySep™ magnet, and automated cell isolation in a closed system using RoboSep™-C.
The Easy 250 magnet allows users to isolate cells from a full leukopak of up to 20 billion cells in under 30 minutes by simply pipetting out their target cells. Negative selection protocols have been optimized to achieve 95.3% T cell, 97.2% CD4+ T cell, 91.9% CD8+ T cell, 99.4% B cell, 96.5% NK cell, and 91.5% monocyte purities. Positive selection protocols obtain 95.4% CD3+ cell, 94.4% CD4+ T cell, 93.9% CD8+ T cell, and 96.2% CD14+ cell purities.
RoboSep™-C automates this cell isolation procedure, along with the cell washing steps for sample preparation, in as little as 50 minutes. The instrument features a scale tower, pump and clamp modules to direct fluid through defined paths, and a magnet for separation of labelled cells. The system uses a sterile single-use tubing set that incorporates a cartridge for cell washing and concentration, and a magnet chamber for cell separation. Starting with fresh leukopaks, we obtained 95.9% T cell, 95.8% CD4+ T cell, and 89.6% CD8+ T cell purities following negative selection, and 92.0% CD4+ T cell and 89.5% CD8+ T cell purities following positive selection.
These approaches offer efficient and user-friendly cell isolation that allow researchers to scale up their operations, and can be easily integrated upstream of existing workflows.
Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry
Authors: Rogers Clare E , Yao Huihui , Cohen Michael , Loh Christina 
Published: January 2023

Abstract
Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) is a 30-marker panel for suspension mass cytometry. This panel provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. The 18 open mass channels in the Maxpar Direct Assay facilitate panel expansion and enable flexibility for higher multiplexity and applications. Among the potential complementary applications, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (103Rh, Cat. No. 201103) included in the Maxpar Direct Assay is in question, as cell permeabilization during ICS can potentially damage the DNA-intercalator bond. In this study, we investigated the compatibility of 103Rh with intracellular staining. We stained either human peripheral blood mononuclear cell or whole blood samples with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to the benchmark Cell-ID Cisplatin-194Pt (Cat. No. 201194). This work was designed to support use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining.
For Research Use Only. Not for use in diagnostic procedures.
Tregs: The Sertoli cell’s secret weapon in transplant survival
Authors: Hibler Taylor , Kaur Gurvinder , Dufour Jannette 
Published: January 2023

Abstract
Advanced germ cells develop after central tolerance is established and express non-self antigen but are not destroyed by the immune system. They owe their survival in part to the physical and immunological barrier created by Sertoli cells (SC). The protective nature of SC has sparked interest in the field of transplant immunology as a way to prevent transplant rejection. Indeed, SC prolong the survival of various co-transplanted tissues. However, exactly how SC achieve this protection remains largely unexplored, as does their effectiveness in female recipients. Data from transplantation studies indicates that T regulatory cells (Tregs) are critical in graft survival. This study aims to determine if SC survive at comparable rates in males and females, and if SC survival is dependent on Tregs. Therefore, we transplanted SC from B6 mice into allogeneic BALB/c male and female immunosuppressive drugs. At day 20, all SC grafts survived in male recipients and at least 67% survived in female recipients. In both males and females, Tregs were detected in high numbers in surviving grafts. To further explore their role in SC graft survival, Tregs were depleted in male graft recipients. Analysis of SC grafts at day 20 showed that 57% of SC grafts survived and saw a return of Tregs, as detected through flow cytometry. Tregs remained depleted in the rejected grafts. Together, this data indicates that Tregs must be present for SC grafts to survive and that SC themselves may induce Tregs. To conclude, SC survive allotransplantation in males and females, possibly through the activation of Tregs.
Supported by CH Foundation, NIAID
Dysregulated regulatory T cell responses target fibroblasts and lead to chronic rejection after heart transplantation
Authors: Dwyer Gaelen K , Fan Lu , Zhang Xue , Li Tengfang , Mathews Lisa , Saba Johnny , Colon Emmanuel , Calderon Michael , Helfrich Katherine , Ross Mark , Watkins Simon , Turnquist Heth Roderick 
Published: January 2023

Abstract
Suppressive and reparative regulatory T cell (Treg) functions are described. However, in transplantation (Tx), recipient immune responses to persistent MHC differences will sustain injury and impact repair. The development of immunosuppressant-resistant graft vasculopathy and fibrosis, or chronic rejection (CR) remains a leading cause of death post-heart Tx (HTx) and may reflect failed tissue repair. We tested the hypothesis that Treg repair responses become dysregulated due to sustained alloreactive immune responses. Our studies utilized IL-33 as a model injury signal, or damage-associated molecular pattern (DAMP) and B6 (H-2b) CD45.1+ mice received heterotopic HTx from MHCII-mismatched Bm12 (H2-I-Abm12) donors that were IL-33-deficient or competent. On day (d)14, scRNAseq and CITE-seq were used to compare CD45.1+ cells from HTx digests (n=4). IL-33 modulated &gt;800 genes in HTx-infiltrating Treg, including the growth factor amphiregulin (Areg; 2.25x; P=0.0241). Areg is implicated in tissue repair, yet when B6 Foxp3YFP-Cre xAregfl/fl and Foxp3YFP-Cre mice receiving Bm12 HTx were assessed at d100 (n=6–10), the deletion of Areg in Treg protected against CR. Using IncuCyte Live-Cell imaging, it was clear that Areg from Treg stimulated fibroblast proliferation and migration. Tregs stimulated with IL-2 alone or with IL-33 were assessed at d14 or d21 for CpG methylation of Treg-associated genes. These data revealed that sustained exposure to IL-33 increased methylation of Foxp3, suggesting that sustained DAMP stimulation makes Treg unstable. In total, our studies identify how a sustained and dysregulated repair response involving interactions between fibroblasts and recipient Treg contributes to the progression of HTx CR.
Supported by grants from NIH (F30AI147437, T32 CA082084) (GKD), NIH (R01HL122489, R01AR073527) (HRT)
Engineering T cells to prevent graft-versus-host disease and leukemia relapse following allogeneic stem cell transplantation
Authors: Mo Feiyan , Watanabe Norihiro , Burkhardt Phillip M , Heslop Helen E , Brenner Malcolm K , Mamonkin Maksim 
Published: January 2023

Abstract
Acute graft-versus-host disease (aGvHD) and leukemia relapse remain major causes of mortality after allogeneic hematopoietic stem cell transplantation (alloHSCT). Prophylaxis and treatment of aGvHD rely on generalized immunosuppression, increasing the risk of tumor relapse and opportunistic infections and emphasizing the need for more targeted therapies.
Alloreactive donor CD4+ T cells play a central role in aGvHD pathogenesis, we thus hypothesized that elimination of activated CD4+ T cells with engineered T cells would mitigate aGvHD while preserving protective CD8+ T cell immunity post alloHSCT. We developed an alloimmune defense receptor (ADR) targeting OX40, a surface marker predominantly upregulated on activated CD4+ T cells. OX40 ADR-expressing T cells eliminated activated CD4+ T cells during coculture but spared the majority of activated CD8+ T cells, including virus-specific T cells, and had no discernible activity against resting lymphocytes. A single infusion of ADR T cells fully protected mice from fatal xenogeneic aGvHD induced by intravenous injection of human PBMC, minimizing signs of aGvHD and maximizing survival. To enable simultaneous activity of engineered T cells against aGvHD and leukemia relapse, we further armed ADR T cells with a CD19-directed chimeric antigen receptor (CAR). In a mouse model of residual leukemia post alloHSCT, administration of T cells co-expressing OX40 ADR and CD19 CAR mediated dual protection against tumor relapse and aGvHD. These results support the feasibility of a bi-functional CAR.ADR T cell product to improve outcomes post alloHSCT and reduce transplant-related mortality.
Supported by grant from Leukemia & Lymphoma Society.
Enhancing the antitumor response of CD8+ T cells by 4-1BB (CD137) co-stimulation with distinct inactivation of the type 2 adenosine receptors.
Authors: Ahn Jihae , Chen Siqi , Fan Jie , Zhang Bin 
Published: January 2023

Abstract
Activating the immune co-stimulatory receptor 4-1BB (CD137) with agonist antibody binding and crosslinking-inducing agents that elicit 4-1BB intracellular signaling potentiates the anti-tumor responses of CD8+ T cells. However, the underlying in-depth mechanisms remain to be defined. The type 2 adenosine receptors (primarily the high affinity receptor, A2AR) predominantly expressed on CD8+ T cells inhibit T cell activation and expansion by blocking TCR signaling in a cAMP-dependent manner. Here, we show that inactivation of the low affinity receptor A2BR rather than A2AR by continuous treatment of antagonists and/or genetic deletion induces superior survival advantage of effector CD8+ T cells with agonistic 4-1BB co-stimulation especially upon chronic TCR stimulation and/or long-term antigen exposure. Mechanistically, A2BR inactivation helps sustain the increased energy and biosynthetic requirements through the accumulation of intracellular glutathione (GSH) in response to agonistic 4-1BB co-stimulation. Importantly, A2BR inactivation in combination with agonistic 4-1BB co-stimulation displays a greater ability to modulate mitochondrial fitness for anti-tumor CD8+ T cell expansion while minimize T cell exhaustion. Thus, the A2BR pathway plays an unexpected role in metabolic reprogramming of GSH upon agonistic 4-1BB co-stimulation that allows the fine-tuning of the anti-tumor responses of CD8+ T cells.
Supported by NCI CCSG P30 CA060553 R01CA258857
Spontaneous class-switched antibody responses at endometrial cancer tumor bed drives superior patients’ outcome
Authors: Mandal Gunjan , Biswas Subir , Anadon Carmen M , Yu Xiaoqing , Gatenbee Chandler D , Prabhakaran Sandhya , Payne Kyle K , Chaurio Ricardo A , Martin Alexandra , Innamarato Patrick , Moran Carlos , Powers John J , Harro Carly M , Mine Jessica A , Sprenger Kimberly B , Rigolizzo Kristen E , Wang Xuefeng , Curiel Tyler J , Rodriguez Paulo C , Anderson Alexander R A , Saglam Ozlen , Conejo-Garcia Jose R 
Published: January 2023

Abstract
The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we report that concomitant accumulation of T, B and plasma cells at tumor beds predicts better survival. However, only B cell markers predict survival specifically in high-grade endometrioid type and serous tumors. Accordingly, immune protection is associated with class-switched IgA and, to a lesser extent, IgG. Notably, expression of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA are superior predictors of outcome, and correlate with defects in methyl mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drives inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and ER stress pathways, while thwarting DNA repair mechanisms. Therefore, coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer, and therefore the potential for effective immunotherapies.
Supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687 and R01CA211913), and from Cancer Center Support Grant (CCSG) CA076292
Inhibition of DNMTs and RNA editing increases immunogenic transposable element RNA to reduce tumor burden and prolong survival in a murine ovarian cancer model
Authors: Gomez Stephanie , Cox Olivia L , Rentia Uzma , Balick Veronica , Hadley Melissa , Grundy Erin E , Kanholm Tomas , McDonald James I , Kobyra Julie , Palmer Erica , Saunthararajah Yogen , Chiappinelli Katherine B 
Published: January 2023

Abstract
Novel therapies are urgently needed for ovarian cancer (OC), the fifth deadliest cancer in women. Preclinical data shows great promise for the treatment of OC with epigenetic therapy. The inhibition of DNMT activates transcription of double-stranded (ds)RNA, including transposable elements. These dsRNAs activate sensors in the cytoplasm and trigger the induction of type I interferon signaling. Adenosine deaminase 1 (ADAR1) is induced by interferon signaling and edits mammalian dsRNA with an A-to-I nucleotide change. These edited dsRNAs cannot be sensed by dsRNA sensors, and thus ADAR1 inhibits the type I interferon response. We show that transposable elements including Alus, LINEs, and ERVs, are edited by ADAR1 in human OC cell lines after DNMTi treatment.
We hypothesized that increased ADAR1 editing reduces the DNMTi immune response and treated Adar1 knockdown cells with DNMT1 in vitro and in vivo. Combining ADAR1 knockdown with DNMTi significantly increased pro-inflammatory cytokine production compared to either perturbation alone. We treated an immunocompetent mouse model of OC with DNMTi after implanting them with Adar1 knockdown cells or control cells (ID8 Trp53−/−) and found that DNMTi treatment and Adar1 loss reduces tumor burden and extends survival in this model, and the combination of both is the most effective. Combining Adar1 loss and DNMTi transforms the immune microenvironment, including increased recruitment of natural killer cells and M1 macrophage polarization. This survival benefit is a result of type I interferon signaling and CD8+ T cells. Thus, epigenetically inducing transposable element transcription, followed by inhibition of RNA editing, is a novel therapeutic strategy to reverse immune evasion in OC.
Research was supported by the National Cancer Institute R00CA204592 and R37CA251270 (to KBC), the DOD Ovarian Cancer Research Program (W81XWH2010273 to KBC), and by the Marlene and Michael Berman Endowed Fund for Ovarian Cancer Research. JIM was supported by an NCI NRSA Institutional Research Training Program grant (T32 CA 247756). SG was supported by a NRSA Predoctoral Fellowship (NIH/NCI 1F31CA254315-01). The authors would like to acknowledge the Institute for Biomedical Sciences at the George Washington University for graduate student support and training (SG, EEG, TK, VB).
Effects of tumor-derived extracellular vesicles on T cell fate and function
Authors: Ma Feiya , Wang Yuqi , Peng Guangyong 
Published: January 2023

Abstract
Immunosuppressive microenvironments created by malignant tumors represent a major obstacle for effective anti-tumor immunity. There are multiple immunosuppressive mechanisms operative in the tumor microenvironment (TME) that interfere with T cell function. Extracellular vesicle (EV) in tumor microenvironment is an important way employed by tumor cells to control T cell fate and function. However, the mechanisms responsible for EV-mediated T cell suppression and dysfunction are still largely unknown. Here, we purified EVs from human melanoma A375 and breast cancer MCF7 cell lines and then explored EV impacts on T cell function and fate in anti-tumor immunity. We found that tumor derived EVs can suppress T cell proliferation and induce T cell senescence, which is a novel and fundamentally important phenomenon that contributes to tumor-induced immunosuppression in the TME. Mechanistically, tumor derived EVs promoted DNA damage responses and cell cycle arrest of T cell that induces T cell senescence. Blockage of DNA damage signaling in T cells via the specific inhibitor KU55933 can prevent T cell senescence and recover T cell impaired effector functions. Importantly, we further performed in vivo studies to investigate that tumor-derived EVs can induce T cell suppression and senescence, which can also be reversed by specific signaling inhibitors or/and targeting signaling molecules via the CRISPR knockdown strategy. These studies facilitate a better understanding of the novel suppressive mechanism utilized by the TME and provide insights relevant to the development of effective strategies to reverse immune suppression for enhanced anti-tumor immunity.
Supported by the grants from the Melanoma Research Alliance and National Institutes of Health.
Spatial whole transcriptome profiling of the tumor microenvironment in FFPE prostate carcinoma using the Visium platform
Authors: Reyes Daniel , Giangarra Valeria , Williams Stephen R , Turkekul Mesruh , Mielinis Paulius , Chell James , Taylor Sarah E B 
Published: January 2023

Abstract
Despite years of prostate cancer research, balancing the risks of treatment complications and tumor progression remains a challenge. The inability to dissect tumor microenvironments (TME) and immune compartments contributes to a knowledge gap. Spatially resolved molecular profiling allows understanding these complexities for potential personalized treatments.
We used 10× Genomics Visium Spatial Gene Expression for FFPE tissue for spatial whole transcriptome analysis of two prostate cancer sample TMEs. The tissues were processed over~5,000 molecularly barcoded, spatially encoded capture probes in spots for spatially resolved transcriptional readout.
We resolved whole transcriptome tumorigenic profiles in sections of normal, stage III adenocarcinoma, and stage IV acinar cell carcinoma FFPE human prostate tissues. Computational clustering identified spatial gene expression patterns that aligned with pathologist annotations. The data revealed spatial disorganization of basal and luminal cells in tumor samples. T lymphocytes were dispersed throughout the whole tissue in the adenocarcinoma, while plasma B cells were in the peritumoral region impacting patient prognosis. Computational methods inferred copy number variation, identifying aneuploidy regions and specific loci driving the genomic profile of the cancerous regions.
We demonstrated that spatial whole transcriptome analysis resolves FFPE prostate samples. Our data rapidly confirms known cell type patterns and tumor region-specific gene expression while enhancing understanding of the TME for drug target or biomarker discovery for tailored therapies and patient stratification.