bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel computational analytics of clinical flow cytometry data identifies difficult-to-resolve leukemia cells for precision diagnosis
Authors: Qian Yu , Nguyen Quang Vinh , Putzel Preston , Bevins Nicholas , Bui Jack D , Wang Huan-You , Smyth Padhraic , Scheuermann Richard H 
Published: January 2023

Abstract

Motivation
Diagnosis of leukemia relies on accurate identification of leukemic cell populations. Flow cytometry (FCM) is a primary diagnostic assay routinely used in clinical practice. The assay workflows consist of multiple manual gating steps performed by technicians, followed by interpretation by hematopathologists. Challenges to this process include technical variability in manual gating, difficulty in identification of the atypical leukemic cells, and the growing number of antigens used for diagnosis.

Methods
Instead of conducting ad hoc analysis of individual samples, our proposed computational approach leverages preexisting clinical FCM data to improve robustness of the computational identification of leukemic cells. Instead of separating cell population identification and sample classification into two steps, our machine learning classification method optimizes them simultaneously, producing gating locations that are recognizable to hematopathologists.

Results
Our study consists of 10-color FCM data from blood or bone marrow samples of 129 random subjects for chronic lymphocytic leukemia (CLL) diagnosis. Our initial automated gating analysis rendered an accuracy of 89% matched the diagnosis of the hematopathologist. In the remaining cases with discrepant results, the misclassified CLL cells had atypical molecular phenotypes, making them difficult to identify. In our improved pipeline, we demonstrate that these atypical types of CLL cells can be clearly captured using a non-linear embedding dimensionality reduction step.

Conclusion
The results demonstrate the power of a novel computational analysis pipeline for improving the identification of aberrant leukemia cells for precision diagnosis.
Evaluation of Peripheral Blood Leukocyte Subsets in Systemic Lupus Erythematosus (SLE) Enabled by a Microfluidic Cell Separator
Authors: Olferiev Mikhail , Greenman Dina , Kulnarni Nitin , Kou Peng Meng , Smith Kyle C , Crow Mary K 
Published: January 2023

Abstract
Although granulocyte signatures have been associated with SLE severity, their role in SLE has been poorly understood due to the suboptimal methods for granulocyte recovery from peripheral blood. An automated microfluidic technology (Sorterra™ prototype) was used to isolate all leukocytes with minimal platelet (PLT) or red blood cell (RBC) contamination to assess the cell subsets.
Leukocytes were isolated from peripheral blood collected from healthy donors (HD) and SLE patients (3 female, 3 male in each group) using Sorterra, RBC lysis, or Ficoll®. Viability and recovery of each leukocyte subset was evaluated by cell counter and flow cytometry.
Sorterra recovered 92±4% of leukocytes from HD and SLE samples with negligible RBC/PLT/microparticle (MP) contamination, whereas Ficoll isolated 35±15% of leukocytes, losing nearly all the granulocytes and retaining a substantial level of RBC/PLT/MP. With RBC lysis, up to 65% of neutrophils and 41% of eosinophils were lost in the process. Sorterra recovered more monocytes (~80%) than Ficoll (p&lt;0.05). The recovery of lymphocytes by Sorterra (~90%) was not different from Ficoll or RBC lysis. The recovery of T and B cell subsets, NK, and NKT cells, was also not different among the isolation methods. The viability of Sorterra isolated leukocytes was significantly higher than the Ficoll isolated cells.
The Sorterra prototype was superior to RBC lysis and Ficoll in recovering total leukocytes with high viability and ease of use and provided a more consistent leukocyte isolate from HD and SLE patients than other methods. The ability to recover granulocytes in their native state is unmatched by existing technologies and enables future studies to better elucidate the role of granulocytes in SLE development.
DNA-scaffolded biomaterials enable modular and tunable presentation of proteins to control immune cell therapies
Authors: Huang Xiao , Williams Jasper Z. , Chang Ryan , Li Zhongbo , Gai Eric , Patterson David M. , Wei Yu , Lim Wendell A. , Desai Tejal 
Published: January 2023

Abstract
Advanced biomaterials can be used to spatially and temporally control immune cell activities to improve the efficacy and safety of cell therapies. Precise immune cell modulation demands multi-modal display of functional proteins on biomaterials with a high level of control. Here, we developed biodegradable immune cell engaging particles (ICEp) with synthetic short DNA as scaffolds for efficient and tunable protein loading. We demonstrate the precise ratiometric control of anti-CD3 and anti-CD28 antibodies on this biodegradable platform can impact the quality of ex vivo expanded human primary T cells. Furthermore, the high-density presentation of antigens, difficult to achieve by other chemistries, is necessary for controlling the activity of engineered T cells in vivo. Antigen-presenting ICEp injected intratumorally can provide a local priming signal for systemically administered AND-gate chimeric antigen receptor (CAR) T cells that recognize dual antigens: the first antigen on ICEp activates CAR expression to target the second antigen on tumor cells. This leads to local tumor clearance while sparing uninjected tumors that model potentially cross-reactive healthy tissues. This modularly functionalized biomaterial thus provides a flexible platform to achieve sophisticated control of cell-based immunotherapies.
Individualized expansion of T cells from CLL patients for immunotherapy
Authors: Yuan Dennis Jinglun , Fernandes Stacey M , Brown Jennifer R , Kam Lance C 
Published: January 2023

Abstract
T cell therapy has shown significant promise in treating blood cancers, yet high proportions of T cells from patients are either unresponsive to stimulation methods or show wide variability in regards to which activation conditions induce optimal expansion. In this work, we explore how expansion of T cells from CLL patients using scaffolds with tunable physical parameters (rigidity, fiber dimensions, pore size) is correlated to patient biomarker data, as well as exhaustion and functional profiles of patient T cells, to gain insight on personalizing T cell production for immunotherapy.
Polymer fiber scaffolds are fabricated via electrospinning with PDMS/PCL polymer blend and coated with activating antibodies to CD3 and CD28 to stimulate T cells isolated from CLL patients with pre-annotated mutation burdens and disease progression. Exhaustion surface markers, memory/effector phenotyping, and cytokine secretion are measured via immunostaining before and after expansion.
Preliminary data has shown that decreasing fiber diameters (from 6 to 1 μm) enhanced T cell expansion and increased the ratio of effector memory to central memory phenotypes. We observe that higher CLL Rai stage leads to decrease in proliferative potential. Interestingly, cells from each CLL patient in our initial study showed different patterns of expansion for the same set of scaffold compositions. Machine learning methods can identify clinical markers that predict exhaustion and functional profiles from starting cell populations as well as expansion with different biomaterial parameters. Collectively, insight into predicting how patient T cells can be robustly expanded in a personalized approach is powerful for translating T cell therapies to more patients.
One step ultra-high-throughput sequencing of the paired antibody VH:VL and TCRβ:α repertoires using cell lysate resistant xenopolymerase in emulsion
Authors: Tanno Hidetaka , McDaniel Jonathan , Stevens Christopher , Voss William , Li Jie , Durrett Russell , Lee Jiwon , Gollihar Jimmy , Tanno Yuri , Delidakis George , Pothukuchy Arti , Ellefson Jared , Goronzy Jörg , Maynard Jennifer , Ellington Andrew , Ippolito Gregory , Georgiou George 
Published: January 2023

Abstract
Antibodies and T cell receptors (TCRs) have critical roles in adaptive immunity and thus the determination of their sequences is essential for understanding immune responses in infections or vaccinations, for the discovery of therapeutic antibodies, and for the engineered T cell therapies. Antibodies are composed of variable heavy (VH) and light (VL) chains, and TCRs are composed of variable TCRα/γ and TCRβ/δ chains. Since those chains are encoded by two different transcripts, the determination of functional sequences requires single-cell sequencing. Emulsification of single-cells followed by barcoding of the transcripts or fusion of VH:VL or TCRβ:α transcripts using overlap extension RT-PCR allows high-throughput single-cell sequencing; however, because cell lysate in emulsion inhibits the RT-PCR reaction, these methods require complex microfluidics devices to bypass the inhibition which most of biomedical laboratories cannot access. We discovered recently engineered xenopolymerase, RTX, has an exceptional cell lysate resistance that can overcome this limitation. By employing this unique feature of RTX, we have developed a one-step emulsion-based ultra-high-throughput sequencing method for paired antibody VH:VL and TCRβ:α repertoires not requiring microfluidics or any other specialized equipment. Using this methodology, we obtained high yields (5,000 to &gt;20,000/sample) of paired VH:VL or TCRβ:α clonotypes at low cost. As a demonstration, we applied the methodology to the peripheral blood plasmablasts and T follicular helper (TFH) cells from a seasonal influenza vaccine recipient and discovered high-affinity influenza-specific antibodies and TCRβ:α.
StraightFrom®MicroBeads: Fast T cell isolation for CAR T cell manufacturing without density gradient centrifugation
Authors: Lennartz Simon , Lock Dominik , Kolbe Carolin , Winkels Gregor , Assenmacher Mario , Kaiser Andrew 
Published: January 2023

Abstract
In recent years, CAR T cell mediated immunotherapy has shown clinical efficacy in the fight against cancer. Increased research efforts to address several challenges associated with CAR T cell therapy for solid tumor treatment for instance, such as poor efficacy and unwanted side effects, are required to progress current approaches. Fast, efficient and flexible CAR T workflows can offer the opportunity to progress such research.
At Miltenyi Biotec we have established an optimized CAR T cell research workflow, combining cell isolation, culture and analysis tailored for CAR T cell generation that can be transferred into clinical setting.
Using our StraightFrom® MicroBeads, the fundamental step of isolating high quality T cells as starting material to generate functional CAR T cells, can be performed directly from whole blood, buffy coat and blood products without density gradient centrifugation. Thereby, experimental procedure is accelerated and allows for automation on platforms such as the MultiMACS™ Cell24 Separator Plus and the MultiMACS™ X, whilst maintaining the quality, functionality and downstream compatibility of the isolated T cells, resulting in an ideal starting population for the generation of fully functional CAR T cells.
In conclusion, our innovative tools can reduce experimental time and increase the reproducibility and quality of data obtained in your CAR T cell research efforts in immuno-oncology and immunotherapy.
Multiomic characterization of T-cell populations at the single-cell level utilizing sensitive dextramers and BD® AbSeq on the BD Rhapsody™ Single-Cell Analysis system
Authors: Sakofsky Cynthia , Jacobsen Kivin , Lopez-Salmeron3 Vadir , Nakamoto Margaret , Brix Liselotte , Mortimer Stefanie 
Published: January 2023

Abstract
Adoptively transferred antigen-specific T cells have shown great efficacy in treatment of some virus-associated diseases and malignancies. A major driver of the development of adoptive T-cell therapy has been our ability to successfully characterize the functional status and antigen specificity of T cells. However, this has been limited by inefficient detection of antigen-specific T cells possibly due to their low frequency and low binding affinities to known MHC-peptide complexes. Here, we aim to combine two powerful technologies, advanced dCODE™ Dextramer® from Immudex and single-cell multiomics analysis using the BD Rhapsody™ Single-Cell Analysis system, to detect and characterize disease-specific CD8+ T cells within thousands of PBMCs. Currently, we are able to identify over 350 mRNAs alongside a panel of over 20 BD® AbSeq cell surface protein markers which can be associated with T cell activation states. These data can be used to define T-cell phenotypes alongside antigen specificity of enriched CD8+ dextramer+ cells from a PBMC population. This study outlines our ability for high-resolution T-cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity.
Evaluation of oligonucleotide conjugated antibodies as reporter molecules in single-cell assays
Authors: Takahashi Chikara , Au-Yeung Amelia , Gutierrez Johnny , Toghi-Eshghi Shadi , Mathews Rod , O’Gorman William 
Published: January 2023

Abstract
Multiplexing in conventional flow and mass cytometric analysis has been constrained by both the number of available reporter molecules as well as signal spillover between these molecules. Monoclonal antibodies(mAbs) conjugated to oligonucleotides have been used in the design of ultrasensitive immunoassays but the multiplexing potential of these reagents have only recently been realized. The Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) method is established and uses oligo-conjugated mAbs and single-cell RNA sequencing technology to simultaneously enable the quantitation of both mRNA and protein molecules. Using the BD Rhapsody™ platform and BD® AbSeq, we examined the expression of 399 genes and 40 proteins in human PBMCs. The ability of single cell RNA-seq (scRNA-seq) to differentiate major and minor subsets of immune cells was evaluated. As in conventional cytometric analysis, immune cell populations could be identified based on the expression of canonical cell surface proteins and we compared the frequency of immune cell populations identified via scRNA-seq to CyTOF technology. Additionally, genes expected to be up- or down-regulated between established major populations such as T- and B-cells were observed thereby encouraging further analysis of gene expression in minor sub-populations. Overall, immune cell frequencies were found to be highly concordant between technologies. Single-cell “multi-omics” in which mRNA, proteins, and immune cell clonality are simultaneously measured on single cells thus stands poised to innovate drug development by providing unprecedented depth in the analysis of cells expressing therapeutic targets and driving disease pathogenesis.
Functional anomaly of CD8 T cells at earlier differentiation stages during HIV infection as established by analysis of synaptic interface and degranulation pattern
Authors: Anikeeva Nadia , Steblyanko Maria , Kuri-Cervantes Leticia , Buggert Marcus , Betts Michael R , Sykulev Yuri 
Published: January 2023

Abstract
It is thought that HIV infection could lead to a global defect in CD8+ T cell function. However, the effect of the infection on CD8+ T cells at various stages of differentiation has not been well studied. To evaluate the functional activity of CD8+ T cells from HIV infected people at various differentiation stages, we assessed their ability to form synaptic interfaces using planar lipid bilayers containing anti-CD3 antibody and soluble ICAM-1 ligand. We analyzed the dynamics of the T cell/bilayer interface formation, size of adhesion area, and kinetics of T-cell degranulation, parameters that are linked to the efficiency of CD8+ T cell functional activity. Unexpectedly, we observed that purified naïve CD8+ T cells (TN) and transitional memory (TTM) from HIV-infected individuals developed mature synapses whereas the same subsets from HIV uninfected individuals did not. While the kinetics of degranulation of naïve T cells was slower, both TN and TTM cells reached a similar extent of degranulation. The fraction and kinetics of CD27-CD45RO+ T cell degranulation were similar to that observed for TTM cells. Treatment with ART led to increase in the fraction of degranulating CD27-CD45RO+ T cells. As opposed to memory cells from healthy donors, a sizable fraction of CD27-CD45RO+ T cells from infected individuals develop lamellopodia and a larger adhesion area after synapse formation. These data suggest that less differentiated CD8+ T cells from HIV-infected people revealed functional anomaly that is likely mediated by aberrant functions of integrins and cytoskeleton causing changes in T-cell plasticity as a result of continuous inflammation during chronic HIV infection.
Development of two novel microglia-specific monoclonal antibodies to discriminate microglia from other CNS-resident macrophages by flow cytometry
Authors: Zahner Sonja Petra , Wilkinson Nicole , Nguyen Chau , Ellsworth Jessica Lynn , Stepniak Dariusz 
Published: January 2023

Abstract
Although microglia are the predominant resident macrophages of the central nervous system (CNS), other macrophages can be found in the CNS. As both populations express CD11b, microglia are generally distinguished from peripheral macrophages by lower expression of CD45. This can introduce bias in the analysis of flow cytometric data and make cell sorting challenging. Other microglia associated markers such as Aif1/Iba1 are less cell specific and therefore of limited use to dissect microglia from other brain residing macrophages. Recent single cell transcriptome studies have identified more specific signature genes of microglia, including surface molecule Tmem119 and transcription factor Sall1.
To allow robust discrimination between microglia and other brain resident/infiltrating macrophages based on the expression of a single marker by flow cytometry, we have developed monoclonal rat antibodies against mouse Tmem119 and Sall1, respectively. Both clones, V3RT1GOsz (Tmem119) and NRNSTNX (Sall1), were validated by flow cytometric analysis of fresh mouse brain. Here we show that both antibodies can be used to distinguish microglia from other macrophages by flow cytometry without reliance on CD11b or CD45 expression. The availability of these specific antibodies against microglia signature molecules will contribute significantly to the ability to isolate mouse microglia and their advanced characterization.
Immuno-phenotyping of tumor-specific CD8 T cells using high-dimensional mass cytometry
Authors: Abel Brian , Fehlings Michael , Nardin Alessandra , Newell Evan , Yadav Mahesh 
Published: January 2023

Abstract
There is strong evidence that tumor-specific T cells play a central role in tumor rejection. immunoSCAPE leverages the high-dimensional immune profiling capabilities of CyTOF for the identification of antigen-specific T cells to support the development of immunotherapy strategies. By applying CyTOF in conjunction with combinatorial peptide-MHC tetramer staining and high-performance dimensional analysis tools, we broadly and sensitively map T cell reactivity against MHC-class I epitopes while concurrently performing in-depth characterization of these rare antigen-specific T cells.
Here, we have applied our unique capabilities to detect and characterize circulating neoantigen-specific T cells in lung carcinoma patient undergoing atezolizumab treatment. Out of 782 neoantigen candidates, T cell reactivity against 13 unique neoantigens was detected within patients who responded to atezolizumab treatment, while T cells within non-responder patients recognized 7 neoantigens. Additionally, neoantigen-specific CD8 T cells from treatment responders exhibit a differentiated effector phenotype similar to CMV or EBV-specific T cells, whereas neoantigen-specific CD8 T cells from non-responders display a memory-like phenotype, suggesting that the properties of tumor-reactive T cells may be associated with clinical response to anti-PD-L1 treatment.
By providing insight into the nature and function of tumor-specific T cells, immunoSCAPE’s unique high-dimensional immune profiling platform is a valuable tool to guide the development of novel immunotherapy strategies through assessment of drug biological activity, definition of mechanism of action, and identification of biomarkers of patient response.
Evaluation of regulatory immune cells that favor engraftment following haploidentical peripheral blood stem cell transplantation for sickle cell disease
Authors: Bhat Deepali K , Gangaplara Arunakumar , Saxena Ankit , Pfeiffer Ruth , McCoy Philip , Fitzhugh Courtney D 
Published: January 2023

Abstract
Haploidentical hematopoietic stem cell transplantation is one of the most widely available curative options for patients with sickle cell disease (SCD). Our previous non-myeloablative haploidentical peripheral blood stem cell transplantation (PBSCT) trial showed improved engraftment with post-transplant cyclophosphamide, but with a high incidence of allograft rejection. Here, our aim was to evaluate mechanisms of engraftment in SCD patients who underwent PBSCT. We analyzed and compared the following cells (Treg, Tr1, Th1, Th17, NK, Breg, pmnMDSCs, mMDSCs, eMDSCs, mDCs, and pDCs) in peripheral blood samples obtained from 20 transplanted patients at baseline (BSL), post-transplant day (PT-D) 30, 60, 100, and 180 using flow cytometry. Because we previously showed that 20% donor myeloid chimerism (DMC) is sufficient to reverse SCD, we classified patients at each time point as engrafted (DMC ≥20%) and rejected (DMC&lt; 20%). Early MDSC frequencies were significantly elevated in engrafted patients (PT-D30). Patients were also evaluated based on engrafted/rejected at the end of study and mMDSC and Th17 cell frequencies were significantly elevated at PT-D30 and PT-D60 respectively in engrafted patients. All other cell populations were not statistically different. Tregs showed an elevated trend in engrafted patients and exhibited direct proportionality in patients with varying DMC levels. Our results reveal that eMDSC and mMDSC frequencies were elevated in engrafted patients and decreasing Tregs were associated with decreasing DMC. Taken together, eMDSCs, mMDSCs and Tregs may have a synergistic association in favoring the development of a tolerogenic immune milieu that promotes stable mixed chimerism after PBSCT in SCD patients.
Dimethyl Fumarate ameliorates cGVHD development by reducing Tfh and Breg differentiation
Authors: Gong Huanle , Ma Shoubao , Yang Bingyu , Wu Depei 
Published: January 2023

Abstract
Our previous study demonstrated that Dimethyl Fumarate (DMF), an activator of Nrf2, could significantly prevent acute graft-versus-host disease (aGVHD) development by inhibiting Treg differentiation after allogeneic hematopoietic stem cell transplantation. However, whether the DMF regulated anti-oxidant response also plays an important role in the setting of chronic GVHD remains unknown.
In this study, we firstly immunized B6 mice with NP-OVA in CFA and found that DMF remarkably inhibited Tfh differentiation. Next, we established SLE-cGVHD (DBA2 to B6D2F1) model to assess the role of DMF in cGVHD development. DMF treatment significantly reduced proteinuria and dsDNA production, leading to a markedly decreased cGVHD clinical score. Moreover, DMF treatment significantly enhanced the percentage of IL-10 positive Breg cells, but not Tregs in cGVHD.
Taken together, we found that DMF treatment significantly inhibited cGVHD by reducing Tfh and Breg cells. Further studies are needed to investigate the detailed mechanisms of DMF in the regulation of Tfh and Breg differentiation.