bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microfluidic device platform for T cell fate analysis (TECH2P.907)
Authors: Falcon Jessica , Conway Daniel 
Published: January 2023

Abstract
Microfluidic systems have enormous potential as investigational devices in biomedical research, in particular immunology. Microfluidic devices can be fabricated with the precise regulation of any number of parameters including controlled surface chemistries, geometrical dimensions, signal input and output and timing. The adjustable nature of microfluidic devices makes them an ideal platform to recreate cellular microenvironments for studying cell-cell interactions, migration, antibody and cytokine production, and differentiation in vitro. Investigations with customized microfluidic devices have the potential for elucidating the mechanism behind T lymphocyte commitment to either the αβ or γδ lineage. Both types of T cells arise from immature CD4-CD8- precursors in the thymus but diverge during development. Recent publications indicate that γδ T cell receptor (TCR) works in concert with ligand to direct progenitors to the γδ fate. We propose to develop a microfluidic device in which immature T lymphocytes can migrate through a thymus-like microenvironment allowing for αβ or γδ lineage commitment. As of yet, the microfluidic device fabrication protocol has been developed and preliminary cell culture experiments with the commonly used murine stromal cell line OP9 have success. Such a device would allow for analysis of specific developmental points in T cell development.
Human regulatory T cell isolation in 55 minutes using EasySep™ Releasable RapidSpheres™ (TECH2P.912)
Authors: Kokaji Andy , Sun C. , MacDonald G. , Clarke Samuel , Fairhurst Maureen , Eaves Allen , Thomas Terry 
Published: January 2023

Abstract
The preferred marker for identifying regulatory T cells (Tregs) is FOXP3 but its intracellular localization currently precludes it from being used for isolating viable cells. Human FOXP3+ Tregs are characterized as CD4+ T cells expressing high levels of CD25 and low levels of CD127. We have developed a novel immunomagnetic approach for the isolation of magnetic-particle-free human Tregs from PBMCs in as little as 55 minutes. This offers a vast improvement in speed over other immunomagnetic Treg isolation approaches, which typically require more than 2 hours. The standard approach for isolating Tregs combines CD4+ T cell negative enrichment followed by CD25 positive selection. By contrast, our novel approach is to first perform the CD25 positive selection step with EasySep™ Releasable RapidSpheres™, a new type of magnetic particle that can be rapidly released from the isolated cells with a no-incubation, no-wash protocol performed at room temperature. Subsequent depletion of CD127high and non-CD4+ T cells is achieved by negative enrichment with EasySep™ Dextran RapidSpheres™. Starting with 5x107 PBMCs, a purity of 87.6% ± 3.1% and recovery of 2.7x105 ± 8.2x104 Tregs (CD4+CD127lowCD25+FOXP3+) can be achieved (n=6). This is the first EasySep™ kit to incorporate the EasySep™ Releasable RapidSpheres™ and it exemplifies the potential of this technology for use in rapidly isolating magnetic particle-free cell populations possessing complex cell surface phenotypes.
Host-influenza proteomics leads to metabolic drug repurposing that reduces viral burden while significantly improving survival after lethal infection (TECH2P.917)
Authors: Smallwood Heather , Morfouace Marie , Duan Susu , Wang Ruoning , Zink Erika , Shulkin Barry , Green Douglas , Pasa-Tolic Ljiljana , Thomas Paul 
Published: January 2023

Abstract
A retrospective survey of pediatric patients infected with respiratory pathogens identified increases in glucose metabolism in the lungs of several individuals. After viral infection the host cell mounts an energetic and resource intensive antiviral response, while the virus exploits the host to generate progeny. Using absolute quantification with Host-Influenza Proteomics (HIP) we sought to characterize changes in host cell homeostasis that contribute to these competing processes. Like in the patients, we found gross metabolic changes occurring after influenza infection in primary human respiratory cells as well as dendritic cells. This new metabolic phenotype is distinct from immune activation and involves increases in glycolysis and glutaminolysis, decreased fatty acid β-oxidation concomitant to non-canonical TCA activity. Based on these findings we developed a metabolic drug repurposing screen in human bronchial epithelial cells and high-throughput viral tittering system. We screened over 160 metabolic substrates and small molecules to identify responsive prophylactic and therapeutic pathways (e.g. lipid and glucose metabolism respectively). One compound known to alter glucose sensitivity, NVP-BEZ235, reduced viral titers and significantly increased survival in vivo after lethal influenza challenge. Human PET scans followed by HIP analysis elucidated a unique metabolic signature in influenza-infection exposing targets for therapeutic intervention.
iRock 2.0 and the iR-Processor: an automated immune repertoire library preparation platform for NGS (TECH2P.919)
Authors: Byrne-Steele Miranda , Pan Wenjing , Brown Brittany , Alleyne Jessica , Caudle David , Yang Qunying , Wang Chunlin , Han Jian 
Published: January 2023

Abstract
Immune repertoire amplification of T and B cell receptors coupled to Next Generation Sequencing (NGS) provides detailed sequence-level insight into the immune system. Previously, we described the automation of a patented, nested multiplex PCR method (arm-PCR) to amplify an immune repertoire, inclusively and semi-quantitatively, on the iR-Processor, an instrument developed by iCubate, Inc. The iR-Processor is an open development platform for diagnostic panels consisting of a closed-cassette and the iR-Processor. The cassette is pre-loaded with the reagents necessary for library preparation, and the iR-Processor instructs the cassette to perform the necessary liquid handling steps and PCR for library generation. Template RNA is added to the cassette with the RT enzyme. iRock 2.0 represents an improved method in which all of the first round cDNA is rescued prior to library generation with a pair of universal primers. Once PCR is complete, residual primer-dimers are removed by magnetic selection of the primary product in the cassette. The amplified library is eluted from the magnetic beads by the user and is directly ready for sequencing using the Illumina MiSeq. The sequencing results from repeat amplification experiments of the TCRβ chain from the same individual and between individuals are compared. The results demonstrate high repeatability among shared CDR3 sequences and an improvement in the percentage of shared CDR3 discovery upon repetition.
Deep profiling of single T cell receptor repertoire and phenotype with targeted RNA-seq (TECH2P.927)
Authors: Ji Xuhuai , Lyu Shu-Chen , Spindler Matthew , Bacchetta Rosa , Goncharov Igor , Han Arnold , Glanville Jacob , Wang Weiqi , Roncarolo Maria , Meyer Everett , Nadeau Kari , Maecker Holden 
Published: January 2023

Abstract
Analysis of TCR repertoires is useful for monitoring T cell responses. Integrating TCR sequencing with expression of targeted genes at the single cell level allows comprehensive analysis of T cell function and specificity. RT-PCR reactions of single T cells were performed using 76 primers from VJC regions and 136 primers for targeted T cell genes. A series of nested PCR reactions incorporated with barcodes and adapters for pooled amplicons sequencing. A bioinformatics pipeline was used data analysis. 2880 single T cells were collected and tested. TCR sequences were obtained in up to 90% of the wells. Based on the CDR3 region, multiple dominant TCR clones were identified. 52 of 68 target genes were validated with sorted target-specific T cells. FOXP3, IL10, PRF1, IL13, and RUNX3 were highly expressed in memory activated cells. The specific CD8+ T cells had high frequency of CCR9, LAG3, CD8, CD62L, and IFNG expression. TGFB, TNFA, IL12, FOXP3, PD1, TBET, CTLA4, MKI67, RUNX1, and GATA3 were major biomarkers in the specific CD4 cells. The gene expression patterns were often associated with TCR repertoire CDR3 variations and/or the TCRβ and TCRα chain usage although the same TCRα/β sequences may have different target genes expressed. We expanded a method enabling sequencing of TCR repertoire and multiple functional genes in single, sorted, T cells through targeted RNA-seq technology. This approach could reveal important insights into T cell functions and clonal development.
High-throughput pairing of T cell receptor alpha and beta sequences (TECH2P.930)
Authors: Sherwood Anna , Howie Bryan , Berkebile Ashley , Berka Jan , Emerson Ryan , Williamson David , McCormick Michael , Kirsch Ilan , Vignali Marissa , Rieder Mark , Carlson Christopher , Robins Harlan 
Published: January 2023

Abstract
Conventional high-throughput immunosequencing methods profile TCR alpha (TRA) and/or TCR beta (TRB) repertoires, but do not enable pairing of the cognate subunits comprising functional TCRs. We developed a method called pairSEQ that can pair hundreds of thousands of TRA and TRB sequences in a single experiment. Our approach works by allocating random samples of T cells to a micro-titer plate, sequencing amplified cDNA from TRA and TRB genes, and DNA bar-coding the wells. Most TCR sequences uniquely identify clonal cell populations, so pairs can be assigned based on TRA and TRB sequences that occupy the same wells. To validate our method we identified cognate pairs from a sample of cells mixed from two people. The cross-sample pairs directly measure false pairing rates, which closely matched predictions from our statistical model. To show our method can be targeted to any clonal frequency range of interest, we designed a 3-plate experiment to capture pairs with repertoire frequencies from 0.5% to less than 1 in 10 million. These experiments yielded over 300,000 confident TCR pairs with one 96-well plate alone providing over 200,000 pairs. This technology is not restricted by material; in a single experiment we used this method to identify 6,349 TCR pairs from tumor infiltrating lymphocytes in 9 tumor samples. Our TCR pairing method is highly scalable, and should be broadly applicable to all somatically rearranged immune receptor loci, including immunoglobulins.
Rapid and cost effective immunoassay development and fit-for-purpose validation of a biomarker assay using Spatial Proximity Analyte Reagent Capture Luminescence (SPARCL) (TECH2P.760)
Authors: Cameron Mark 
Published: January 2023

Abstract
A novel detection technology was used for rapid immunoassay development and validation. SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) is a proximity dependent and homogeneous chemiluminescent detection method. In a SPARCL assay, a chemiluminescent substrate (acridan) is brought into the proximity of an oxidative enzyme (horseradish peroxidase) through the specific antigen/antibody interaction. A flash of light is generated and is proportional to the amount of IL-8 bound. Assay development was completed in less than 3 days. The validated assay features a wide dynamic range (2 - 4,000 pg/mL), low MRD, short assay run time (60 min.), and sensitivity (2 pg/mL). Quality control samples (QC’s) run in plasma resulted in statistics that show the assay is precise and accurate, with a total error of all QC levels of under 12%. Matrix spiked with IL-8 diluted in a linear manor. The SPARCL technology enables rapid assay development, delivers an assay with desirable performance characteristics and allows for considerable savings in labor, disposables and capital equipment compared to popular platforms. Existing software and luminometers may be used for a SPARCL data acquisition. As a result, there may not be a need to validate new software or plate readers already running in existing Laboratory Information Management Systems or in a regulated environment. The SPARCL assay is microtiter plate-based and is suitable for use with robotics and in HTS applications.
PrimeFlow™ RNA assay as a method to elucidate the effect of protein transport inhibitors on cytokine gene expression in whole blood (TECH3P.941)
Authors: Frank Richard , Scott Bradley , Lai Chunfai , Sias Leslie , Funatake Castle 
Published: January 2023

Abstract
Intracellular antibody staining followed by flow cytometric analysis is commonly used to study cytokine expression in leukocytes. Detection of cytokines typically requires that cells be treated with protein transport inhibitors such as Brefeldin A and Monensin in order to accumulate sufficient levels of intracellular protein. However, it is not known whether the use of these inhibitors affects gene expression. PrimeFlow™ RNA is a new flow cytometric assay that allows the simultaneous interrogation of RNA and protein levels within single cells. We have used this assay to investigate the effect of protein transport inhibitors on the expression of human IL-2, TNF alpha and IFN gamma during the course of a 5 hour stimulation of whole blood with PMA and ionomycin. This represents first time that the effect of protein transport inhibitors on gene expression in whole blood has been examined in single cells.
Identification of predictive protein biomarkers for treatment efficacy and clinical relapses of multiple sclerosis (THER7P.950)
Authors: Raphael Itay , Forsthuber Thomas 
Published: January 2023

Abstract
Despite extensive research, MS remains a disease that lacks a definitive prognostic test to predict imminent disease relapses. Thus, patients may undergo years of unnecessary treatments. Additionally, current treatments for MS can produce dramatically different outcomes in different individuals and therefore there is a critical need to develop biomarkers for treatment efficacy and resistance. We have recently developed a novel quantitative proteomics method to measure changes in proteome expression over the course of experimental autoimmune encephalomyelitis (EAE). Our statistical analyses indicate a strong correlation to EAE severity and/or clinical-phase. Interestingly, we revealed characteristic CNS-specific protein expression waves prior to the onset of clinical symptoms. We are currently testing whether these protein expression waves allow us to predict the onset of clinical episodes and forecast the severity of the disease to guide treatment strategies. Additionally, we have identified changes in the CNS proteome that can be measured in serum during EAE that correlate with the therapeutic efficacy of glucocorticoid treatment. Our studies will provide proof-of-principle for developing homologous human biomarkers that may be useful to predict disease onset and treatment efficacy. Finally, the detected changes in the CNS proteome may provide insights into key mechanisms that contribute to the disease pathology and may be useful to develop new therapeutic targets for MS.
Understanding the opposing roles of the adaptive immune system in stroke (THER7P.955)
Authors: Ortega Sterling , kong xiangmei , Meeuwissen Anouk , Noorbhai Ibrahim , Anderson Ashley , Chen Ding , Monson Nancy , Stowe Ann 
Published: January 2023

Abstract
Stroke is caused by a loss of blood flow to the brain, which results in permanent neurological injury. Murine models of ischemic stroke implicate the immune system as both contributor and suppressor of neuro-inflammation following brain ischemia. Using a 60-min transient middle cerebral artery occlusion model and a recall response assay, we have detected autoreactive immune responses to myelin, neuronal and glial peptides, suggesting a possible immune involvement in further brain inflammation. We previously found that repetitive hypoxic preconditioning (RHP) prior to stroke endogenously decreases infarct volumes and creates a unique, anti-inflammatory B-cell phenotype that may contribute to RHP-mediated reversal of neuropathology. Now, we show that B(RHP)cell adoptive transfer significantly reduces infarct volumes and CNS-antigen autoreactivity in CD4+ T-cells compared to PBS- or control B-cell treatments. B(RHP)cells exhibited increased production of IL10, a neuroprotective cytokine, and IFN-γ, a cytokine that potentiates the conversion of effector to regulatory T-cells that suppress neuropathological immune cells. Co-culture of B(RHP)cells with T-cells significantly converted naïve CD4 T-cells to Foxp3+CD25+ regulatory CD4 T-cells. Based on these observations, we hypothesize that RHP induces a novel subset of neuroprotective B-cells as an important regulatory arm of the adaptive immune system that can be potentially harnessed for immunotherapeutic strategies.
Cbl-b-deficient mice demonstrate alterations in T cell trafficking but retain sensitivity to the Multiple Sclerosis therapeutic agent FTY720 (THER7P.957)
Authors: Fujiwara Mai , Anstadt Emily , Khanna Kamal , Clark Robert 
Published: January 2023

Abstract
The variable response to treatment options in multiple sclerosis (MS) suggests the need for genetic-based personalized therapeutic approaches. CBLB gene polymorphisms have been identified in MS, and altered T cell function and aberrant responses to interferon-β in MS patients with a CBLB mutation have been recently reported. Cbl-b is an E3 ubiquitin ligase that regulates T cell activation and Cbl-b-deficient (Cbl-b-/-) mice show many T cell abnormalities also described in MS patients. This suggests that Cbl-b-/- mice may provide a novel approach for analyzing treatment options in patients with MS-associated CBLB mutations. We now report that Cbl-b-/- CD4+ T cells show significant trafficking-related abnormalities: decreased lymph node accumulation after adoptive transfer into RAG-1-/- mice; increased sphingosine-1-phosphate receptor 1 (S1P1) expression; and decreased CD69 expression compared to wild-type cells. These data imply that CBLB mutations may compromise the therapeutic efficacy of FTY720, an MS-approved drug that modulates S1P1 expression. Despite altered Cbl-b-/- T cell trafficking, we observed robust inhibition of EAE by FTY720 in Cbl-b-/- mice. These results not only document a novel role for Cbl-b in T cell trafficking but also indicate that FTY270 may be effective in EAE/MS through mechanisms other than T cell lymph node trapping. Moreover, our findings suggest that FTY720 treatment is still an appropriate choice in patients with MS-associated CBLB mutations.
Impaired expansion of CD11b+Gr-1+ cells from MyD88-deficient donor bone marrow cells aggravates graft-versus-host disease (TRAN2P.966)
Authors: Lee Young-Kwan , Ju Ji Min , Shin Dong-Mi , Min Chang-Ki , Choi Eun Young 
Published: January 2023

Abstract
Myeloid differentiation 88 (MyD88), an adaptor molecule in TLR-associated signaling, has recently been known to play a role in expansion/activation of CD11b+Gr-1+ myeloid-derived suppressor cells. To understand role of MyD88 expressed by donor-originating myeloid cells in GVHD development, we investigated dynamics of T cell-depleted bone marrow (TCD-BM) cells and gene-expressions of CD11b+Gr-1+ cells originated from Myd88-/- donor mice after transplantation into allogeneic BALB.B hosts with WT T cells. The allogeneic recipients of Myd88-/- TCD-BM and WT T cells showed aggravated GVHD, with significant loss of CD11b+Gr-1+ population, compared with BALB.B recipients of WT TCD-BM and T cells. Impaired expansion of Myd88-/- TCD-BM cells in BALB.B GVHD hosts was confirmed via in vivo tracking analysis. However, their expansion was intact in syngeneic C57BL/6 recipients, indicating that impaired CD11b+Gr-1+ cell expansion from Myd88-/- TCD-BM cells was specific to GVHD circumstance. In transcriptome analysis, CD11b+Gr-1+ cells of MyD88-/- TCD-BM origin exhibited higher expression of genes related to apoptosis and antigen-presentation than did those of WT TCD-BM origin. These results suggest that impaired expansion of the CD11b+Gr-1+ cells from Myd88-/- TCD-BM cells in allogeneic hosts is related to enhanced apoptosis in combination with enhanced differentiation into DC-like cells in the presence of activated allo-reactive T cells, and provide insight into the control of GVHD.
CCR5+ regulatory T cells induced by metastatic breast carcinomas migrate toward a gradient of CCL8 and accumulate in metastatic lung tissue (TUM9P.1016)
Authors: Halvorsen Elizabeth , Hamilton Melisa , LePard Nancy , Bosiljcic Momir , Young Ada , Wadsworth Brennan , Bennewith Kevin 
Published: January 2023

Abstract
Under homeostatic conditions, regulatory T cells (Tregs) mediate peripheral tolerance and prevent autoimmunity. Despite their crucial role in the regulation of immunity toward innocuous antigens, Tregs may also contribute to the growth and metastasis of breast cancer by suppressing anti-tumor immune responses. We are interested in developing therapies to inhibit intratumoral Treg infiltration to decrease the growth of primary and metastatic tumors. Using mammary carcinomas syngenic to BALB/c mice, we identified elevated levels of CCR5+ Tregs in the primary tumour and metastatic lungs relative to naive control tissues. Interestingly, we observed that C-C chemokine receptor type 5 (CCR5) is highly and selectively expressed by Tregs in metastatic lungs relative to other immune cell populations. CCR5+ Tregs largely co-express CTLA-4 and CD103, indicating a suppressive phenotype. The production of CCL8, an endogenous ligand of CCR5, was increased in the tumour and lungs, and CCL8-mediated migration of Tregs ex vivo was inhibited by the CCR5 antagonist Maraviroc. These data suggest that targeting CCR5 or CCL8 may be a viable therapeutic strategy to inhibit Treg accumulation during tumour progression to decrease metastatic tumour growth. Currently, there is a paucity of research involving the role of CCR5+ Tregs in tumour progression. We anticipate that this work will advance the generation of targeted, immune-based therapeutics for the treatment of metastatic breast cancer.
Development of vaccination approaches that reverse leukemia-induced tolerance and reduce leukemic burden (TUM9P.1020)
Authors: Manlove Luke , Vrieze Katherine , Manlove Kezia , Pauken Kristen , Schenkel Jason , Williams Richard , Jenkins Marc , Farrar Michael 
Published: January 2023

Abstract
Current therapies induce only transient responses in patients with BCR-ABL+ B Acute Lymphoblastic Leukemia. Thus augmenting immune responses to the BCR-ABL fusion peptide could be therapeutically beneficial. The fusion between BCR and ABL encodes a neo-antigen (called BAp) that stimulates an immune response, but this response does not eliminate leukemia. To characterize the immune response to BAp we examined BCR-ABL+ leukemic cells in mice. Principal Component Analysis demonstrated that a CD4+ T cell response driven by high CD40 and low PDL1 on leukemic cells inversely correlated with leukemic burden. Using BAp:I-Ab-specific tetramers we tracked BCR-ABL-specific CD4+ T cells in response to leukemia. Naive mice had a small population of BAp:I-Ab-specific T cells. Transfer of live leukemia resulted in modest expansion (55-fold) of BAp:I-Ab-specific T cells, which were largely diverted into FOXP3+ Tregs. Depletion of Tregs led to increased expansion of BAp:I-Ab specific T cells and protected mice from leukemia. Likewise, acute viral vaccination in the presence of BAp (but not infection with Listeria monocytogenes expressing BAp) induced expansion of Ly6C+ BAp:I-Ab-specific T cells, prevented Treg induction, and significantly reduced leukemic burden. Thus, BCR-ABL-specific T cells exist in non-leukemic mice but are tolerized in the presence of leukemia; this tolerance can be broken by Treg ablation or specific vaccination approaches, leading to protection against leukemia.