bluuebunny/crossref_metadata_2025_split huggingface dataset:
SimpleStep ELISA™: Innovative immunoassays in less than 90 minutes (TECH2P.918)
Authors: Campbell Adam , Constable John 
Published: January 2023

Abstract
Here we introduce the novel SimpleStep ELISA™ platform which has improved features over the traditional sandwich assay. The SimpleStep ELISA format is a novel, streamlined protocol that can be completed in less than 90 minutes, requires no additional training and uses standard laboratory equipment. In one step, the complete sandwich complex forms in solution and is anchored to the plate through an immunoaffinity tag. Only a single incubation and wash step is required, compared to multiple incubation and wash steps with standard ELISAs, that can take up to five hours. Fewer steps mean less time is needed and there are fewer opportunities for errors with no loss on sensitivity or increased background. While simple in concept, ELISA performance can be affected by many factors, including the sample type, as many antibody pairs and immunoassays that work well on purified proteins fail to perform acceptably when a complex biological sample, such as plasma or serum, is used. The foundation of SimpleStep ELISA performance is an extensive screening program to identify antibody pairs that perform well in a range of biological samples, and the inclusion of specially-designed buffers to ensure accuracy and minimize the effect of non-specific binding, for example by human serum antibodies reactive against mouse antibodies. Supporting data for each SimpleStep ELISA includes sensitivity, range, precision, linearity, recovery and cross-reactivity.
Quantification of 500 human protein biomarkers using antibody arrays and its applications in immunology research (TECH2P.921)
Authors: Huang Ruo-Pan , Mao Yingqing , Huang Ruochun , Yen Haw-Han , Sun Yanni 
Published: January 2023

Abstract
Proteomic approach continues to be the trend for biomarker discovery through profiling as many proteins as possible in a biological sample. Though LC-MS technology can accurately detect medium-to-high abundance proteins (&gt; ng/ml), many cellular modulators such as cytokines are present at pg/ml level. Immunoassays continue to be the most utilized tools for biomarker discovery and validation due to their convenience, sensitivity and specificity. Here we validated the sandwich detection of 500 most analyzed human protein biomarkers with a pair of antigen-specific antibodies. Subsequently, capture antibodies were fixed on glass slides with non-contact arrayer, and multiplex ELISA detection of these protein biomarkers was achieved through planar antibody array technology with laser fluorescence detection. To overcome the potential cross reactivity among different reagents, multiple non-overlapping arrays were developed based upon their cellular abundance, functionality, and cross reactivity. Most of the markers can achieve pg/ml detection - similar to single ELISA detection but with much wider dynamic range. These arrays were further validated with normal human sera, where over two thirds of these proteins are within the detection range. Separate array panels were developed for those proteins with high abundance in sera, where high sample dilutions are needed. In summary, our system provides a powerful, efficient, and low-cost tool for immunology research and biomarker discovery.
Rapid multiparametric analysis of activation and cytotoxicity in lymphocyte subpopulations (TECH2P.928)
Authors: Gillis Katherine , Clor Julie , Tyagarajan Kamala 
Published: January 2023

Abstract
Multiplexing cell health parameters with phenotyping markers provides greater understanding of time and sequence for activation and cellular stress pathways in immune cells. Sample heterogeneity often makes this type of assessment complex to perform and evaluate. The development of the easyCyte™ 12 instruments, which combines 3 lasers and 12 parameters, has greatly facilitated 6-9 color multiparametric analysis of immune cell populations using small amounts of sample without compensation. In this study, we evaluated the impact of activation agents and cytotoxic treatment on PBMCs using a multiparametric assay which identifies lymphocyte subsets in combination with cell health or activation parameters. Our studies demonstrate that overnight treatment with anisomycin resulted in more sensitive impacts on mitochondrial depolarization on all lymphocyte subsets with lower apoptotic and cell death effects being observed. Overnight treatment with celastrol demonstrated almost parallel changes in mitochondrial potential and annexin V based apoptosis effects in lymphocyte subpopulations. The methodology was also utilized to study dose and time course treatments of activation, cellular stress, and cell death caused by PHA or PMA/ionomycin with PBMCs. Which allowed for dissection of sequence of changes in multiple subpopulations. Such studies can greatly enhance our understanding on response of immune cell subpopulations to compounds that cause activation or cytotoxicity.
Study human antibody repertoire in malaria using ultra accurate immune repertoire sequencing (TECH2P.931)
Authors: Jiang Ning , Wu Di , Qu Mingjuan , Wendel Ben , He Chenfeng 
Published: January 2023

Abstract
Recently developed Immune repertoire sequencing (IR-seq) has become a useful tool to quantify the composition of the various antigen receptor repertoires, such as antibody and T cell receptor. However, early versions of IR-seq suffer significantly from high amplification bias and high sequencing errors. In this study, we developed an improved IR-seq method that clusters antibody sequences within each molecular identifier group to further separate different Ig transcripts that have been tagged with the same molecular identifier. We first validated this method by using naïve B cells sorted to different amounts. Analysis showed that our methods has significantly improved the sequence accuracy and diversity coverage. In addition, it enables us to examine small number of cells, such as memory and plasmablasts. We used this method to study antibody repertoire in children who experienced malaria in Africa. Epidemiology studies showed that children under 5 are most vulnerable to malaria with more than 460,000 cases of malaria caused death in children younger than 5 in Africa in 2012. However, little is known on how immunity toward malaria is established. We sequenced children from as young as 3 month to 10 year old and observed many traits in antibody diversity and mutations that correlate with age and confer protection, demonstrating the power of systems immunology approach to study infection.
Enhancements in mass cytometry for multiplexing samples and increasing cell detection rate (TECH3P.938)
Authors: Bandura Dmitry , Ornatsky Olga , Majonis Daniel , Wei Yuhong , Baranov Vladimir , Tanner Scott 
Published: January 2023

Abstract
Mass Cytometry uniquely enables high dimensional single cell proteomic analysis for systems-level discovery and comprehensive functional profiling applications. At the core of the technology are the ICP ion source and a fast elemental analyzer designed for metal-conjugated affinity reagents. We show how recent advancements in reagents, sample introduction system and the ion source have simplified data acquisition and improved data quality with the mass cytometry platform. Up to 20 samples from an experiment are barcoded with palladium-based cell tagging reagents, combined into one tube, and stained and run as one sample. This multiplexing improves data quality by eliminating sample-to-sample staining and running variation, increases experimental throughput by reducing the number of tubes that need to be processed and run, and reduces reagent consumption. Since each barcode consists of a unique set of 3 Pd isotopes, the de-barcoding algorithm also filters out most doublets (with more than 3 isotopes). The multiplexed sample is run on the CyTOF using the novel large volume sample introduction system. We will discuss results of modifications to the sample introduction system and the inductively coupled plasma ion source, which enable analysis of large samples at high cell detection rate. Examples of barcoded PBMCs from multiple donors, stained with high dimensional antibody panels that combine deep phenotyping with multiple cytokine or signaling targets will be demonstrated.
Residual EAE severity following B cell depletion is due to treatment-resistant plasma cells (THER7P.953)
Authors: Monson Nancy , Chen Ding , Blazek Monica , Ireland Sara , Ortega Sterling , kong xiangmei , Stowe Ann , Wang Yue , Herbst Ronald 
Published: January 2023

Abstract
Plasma cells and the autoreactive antibodies they produce are suspected to contribute to the pathogenesis of Multiple Sclerosis, but recent attempts to target these components of humoral immunity have failed. Recently we reported that MEDI551, an anti-CD19 antibody, disrupts development of EAE, the mouse model of MS. Leukocyte infiltration into the spinal cord is significantly reduced as well as short- and long-lived autoreactive CD138+ plasma cells in the spleen and bone marrow. In addition, potentially protective CD1dhiCD5+ regulatory B cells show resistance to depletion, and myelin-specific FoxP3+ regulatory T cells are expanded. Thus, MEDI551 may offer a compelling alternative that reduces pathogenic adaptive immune responses while preserving regulatory mechanisms. The sparing of long-lived plasma cells by anti-CD20 therapies and targeting of this population by MEDI551 prompted us to compare the efficacy of anti-CD19 Ab and anti-CD20 Ab in our EAE model. Our data demonstrated that anti-CD19 depletion is more effective than anti-CD20 depletion to ameliorate EAE. In fact, the frequency of residual antibody-producing cells correlated with residual disease severity in both the anti-CD19 and anti-CD20 treatment groups. No other correlations with residual EAE severity were found. We conclude that CD19+ plasma cells remaining after B cell depletion therapy contribute to residual EAE severity by producing autoreactive antibodies and pro-inflammatory factors.
Identifying biomarkers for monitoring disease progression in experimental autoimmune encephalomyelitis (THER7P.942)
Authors: Ji Niannian , Chase Carol , Saenz-Trevino Erica , Raphael Itay , Forsthuber Thomas 
Published: January 2023

Abstract
Multiple sclerosis (MS) is the most common demyelinating neuroinflammatory disorder which afflicts over 400,000 Americans. Currently, determining clinical or sub-clinical disease progression in MS patients and development of novel treatments has been hampered by the lack of specific and sensitive laboratory tests. Recently, our lab has developed a technique called M2 proteomics, which is a rapid quantitative approach for identifying putative protein biomarkers and therapeutic targets of experimental autoimmune encephalomyelitis (EAE), a commonly used animal model for MS. Notably, we identified several putative-biomarkers which correlate with different stages of monophasic EAE. The objective of this study is to identify protein biomarkers of disease progression in a progressive EAE model using non-obese diabetic (NOD) mice. We hypothesize that during progressive EAE key central nervous system (CNS) disease-specific proteins will be released into blood and changes of these proteins can be used to determine disease progression. Using the progressive NOD EAE model, we determined the expression of putative CNS-specific protein biomarkers by immune assay in brain homogenate and serum longitudinally over the course of disease. We identified several CNS-specific potential biomarkers in serum that correlated with the progression of disease. The results of this study could help development of biomarkers for disease progression and testing of novel treatments for progressive MS.
Infusion of SMCC-conjugated MOG-coupled spleen cells effectively prevents and reverses experimental autoimmune encephalomyelitis: induction of antigen-specific Foxp3+ regulatory CD4+T cells and suppression of antigen-specific Th17 cells (THER7P.949)
Authors: Xia Changqing , Zhang Langfang , Guo Yixian 
Published: January 2023

Abstract
Experimental autoimmune encephalitis (EAE) is an induced autoimmune disorder in rodent animals with the development of autoimmune T cells attacking axonal myelin protein causing demyelination. This model has been widely used to develop preventive or therapeutic approaches for human multiple sclerosis (MS). In current study, we report that infusion of SMCC-conjugated MOG35-55 coupled autologous spleen cells (either live, or ultraviolet B-irradiated apoptotic) significantly prevents and reverses EAE in mice. Further studies show that the protected animals resist EAE re-induction by MOG35-55 antigen challenge. Moreover, adoptive transfer of CD4+ T cells from the protected mice to naïve syngeneic mice renders the recipients resistant to EAE induction, suggesting that MOG35-55-specific regulatory CD4+ T cells may be induced. Unexpectedly, CD4+T cell proliferation is similar upon ex vivo stimulation by MOG35-55 amongst all groups. However, further analysis of those proliferating CD4+ T cells shows remarkable difference in Foxp3+ cells (70% in MOG-SP groups versus 10-25% in control groups, p&lt;0.05), and in IL-17+ cells (2-3% in MOG-SP groups versus 6-9% in control groups,p&lt;0.05). These findings indicate that MOG35-55 coupled spleen cell treatment induces MOG35-55 specific regulatory T cells, and suppresses EAE pathogenic Th17 cells. Our study provides a novel approach for antigen-based immunotherapy for EAE, which has a great potential for developing immunotherapies for MS .
Computational prediction and functional identification of HLA-A2-restricted T lymphocyte epitopes derived from swine leukocyte antigen (TRAN2P.962)
Authors: Lee Chong-Kil , Park Chan-su , Im Sun-A , Park Young-Jun , Lee Jae Hee , Kim Kyungjae 
Published: January 2023

Abstract
Xenotransplantation using porcine organs is a potential solution to the problem of the shortage of allografts. However, immunological rejection of xenografts remains as a major hurdle to success. Little information is available regarding the precise swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules. The SLA-derived peptides that bind to HLA-A*0201, a representative of the A2 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and the stabilities of complexes formed between the peptides and HLA-A*0201 were compared using MHC stabilization assays. The cytotoxic T lymphocyte (CTL)-inducing activity of the selected peptides was examined in HLA-A*0201-transgenic mice. Among 15 candidate peptides synthesized, two peptides, peptide-35 (YLGPDGLLL) and peptide-43 (TLICHVDSI), were selected to have high affinity and stability with HLA-A*0201. Examination of the CTL-inducing activity of the two peptides in HLA-A*0201-transgenic mice showed that immunization with peptide-35, but not peptide-43, elicited potent CD8-specific-CTL responses. The Peptide-35 is present in non-polymorphic a2 domains of 34 SLA-1 alleles, 18 SLA-2 alleles, and 1 SLA-3 allele. These results show that Peptide-35 is a HLA-A2-restricted immunogenic peptide derived from SLA.
PD-1 and IL-10: partners in crime against anti-tumor immunity in ovarian cancer. (TUM9P.1011)
Authors: Lamichhane Purushottam , Karyampudi Lavakumar , Shreeder Barath , Knutson Keith 
Published: January 2023

Abstract
PD-1: PD-L1 is a major inhibitory axis that acts to suppress tumor-rejecting effector responses. In addition to its expression and inhibitory functions in T and B cells; PD-1 has also been shown to be expressed on and thereby impact the functions of cells of innate arm such as dendritic cells (DCs) primarily at the tumor site but not in the periphery. We show here that one of the culprits in the tumor microenvironment responsible for enhancing PD-1 expression on the tumor-associated DCs is the cytokine IL-10; which is expressed in abundance in many malignancies including ovarian cancer. Based on inhibition and knockdown studies, we show that IL-10 mediated PD-1 expression depends on STAT3 activation. Using murine model of ovarian carcinoma (ID8), we show that tumor-associated DCs express not only high levels of PD-1 on the surface but also have increased expression of STAT-3 and p-STAT3 compared to splenic DCs. Blockade of PD-1 on DCs led to increased production of IL-10 which correlated with a further increase in PD-1 expression. In in vivo studies, blockade of PD-1 and IL-10 neutralization as combination therapy augmented the anti-tumor response in ID8 ovarian tumor bearing mice; leading to a decrease in tumor mass and a significant increase in survival. These results show that IL-10 and PD-1 pathway intersect in DCs in theTME hence making them attractive targets for therapy of ovarian cancer.
Regulatory rebound in Interleukin-12-treated tumors is driven by uncommitted peripheral Treg (TUM9P.1012)
Authors: Li Qingsheng , Virtuoso Lauren , Egilmez Nejat 
Published: January 2023

Abstract
Interleukin-12 (IL-12) promotes a rapid reversal of immune suppression in the tumor microenvironment. However the adjuvant activity of IL-12 is short-lived due to T-regulatory cell (Treg) re-infiltration. Quantitative analysis of Treg kinetics in IL-12-treated tumors and tumor-draining lymph nodes (TDLN) revealed a transient loss followed by a rapid 4-fold expansion of tumor Treg between days 3 and 10. Subset-specific analysis demonstrated that the post-treatment rebound was driven by the CD4+ CD25+ Foxp3+ Neuropilin-1(Nrp-1)low peripheral Treg (pTreg) resulting in a 5-fold increase in the pTreg to CD4+ CD25+ Foxp3+ Nrp-1high thymic Treg (tTreg) ratio by day 10. The expanding pTreg displayed hypermethylation of the CpG islands in Treg-specific demethylated region (TSDR), CTLA-4 exon 2 and GITR exon 5; were phenotypically unstable; and exhibited diminished suppressive function consistent with an uncommitted in vitro-induced Treg (iTreg)-like phenotype. In vitro culture of post-therapy Treg populations under TH1-promoting conditions resulted in the production of IFNg by pTreg but not by tTreg, confirming their transitional state. Blockade of selected molecular mechanisms that are known to promote Treg expansion identified Indoleamine 2,3 dioxygenase (IDO)-positive Dendritic cells as the primary mediator of post-IL-12 pTreg expansion. Clinical implications of these findings are discussed.
The role of host B7-H1 in the recurrence of B16 melanoma. (TUM9P.1013)
Authors: Goding Stephen , Wilson Kyle , Rosinsky Carolyn , Antony Paul 
Published: January 2023

Abstract
Our B16 melanoma mouse model, in which ~50% of mice receiving adoptive cellular therapy with cytolytic CD4+ T cells recognizing the tumor/self antigen tyrosinase protein 1 (TRP-1), exhibits tumor relapse following initial tumor regression. CD4+ T cells from tumor-bearing TRP-1 antigen knockout (tyrp1BwRAG-/-) transgenic mice, or tumor sensitized (t.s.) T cells, expressed low levels of the exhaustion marker PD-1 and were 15-25% Foxp3+. Due to this presensitization, B16 tumors recurred faster and more predictably in RAG-/- mice with t.s. CD4+ T cells than naïve TRP-1-specific CD4+ T cells. However, in RAG-/- mice lacking B7-H1 (i.e. PD-L1, the ligand for PD-1), relapses occurred more rarely and developed at slower rates following adoptive transfer of t.s. CD4+ T cells. When CD4+ T cells from mice bearing relapsing tumors, expressing high levels of PD-1 and Foxp3+, were transferred into either RAG-/- or B7-H1-/-RAG-/- mice, the treatment effect was completely abrogated. We previously reported that primary tumors required B7-H1 blockade or Treg depletion to be treated, and relapsing tumors required both treatments. Here we show that Treg cell depletion in B7-H1-/-RAG-/- mice partially restored treatment with fully exhausted CD4+ T cells and relapses took longer to occur. These data indicate that although multiple mechanisms are involved in tumor recurrence (Treg cells and PD-1), the expression of B7-H1 in the host rather than the tumor plays a major role in B16 melanoma relapse.
Cancer mediates effector T cell dysfunction by targeting microRNAs and EZH2 via glycolysis restriction (TUM9P.1007)
Authors: Zhao Ende , Maj Tomasz , Kryczek Ilona , Wei Shuang , Crespo Joel , Wan Shanshan , Vatan Linda , Szeliga Wojciech , Chinnaiyan Arul , Welling Theodore , Marquez Victor , Wang Guobin , Zou Weiping 
Published: January 2023

Abstract
Aerobic glycolysis regulates T cell function. However, if and how primary cancer alters effector T cell glycolytic metabolism and affects tumor immunity remains a question in cancer patients. Here we report that ovarian cancer imposes glucose restriction on effector T cells, dampens their function via maintaining high expression of microRNA101 and microRNA26a, which subsequently constrains EZH2 expression. EZH2 activates the Notch pathway by suppressing Notch repressors via H3K27me3, and consequently stimulates T cell polyfunctional cytokine expression and promotes their survival via Bcl-2 signaling. Moreover, EZH2-/- T cells elicit poor anti-tumor immunity and EZH2+CD8+ T cells are associated with improved long-term cancer patient survival. Together, our data unveil a novel metabolic target and mechanism of cancer immune evasion.
Organ specificity of cancer metastasis depends on the adaptive immune surveillance and the neuronal chemokine fractalkine (TUM10P.1030)
Authors: Nwajei Felix , Beceren-Braun Figen , Gabrusiewicz Konrad , Shanmugasundaram Meenakshi , Zal Anna , Wu Wuenye , Heimberger Amy 
Published: January 2023

Abstract
The phenomenon of organ specificity in cancer metastasis has been traditionally interpreted in terms of the “seed and soil” hypothesis. However, the role of the adaptive immune system in this phenomenon is largely unknown and controversial. We found that MCA-fibrosarcoma cancer cells formed lethal tumors in the lungs, but not in the brain thereby representing a model of organ-specificity of cancer metastasis. Using this model, we assessed the role of the adaptive immune system. In immune competent multi-color fluorescent reporter mice, longitudinal intravital imaging via cranial windows revealed initial engraftment and growth of MCA cancer cells that was followed by tumor regression in concordance with T cell infiltration. However, MCA cancer cells formed lethal tumors in the brains of Rag1-KO mice indicating a key role for the adaptive immune system in the organ specificity of MCA cancer cell metastasis. In contrast, T cells were recruited to pulmonary MCA lesions but were ineffective in tumor rejection in that organ. Interestingly, T cell recruitment to MCA micrometastases in the brain was dramatically impeded in mice lacking the receptor for the neuronal chemokine fractalkine, and MCA tumors progressed in the brains of those mice. Our results reveal a role for brain-specific adaptive immunity to cancer metastasis and implicate fractalkine in regulating this process thereby broadening the “seed and soil” concept.