bluuebunny/crossref_metadata_2025_split huggingface dataset:
MojoSort: a versatile nanoparticle for magnetic isolation of cells with high purity, yield, and preserved functionality (TECH2P.926)
Authors: Zhang Hong , Tam Miguel , Gohel Dhanesh , Zhao Xinfang , Koury Jeffery , Wiethe Carsten , Yang Xifeng , Ransom John , Monell Craig , Jiang Nan , Lay Gene 
Published: January 2023

Abstract
The isolation of specific cells from a complex population is a technical challenge that researchers face very often. Several strategies have emerged over the years, including gradient centrifugation, Fluorescent Activated Cell Sorting and Magnetic Activated Cell Sorting. Magnetic labeling is the most common approach, as it constitutes a fast, reliable, and convenient method to obtain populations with high purity and yield, and the cells are suitable for downstream applications. Here we present a new type of magnetic nanoparticles that may be used with current separation methods. To illustrate the ease and utility of the system, while maintaining functionality of the isolated cells, bone marrow cells were isolated from C57BL/6 mice using magnetic particles, and characterized by flow cytometry. The cells were then differentiated towards a macrophage-like phenotype in vitro. After culture, the cells were stimulated with LPS and CpG and the activated phenotype was compared to non-stimulated cells. Surface markers analyzed include CD80, CD86 and MHC II. The supernatant was collected and screened for the presence of cytokines, such as IFN-b, GM-CSF, IL-1b, IL-12, among others. The isolated cells show a differential activation phenotype, as well as a defined cytokine profile, in accordance with the stimulation used when compared to non-stimulated cells. Thus, we validated the high quality performance of BioLegend’s new low-cost magnetic cell separation system.
A new protein crosslinking method for labeling and modifying antibodies (TECH2P.758)
Authors: Liao Jinfang , Diwu Jack , Zhao Qin , Guo Haitao , Han Xing 
Published: January 2023

Abstract
The modification and labeling of a protein with a tag macromolecule is an important task in biological research. There are a few methods available for crosslinking two macromolecules. The direct crosslinking of two macromolecules suffer extremely low yield and difficult purification. A common method is to use a small crosslinker (such as SMCC) in numerous bioconjugations, but SMCC-modified protein is extremely unstable and often self-reactive during storage since proteins often contain both amine and thiol groups that cause significant amount of homo-crosslinking. We have recently developed an effective crosslinking method to selectively link two macromolecules. The new crosslinking method uses two unique crosslinkers that readily react upon mixing. Specifically, a pair of crosslinkers is used, including Buccutite FOL and Buccutite MTA. A protein (e.g., antibody) is first labeled with Buccutite FOL, and the tag macromolecule is labeled with Buccutite MTA. The two resulted Buccutite-modified conjugates can be readily purified by a desalting column or dialysis. Buccutite FOL-labeled protein and Buccutite MTA-tag macromolecule are mixed under extremely mild neutral conditions to give the desired protein-tag macromolecule conjugate. The Buccutite bioconjugation system is run at neutral pH, and completed within 1 hour. The conjugation can be run at low concentration, thus eliminating the pretreatment step for some diluted proteins. No catalyst is required.
Development of detection tools for health monitoring in new world and old world camelid (TECH2P.762)
Authors: Holzloehner Pamela , Schliebs Erik , Maier Natalia , Naumann Friederike , Füner Jonas , Micheel Burkhard , Hanack Katja 
Published: January 2023

Abstract
Keeping and breeding of new and old world camelids in Europe is a very complex process influenced by altered climate conditions in comparison to the natural habitat in desert ecosystems. Especially the veterinary care for these animals is lacking specific detection systems for general health monitoring, diagnosis of certain diseases or vaccination. In order to build up efficient tools for detecting important parameters in this field highly affine monoclonal antibodies were developed reacting against camelid immunoglobulins. The humoral immune response of New and Old World Camelids is characterized by generating conventional as well as heavy chain only antibodies which is a unique feature in mammals. To investigate the immunological status of Camelids after vaccination murine monoclonal antibodies were produced by hybridoma technology, characterized and used for assay development. We were able to generate monoclonal mouse antibodies that bind (I) to all subtypes, (II) only the conventional subtype IgG1 and (III) all heavy chain subtypes. All generated monoclonal antibodies showed no cross reactivity with immunoglobulins from other species. Specific monoclonal antibodies were used for assay development to monitor vaccination success against e.g. Coronavirus, Rotavirus and Clostridium. Further the detection systems were extended for disease control as e.g. erysipelas. With these tools it is now possible to develop efficient diagnostic systems for health monitoring in Camelids.
Performance qualification of a 13-color intracellular cytokine staining assay for the assessment of T cell responses in human PBMCs (TECH3P.932)
Authors: Graves Andrew , Henson Dawn , Padilla Marcelino , Schaeffer Jacqueline , Hokey David 
Published: January 2023

Abstract
One challenge to tuberculosis vaccine research is the lack of a correlate of immunity, which researchers are attempting to address through use of more high-throughput, high-data assays for assessing trial specimens. Multiparameter intracellular cytokine staining (ICS) assays have proven to be one of the most cost-effective tools available. Previously, we reported the development of a 13-color ICS panel for use in assessing T cell responses from human PBMCs. In an effort to better understand the metrics of the assay performance, a qualification study under GCLP conditions was executed. The parameters assessed in this performance qualification included specificity, sensitivity, selectivity, linearity, and intermediate precision. The lower limits of quantification for cytokine detection were shown to be under 0.03%. Using Fisher’s exact test to determine positivity, the false positive and false negative rates were shown to be 0% in this performance qualification. Furthermore, assessment of intermediate precision suggested that the CD3, CD4, CD8, CCR7, and CD45RO markers each showed a percent coefficient of variation (%CV) of 25% or under, while intracellular and functional markers were generally under a 30% CV as measured via responses to Staphylococcal enterotoxin B. The results of this performance qualification study illustrate the capabilities of this panel for assessing clinical trial responses and suggest it is a reliable assay for the assessment of T cell responses.
Incorporation of BD HorizonTM BV421 and BD HorizonTM BV480 immunofluorescence reagents improves immunofluorescence microscopy (TECH3P.933)
Authors: Rohrer Jurg , Wright Adam , Martin Jody 
Published: January 2023

Abstract
New fluorophores offered by BD Biosciences, BD HorizonTM BV421 and BD HorizonTM BV480, have unique spectra, are very bright, photostable, and work well in immunofluorescence microscopy. With narrow excitation and emission spectra these 2 fluorophores are compatible with most fluorescent protein models (eGFP, YFP, RFP, and tdTomato). Additionally, they are compatible with the standard green, yellow, red, and far-red immunofluorescence imaging reagents, (Alexa Fluor® 488, Alexa Fluor® 568, and Alexa Fluor® 647). These new fluorophores enable robust 5-color imaging. The workflow is easy and the dyes are functional (with simple optimization) on most fluorescence microscopes. No spectral unmixing is required for analysis. Because BV421 and BV480 are brighter than standard imaging fluorophores, there is the potential to lower the limit of detection with improved resolution. Side by side experiments with standard fluorophores on an epifluorescence microscope demonstrate improved resolution, via increased signal to noise, using both BV421 and BV480. Brighter fluorophores improve ease of use during microscope focusing and data acquisition by decreasing exposure time. In addition to enhanced sensitivity, these fluorophores enable easy and routine 5 color staining. Examples from human and mouse model systems using directly conjugated primary antibodies, show how these dyes enable simple multi-parameter imaging thereby permitting correlation of more parameters per sample
Tolerogenic synthetic nanoparticles for the prevention of anti-drug antibodies against biologic therapies (TECH3P.936)
Authors: Ferrari Joseph , LaMothe Robert , Kolte Pallavi , Griset Aaron , O'Neil Conlin , Browning Erica , Fu Fen-ni , Altreuter David , Johnston Lloyd , Farokhzad Omid , Langer Robert , von Andrian Ulrich , Maldonado Roberto , Kishimoto Takashi 
Published: January 2023

Abstract
The development of anti-drug antibodies (ADAs) is a common cause for treatment failure and adverse events, such as hypersensitivity reactions, associated with biologic therapies. Therefore, prevention of ADAs in an antigen-specific manner would be highly desirable in order to improve the safety and efficacy of marketed products. Here we describe the use of SVP: polymeric, synthetic, biodegradable nanoparticles carrying a tolerogenic immunomodulator, rapamycin, to induce durable, antigen-specific immune tolerance. In mice, intravenous or subcutaneous co-injections of SVP with free antigen results in robust CD4+ T cell and B cell tolerance (i.e. the inhibition of their activation over multiple challenges), an increase in regulatory cells and the inhibition of antigen-specific hypersensitivity reactions. Only encapsulated rapamycin, not the free form, could induce immunological tolerance to a variety of antigens. Co-injections of SVP and antigen in both rats and cynomologous monkeys also results in B cell tolerance. In mice that spontaneously develop rheumatoid arthritis (RA), we show that treatment with SVP and adalimumab prevents the formation of anti-adalimumab ADAs therefore normalizing adalimumab pharmacokinetics and improving the clinical and histological manifestations of RA. SVP therapy represents a novel antigen-specific approach for the prevention of ADAs against biologic therapies, as well as the treatment of allergies and autoimmune diseases.
MHC-mismatched mixed chimerism augments thymic regulatory T cell production and prevents relapse of EAE (THER7P.960)
Authors: Wu Li-Min , Li Nainong , Zhang Mingfeng , Xue Shengli , Riggs Arthur , Zeng Defu 
Published: January 2023

Abstract
Multiple sclerosis (MS) is an autoimmune inflammatory disease in central nervous system with demyelination, axon damage, and paralysis. There is no yet curative therapy. Induction of MHC-mismatched mixed chimerism is proposed to be a potential cure for autoimmune diseases, but this has not yet been tested in animal models of MS, experimental autoimmune encephalomyelitis (EAE). Here, we report that MHC-mismatched mixed chimerism with C57BL/6 (H-2b) donor in SJL/J (H-2s) EAE recipients eliminates symptoms and prevents relapse. This cure is demonstrated by not only disappearance of clinical signs but also reversal of autoimmunity and elimination of infiltration of T, B and macrophage cells in the spinal cord, as judged by HE, immunofluorescent staining and flow cytometry analysis. We also see regeneration of myelin as judged by fast-blue staining and prevention of axon damage as judged by electronic microcopy. The reversal of autoimmunity is associated with a marked reduction of autoreactivity of CD4+ T cells and significant increase of Foxp3+ regulatory T percentage among host-type CD4+ T cells in the spleen and lymph nodes. We have also found a marked increase of Foxp3+ regulatory T percentage among host-type CD4+ and CD4+CD8+ thymocytes in the thymus. Thymectomy leads to loss of prevention of EAE relapse by the mixed chimerism. These results indicate that induction of MHC-mismatched mixed chimerism can augment thymic production of Foxp3+ regulatory T cells and cure EAE.
Targeting epidermal fatty acid binding protein for treatment of experimental autoimmune encephalomyelitis (THER7P.947)
Authors: Rao Enyu , Singh Puja , Li Yan , Zhang Yuwen , Chi Young-In , Suttles Jill , Li Bing 
Published: January 2023

Abstract
Multiple sclerosis (MS) is an autoimmune disease in which dysregulated immune cells attack myelin in the central nervous system (CNS), leading to irreversible neuronal degeneration. Our previous studies have demonstrated that epidermal fatty acid binding protein (E-FABP) widely expressed in immune cells, in particular in dendritic cells (DCs) and T lymphocytes, fuels their overactive immune responses in the mouse model of experimental autoimmune encephalomyelitis (EAE). In the present study, we conducted an intensive computational docking analysis to identify novel E-FABP inhibitors for regulation of immune cell functions and for treatment of EAE. We demonstrate that compound [2-(4-acetylphenoxy)-9,10-dimethoxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one; designated as EI-03] binded lipid binding pocket of E-FABP and enhanced the expression of peroxisome proliferator-activating receptor (PPAR) γ. Further in vitro experiments showed that EI-03 regulated DC functions by inhibition of TNFα production while promoting IL-10 secretion. Moreover, EI-03 treatment counterregulated T cell balance by decreasing effector T cell differentiation (e.g. Th17, Th1) but increasing regulatory T cell development. Most importantly, mice treated with this newly identified compound exhibited reduced clinical symptoms of EAE in different mouse models. Taken together, we have identified a new compound which displays a potential therapeutic benefit for treatment of MS by targeting E-FABP.
Targeting the anti-tumor function of B cells in non-small cell lung cancer patient tumors (TUM9P.1008)
Authors: Bruno Tullia , Moore Brandon , Munson Daniel , Kern Jeffrey , Slansky Jill 
Published: January 2023

Abstract
Understanding the function of tumor infiltrating lymphocytes (TILs) in NSCLC patient tumors will contribute to the development of rationally designed treatments and an increase in the current 18% survival rate. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer. We used un-manipulated, primary human B cells from fresh tumor and tumor adjacent lung tissue to determine the phenotype and function of TIL-Bs in NSCLC patients. The total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. In analyzing the markers of activation and exhaustion, we observed a spectrum of activation of TIL-Bs. Further, we observed that TIL-Bs present autologous tumor antigens to CD4 TILs in a subset of NSCLC patients. These data suggest that some patients have functional TIL-Bs and some patients have nonfunctional TIL-Bs; TIL-Bs that could not present tumor-specific antigens had higher expression of B cell exhaustion markers. Lastly, we are combining blockade of the immune inhibitory PD-1:PD-L1 pathway in our antigen presentation assays because PD-1 expression is increased on CD4 TILs in our NSCLC patient tumors. Results from this study will advance the understanding of the anti-tumor function of TIL-Bs in solid tumors and will develop a model of B cell exhaustion in human cancer. Ultimately, we will predict which TIL-B functions to target in future NSCLC immunotherapies.
PTPROt maintains T cell immunity in the microenvironment of hepatocellular carcinom (TUM9P.1002)
Authors: Sun Beicheng 
Published: January 2023

Abstract
Background & Aims: Intratumoral T cells play a central role in anti-tumor immunity, and the balance between T effector cells (Teff) and regulatory T cells (Treg) affects the prognosis of cancer patients. However, educated by tumor microenvironment, T cells frequently fail in their responsibility. In this study, we aimed to investigate the role of truncated isoform of protein tyrosine phosphatase receptor - type O (PTPROt) in T cell mediated anti-tumor immunity. Methods: We recruited 70 hepatocellular carcinoma (HCC) patients and 30 healthy volunteers for clinical investigation, and analyzed cellular tumor immunity by using ptpro-/- C57BL/6 mice and NOD/SCID mice. Results: PTPROt expression was significantly down-regulated in human HCC infiltrating T cells due to the hypoxia microenvironment (***P&lt;0.001); PTPROt expression highly correlated with the intratumoral Teff/Treg ratio (**P&lt;0.01) and clinicopathologic characteristics (*P&lt;0.05). Moreover, PTPROt deficiency attenuated T cell mediated anti-tumor immunity and remarkably promoted mouse HCC growth (***P&lt;0.001). Mechanistically, deletion of PTPROt decreased Teff quantity and quality through phosphorylation of lymphocyte specific tyrosine kinase (Lck), but increased Treg differentiation through phosphorylation of signal transducer and activator of transcription (STAT) 5. Conclusions: In support of the Teff/Treg homeostasis, PTPROt serves as an important tumor suppressor in HCC microenvironment.
Intestinal microbiota alters omental tumor growth (TUM9P.1004)
Authors: Meza-Perez Selene , Silva-Sanchez Aaron , Garcia-Hernandez Maria de la Luz , Mudunuru Uma , Rangel-Moreno Javier , Simpler Scott , Sinclair Stacey , Weaver Casey , Schoeb Trenton , Lord Edith , Randall Troy 
Published: January 2023

Abstract
The omentum is an adipose tissue that contains milky spots (MS). The MS are similar to secondary lymphoid organs and can generate B and T cell immune responses to peritoneal antigens. Additionally, omentum CD4+CD25+FoxP3+ T regulatory cells (Tregs) display an activated phenotype CD44hiCD62Llow. Moreover, the omentum also collects metastasizing tumor cells in the peritoneal cavity and tumors growing in the omentum are associated with poor clinical prognosis. Here we show that intestinal microbiota affects Tregs activation profile and impairs tumor growth in omentum. Our transplantable tumor model in wild type (WT) mice shows that peritoneal tumors grow progressively in the omentum and peritoneal cavity in WT mice, whereas in germ free (GF) mice tumors do not grow. Omental tumor growth in WT mice is associated with an increase in PD-1+ Tregs, while tumor-specific CD8+ T are reduced. However, in GF mice numbers of PD1+ Tregs remain unaltered but tumor specific CD8+ T cells are still present. After co-housed GF mice with WT mice, ex-GF mice showed tumor growth and restore the increase in PD-1+ Tregs with low number of specific CD8+ T cells. The induction of tolerance requires PD-1+ Tregs. These data suggest that intestinal microbiota has a role in omental Tr activation and in the tolerance to peritoneal tumors.
MicroRNA-422a downregulates DNAM-1 expression in NK cells and impairs DNAM-1-mediated cytotoxicity (TUM10P.1033)
Authors: zhu min , Zhang Wei , Sang Hai-wei , Fu Xiang-ning 
Published: January 2023

Abstract
Natural killer (NK) cells play a crucial role in innate immunity against cancer. Activation of NK cells is regulated by a balance between activating and inhibitory signals from cell surface receptors. Among these receptors, DNAM-1 and its ligands ( PVR and Nectin-2) play a vital role in NK cell-mediated recognition and lysis of tumor cells. microRNAs (miRNAs) are key regulators of gene expression that may be involved in NK function of immune surveillance against cancer. Several miRNA have been identified as modulators of NK activating receptor and its ligands expression, such as NKG2D and MICA. However, little is known about DNAM-1 gene regulation by miRNA. Based on bioinformatics, we identified miR-422a with putative docking sites in the 3'UTR of DNAM-1. Furthermore, we investigated the potential interaction between the 3'UTR of DNAM-1 gene and miR-422a as well as its functional role in the regulation of DNAM-1 expression and cytotoxicity in NK cells both in vitro and in vivo. We found that TGF-β1 but not L-kynurenine upregulated miR-422a expression in NK cells, resulting in markedly inhibiting NK cytotoxicity against lung cancer cells, which was mediated by suppression of DNAM-1. Conversely, miR-422a downregulation significantly increased DNAM-1 expression in NK cells, resulting in more efficient DNAM-1-mediated cytotoxicity. Our findings indicate that miR‑422a is a novel regulator for DNAM-1. Therapeutic control of miR-422a may provide new strategies against lung cancer.
CXCL10 within the tumor microenvironment induces gemcitabine resistance in pancreatic cancer cells (TUM10P.1037)
Authors: Delitto Daniel , Black Brian , Sorensen Heather , Knowlton Andrea , Thomas Ryan , Behrns Kevin , Moldawer Lyle , Trevino Jose , Wallet Shannon , Hughes Steven 
Published: January 2023

Abstract
The systemic treatment of pancreatic cancer (PC) is hindered by the rapid development of chemoresistance to current cytotoxic therapies. Mechanisms governing the development of chemoresistance remain poorly characterized, particularly with respect to contributions from the tumor microenvironment. Here, intratumoral soluble mediator concentrations from resected PC specimens (n=26) as well as supernatants from co-cultures of PC cell lines and primary tumor-associated pancreatic stellate cells (PSCs) (n=16) were evaluated using a panel of 43 growth factors, chemokines and cytokines. CXCL10 levels were significantly increased in PC specimens as well as upon co-culture of PC cells with tumor-associated PSCs. In addition, high intratumoral CXCL10 concentrations correlated with reduced overall survival (HR 6.9; P = .006). Thus, the effect of CXCL10 on viability, proliferation, and apoptosis of PC cell lines was evaluated with and without gemcitabine treatment. While CXCL10 treatment had a small effect on the viability of PC cell lines, it significantly increased viability of these cells in the presence of gemcitabine. In addition, gemcitabine treatment induced the expression of the CXCL10 receptor, CXCR3. Thus, paracrine CXCL10 signaling between stromal, PC and immune cells within the pancreatic cancer microenvironment the may be responsible for chemoresistance to gemcitabine.
VEGF neutralization alters the innate immune cell compartment and enhances intratumoral T cell activation (TUM10P.1040)
Authors: Walsh Kevin , de Almeida Patricia , Mak Judy , Hernandez Genevive 
Published: January 2023

Abstract
Although targeting the vascular endothelial growth factor (VEGF) pathway has been used in clinic as an anti-angiogenic therapy to combat cancer for almost a decade, the precise mechanisms of action have yet to be fully defined. Here we demonstrate in a panel of syngeneic tumor models that in addition to it’s anti-angiogenic activity, VEGF blockade augmented inflammation within tumors. Enhanced inflammation coincided with altered myeloid cell composition and modulation of dendritic cell activation. Modifications within the innate immune cell compartment mediated by anti-VEGF treatment correlated with enhanced functional capacity of tumor infiltrating T cells. This was characterized by increased cytokine production of total as well as antigen-specific T cells and decreased surface expression of negative immune regulators. VEGF receptors are detected on both innate immune cells and T cells suggesting that VEGF neutralization has a direct effect on these compartments. Based on these data, VEGF neutralization increased tumor inflammation by modulation of the innate immune cell compartment as well as promotion of anti-tumor T cell responses. Therefore, therapeutics that target the VEGF pathway have potential to work in concert with immunotherapeutics.