bluuebunny/crossref_metadata_2025_split huggingface dataset:
IL-23 induces apoptosis of self-reactive thymocytes in thymic negative selection by an Rorc dependent mechanism (64.19)
Authors: Li Hao , Wu Qi , Yang PingAr , Cua Daniel , Hsu Hui-Chen , Mountz John 
Published: January 2023

Abstract
Thymocyte apoptosis can occur by over-induction of a specific transcription factor, RORγt (or its gene, Rorc); however, the role of RORγt in thymocyte selections and the upstream signal that drives the expression of Rorc are largely unknown. We have observed that interleukin-23 (IL-23), a cytokine which has been implicated as a key regulator of Rorc, was expressed by thymic dendritic cells. To determine if the IL-23-RORγt signaling axis is involved in thymocyte selections, we have determined the expression of Il23r and Rorc in thymocytes from the male antigen-specific Db/H-Y T-cell receptor (TCR) transgenic (Tg) and the MOG35-55 antigen-specific 2D2 TCR Tg mice. Specific antigen activation induced Il23r expression on CD4+CD8+ double positive (DP) thymocytes and then upregulated the expression of Rorc with IL-23 stimulation, leading to dramatic induction of DP thymocyte apoptosis in vitro in TCR Tg+ mice. In vivo over-expression of IL-23 in B6 mice by adenovirus (AdIL-23) efficiently depleted DP thymocytes by promoting apoptosis (from 1.5% to 10%) as determined by in situ TUNEL staining. This AdIL-23 induced thymocyte apoptosis was partially blocked in Rorc+/- mice. Consistent with these, there was an increased percent of Db/H-Y Tg+ thymocytes in Il23 p19-/- mice, compared with that in p19 intact male Db/H-Y Tg mice. Taken together, our work suggests an important role for the IL-23-RORγt axis in the induction of TCR-mediated apoptosis and negative selection in thymocytes.
Notch signaling requires GATA-3 for the survival of cells entering the T-lineage development program (64.4)
Authors: Thompson Patrycja , de Pooter Renée , Zúñiga-Pflücker Juan Carlos 
Published: January 2023

Abstract
GATA-3 plays a crucial role in T cell development, however its role and collaboration with the Notch signaling pathway in the induction of T cell specification and commitment have not been fully elucidated. Work in our laboratory using both, in vitro and in vivo models of T cell development, has shown that GATA-3 deficiency arrests this development at the DN2 stage but does not result in B-lymphopoiesis in the presence of Notch ligand Delta-like 1 (Dll1). These GATA-3-/- progenitors express T-lineage associated genes but are unable to survive the T-specification event as indicated by their increased rate of apoptosis. Cell cycle regulation genes, mainly p19Arf and p16Ink4a (Arf/Ink), are upregulated in these cells as indicated by gene expression microarray and qRT-PCR analyses. We have thus sought to address the possibility that GATA-3 represses Arf/Ink following T cell specification/commitment in the presence of Notch signals. We are employing ChIP assay to show whether potential GATA-3 recognition sequences present in the Arf/Ink promoter region are occupied and regulated by GATA-3. Finally, using GATA-3-/-Arf-Ink-/- bone marrow progenitors, we will address a possible rescue of the early T cell developmental block observed in the absence of GATA-3. These experiments will help to identify a potential mechanism of how GATA-3 and the Notch signaling pathway collaborate in regulating T-lineage specification, commitment and survival during T cell development.
Knock-down of IL-12 secretion in dendritic cells leads to Th2 polarization of T helper cells in vitro (65.12)
Authors: Heilmann Katja , Völker Christina , Wand Inga , Micheel Burkhard , Rohayem Jaques 
Published: January 2023

Abstract
Dendritic cells, T lymphocytes and B lymphocytes were co-culativated in vitro in order to induce an in vitro immunization process resulting in the activation of the involved cell populations and in the production of antigen-specific antibodies. Bone-marrow derived dendritic cells were treated with IL12-siRNA to inhibit the secretion of IL-12 which is mainly responsible to induce Th1 directed immune responses. The siRNA-treated dendritic cells were then loaded with ovalbumin (OVA) as antigen and co-cultured with naive OVA-TcR-transgenic T helper cells and naive B lymphocytes. The activation steps and the production of OVA-specific antibodies were investigated by FACS, RT-PCR and ELISA. FACS and RT-PCR analysis revealed a Th2 polarization of the involved T helper cells according to their cytokine patterns and c-MAF expression. Ovalbumin-specific antibodies could be detected in the culture supernatants after co-cultivation of naive B cells with T cells and siRNA-treated dendritic cells. These studies indicate that the silencing of IL-12 can lead to a Th2 polarization of in vitro cultivated T helper cells. This step could allow an in vitro induction of antigen-specific antibody molecules in shorter time frames than the conventional hybridoma technology and can simplify the generation of antibodies of non-murine origin.
A novel imaging cytometry method for quantitative cell viability assays (65.13)
Authors: Lai Ning , Lyettefi Emily , Pirani Alnoor , Smith Tim , Chan Leo , Lin Bo 
Published: January 2023

Abstract
Accurate measurement of viability is essential for many cell based research. The traditional cell viability analysis instrumentation includes microscopy, flow cytometry and plate reader. However, these systems are often expensive to purchase and maintain. Also, the operational processes are often tedious or complicated. Here we present a novel Cellometer Vision system, which incorporates image based cell counting and fluorescence detection in a compact and easy-to-use instrument. This method utilizes both bright-field and fluorescence imaging of a disposable cell counting chamber to quickly provide concentration and viability measurements of cell populations. In this poster, we demonstrate the application of Cellometer Vision system for cell viability assays that test for different characteristics of cells death. Jurkat cells were treated with cytotoxic compounds and heat treatment. Cell viability was assessed by mitochondria function assay using JC-1 dye, apoptosis assay using Annexin-V, membrane integrity assay using trypan blue and propidium iodide, metabolic assay using calcium AM and alamrBlue, and cell proliferation assay. Differences in cell viabilities were observed from different assays for cells intermediate stages of cell death. Cellometer Vision provides a simple, rapid and cost-effective tool for cell viability measurements. This novel imaging cytometry method is demonstrated here to support viability assays that test different stages of the cell death process.
Quantitative analysis of cellular interactions using imaging flow cytometry (65.15)
Authors: Friend Sherree , Burbach Brandon , Jacobs Evan , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia , George Thaddeus 
Published: January 2023

Abstract
Direct cell to cell contact is critical for an effectively coordinated immune response to pathogens. Cells of the innate immune response are specialized to bind, process, and present antigen in the context of appropriate co-stimuli to direct specific responses from cells of the adaptive immune system. T cell dependent antibody responses require B-T cell interaction. Nibbling can directly transfer proteins from an infected cell to an APC for antigen processing and presentation to a lymphocyte. Interactions between virus-infected cells and antigen presenting cells can induce specific cell-cell fusion. Discrimination amongst these types of cellular interactions requires visualization, but these events are often rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. The ImageStream automatically and rapidly collects large numbers of high resolution cell images per sample and is thus ideally suited for robust statistical image-based analysis of cellular interaction. Multispectral images of each conjugate provides objective measurement of conjugate numbers and the location information needed to determine the type of interaction on a per cell basis. We will demonstrate three types of conjugate analysis using the ImageStream. 1. The measurement of a protein in the immune synapse between a T cell-APC. 2. The specific nibbling that occurs between an APC and a virally infected cell. 3. Cell fusion between an APC and HIV-infected cell.
A novel multiplexed immunoassay using salivary antibodies to detect human exposure to waterborne pathogens in drinking and recreational water (65.19)
Authors: Simmons Kaneatra , Curioso Clarissa , Eason Tarsha , Griffin Shannon , Fout G. , Wade Timothy , Egorov Andrey , Augustine Swinburne 
Published: January 2023

Abstract
Water is our most important resource and the world’s supply is becoming alarmingly limited with rising concern about the high levels of microbial contamination. We are developing a novel strategy to assess human exposure to microbial contaminants in drinking and recreational water using a multiplexed, high-throughput immunoassay applying the Luminex xMAP platform. In this assay, antigens from waterborne pathogens are coupled to spectrally unique microspheres that are exposed to either pathogen-specific antibodies or human saliva. Our results indicate that multiplexed microsphere sets coated with antigens to Helicobacter pylori, Salmonella typhimurium, Escherichia coli O157:H7, Campylobacter jejuni, Toxoplasma gondii, Cryptosporidium parvum, and Giardia lamblia are able to capture both pathogen-specific antibodies and human salivary antibodies at the lowest-limit of detection of the assay. Additionally, we implemented a Design of Experiment (DoE) approach to evaluate the impact of antigen and antibody concentration on the detection level of H. pylori. From this work, we were able to generate a mathematical model describing the detection level as a function of critical process parameters to determine optimal conditions for protein coupling to Luminex beads. This immunoassay will allow for a sensitive, rapid, and cost-effective analysis of human exposure to waterborne pathogens with the potential to reduce the cost of epidemiological surveillance of waterborne infections.
Ethanol-dispersed polymer nanofiber net for highly-selective capture of lymph node CD4+ T cells (65.21)
Authors: Kim Kwanghee , Jun Seung-Hyun , An Hyo Jin , Kim Byoung Chan , Sonn Chung Hee , Kim Tae-Jin , Lim Seon Ah , Kim Jungbae , Lee Kyung-Mi 
Published: January 2023

Abstract
The hydrophobic polymer NFs could be dispersed in the aqueous solution easily by simple ethanol treatment, which increases the loading of enzyme or antibody effectively. The degree of dispersion of nanofibers was somewhat proportional to the concentration of ethanol, the loading and activity of immobilized enzymes were proportional increased to the degree of dispersion of nanofibers. Next, the feasibility of anti CD4 monoclonal antibodies conjugated on ethanol dispersed nanofibers (anti CD4 mAbs/EtOH-NF) are developed as a useful vehicle for biomedical application such as specific cell capture and separation. The anti CD4 mAbs/EtOH-NF were examined for their ability to bind CD4+ T cells, bound to anti-CD4 mAbs, isolated from the mixture of lymphocytes. The results show that the anti CD4 mAbs/EtOH-NF successfully isolated CD4+ T cells from the mixture of lymphocytes with high specificity. Therefore, biocompatible of alcohol treated nanofibers provide an efficient tool for the cell separation process and further present the potential to be applied to other areas of biomedical application.
An HLA allele frequency-based approach to resolving allelic and genotypic ambiguity in HLA typing data and its implementation in ImmPort (65.22)
Authors: Guidry Paula , Marthandan Nishanth , Smith Thomas , Campbell John , Dunn Patrick , Erlich Henry , Karp David , Single Richard , Thomson Glenys , Wiser Jeffrey , Scheuermann Richard , Mack Steven 
Published: January 2023

Abstract
The classical human leukocyte antigen (HLA) loci are the most polymorphic loci in the human genome, with hundreds of new alleles reported yearly. HLA polymorphisms in exons 2 and 3 of class I loci and in exon 2 of class II loci distinguish the peptide binding regions of HLA proteins. Current HLA typing methodologies focus on identifying variant positions in these exons, and most do not detect allelic variation in other exons or non-coding regions of HLA genes. Because of the limitations in detecting polymorphic variants and the pace of new allele discovery, commonly used HLA typing methodologies cannot unambiguously identify all known HLA alleles or genotypes. Consequently, many HLA genotyping results are ambiguous in that both alleles of each HLA locus for a given subject may have multiple possible identities, and each subject may have multiple possible genotypes. Here, we describe an approach to allelic and genotypic ambiguity resolution that uses published HLA allele frequencies in 10 world regions to determine the most likely diploid alleles and genotype for each subject in an HLA typing study. This method has been implemented in an online tool at www.ImmPort.org, and is freely available for use by the immunology community. This ambiguity reduction tool will allow investigators studying autoimmune disease, adverse drug reactions, transplantation outcome and other HLA associated conditions to more accurately determine the contribution of specific HLA alleles to disease.
A novel assay for imaged-based analysis of leukocyte migration in a stable chemoattractant gradient (65.36)
Authors: Vu Elizabeth , Meyvantsson Ivar , Lamers Casey , Skoien Allyson , Echeverria Victoria , Hayes Steve 
Published: January 2023

Abstract
Current approaches to study chemotaxis are limited by gradient instability, low throughput, cumbersome manual manipulations, high cell number requirements, and inability to acquire high-content data. We present here a new microfluidics-based approach to study leukocyte chemotaxis. The device features strong miniaturization of the use of cells (&lt;12,000 cells per data point). The microfluidic channels in the device are arrayed in 96 well format and can be operated by standard liquid handling robotics or hand pippettes. Cells migrate along a surface in the presence of a stable gradient of chemoattractant that is maintained for at least three hours. We show the ability to obtain consistent dose response curves to chemoattractants as well as chemotactic inhibitors with the device. Because cells are microscopically accessible, the information content is high. Not only can the number of cells migrated be assessed, but also migration distances of individual cells, and even scoring of cell activation and polarization. In addition, we have developed a smaller slide-sized device embedded within an enclosure that prevents evaporation. This feature allows live cell tracking which can deliver highly detailed information on cellular behavior during chemotaxis.
A bioanalytical method for detection of phosphorodiamidate morpholino oligomers in cells. (65.44)
Authors: Crumley Stacy , Mourich Dan , Iversen Patrick 
Published: January 2023

Abstract
Phosphorodiamidate morpholino oligomers (PMOs) are neutrally charged antisense agents that interfere with target gene expression by inhibition of translation or by altering splicing of pre-mRNA. PMOs have been shown to be highly specific and potent therapies after cellular uptake, yet a method to detect PMO inside the cell has not been developed. We offer in this report a novel method for the detection of cellular resident PMO using flow cytometry-fluorescence in situ hybridization (flow FISH) which takes advantage of locked nucleic acid (LNA) oligonucleotides as probes. A biotinylated LNA probe complementary to an Influenza A specific PMO (IA-PMO) was used to specifically detect and quantify intracellular IA-PMO after in vitro or in vivo administration with or without Influenza A infection. While IA-PMO was detected in non-hematopoietic and hematopoietic cells of the lung regardless of influenza virus infection, it was detected at higher levels in influenza infected hematopoietic lung cells as compared to uninfected cells. Coincident measurement of target knock down to intracellular IA-PMO concentration allowed for the calculation of an EC50. Furthermore, it was observed that an increase in IA-PMO uptake occurs in infected hematopoietic cells.
4-1BB agonist antibody treatment causes intra-tumoral accumulation of KLRG1+ CD8+ and CD4+ T-cells with a novel highly cytotoxic phenotype driven by the master-regulator Eomesodermin (66.17)
Authors: Curran Michael , Kim Myoung-Joo , Al-Shamkhani Aymen , Allison James 
Published: January 2023

Abstract
Antibodies which block the co-inhibitory receptor CTLA-4 or which activate the co-stimulatory receptor 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma. We find that combining these two antibodies in the context of a Flt3-ligand based B16 melanoma vaccine promoted high CD8+ and CD4+ effector to FoxP3+ Treg ratios in tumors, highly elevated TH1 type cytokine production, increased T-cell proliferation and synergistic levels of tumor rejection. A hallmark of 4-1BB agonist antibody treatment is the upregulation of the lectin KLRG1 on tumor infiltrating CD8+ and CD4+ T-cells. We find that these KLRG1+ T-cells in the tumor express high levels of multiple cytotoxic molecules, kill tumor cells better than their KLRG- counterparts and may be responsible for the increased anti-tumor cytotoxicity attributed to α4-1BB treatment. We found that formation of these cells is driven by high-level expression of the master-regulatory transcription factor Eomesodermin (Eomes) in both the CD8+ and CD4+ T-cell compartments. These Eomes+KLRG1+ cytotoxic CD4+ and CD8+ T-cells represent a novel phenotype distinct from any described in the literature (KLRG1expression is not IL-12/T-bet dependent, they are not exhausted, and the Eomes expression exceeds that of normal TH1). The origins and applications of these novel T-cells have broad implications for tumor immunotherapy as well as basic T-cell immunology.
Effect of BCNU on myeloid-derived suppressor cells in anti-tumor immune response (66.20)
Authors: Zhou Juhua , Nagarkatti Prakash , Zhong Yin , Nagarkatti Mitzi 
Published: January 2023

Abstract
We attempted to understand if 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) may prevent the generation of myeloid-derived suppressor cells (MDSCs) during tumor growth and modulate anti-tumor immunity. After intraperitoneal injection of EL4 tumor cells into mice, tumor cells grew rapidly in the peritoneal cavity, and metastasized into liver and lung. Mice died around day 14 after tumor challenge. MDSCs appeared at day 7 and massive MDSCs accumulated in spleen, liver and lung at day 11. In vitro assay indicated that MDSCs from tumor-bearing mice significantly inhibited T cell proliferation and anti-tumor immune response. After mice received BCNU injection (20mg/kg body weight) at day 5, however, tumor cell growth was completely inhibited and all of mice survived after BCNU treatment and tumor challenge. FACS analysis showed that MDSC accumulation was completely inhibited in tumor-challenged mice after BCNU injection, suggesting that BCNU may inhibit MDSC development and thus induce tumor rejection in mice. TUNNEL assay demonstrated that BCNU induced MDSC apoptosis. Interestingly, BCNU-cured mice rejected tumor after EL4 tumor cell re-challenge, suggesting that BCNU may not only inhibit MDSC production but also induce anti-tumor immune memory. The results indicate that BCNU may inhibit the production of MDSCs, increase anti-tumor immune response and thus facilitate tumor rejection.
Costimulation through 4-1BB/CD137 improves the expansion and function of CD8+ melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy (66.21)
Authors: Chacon Jessica , Wang Yijun , Wu Richard , Hwu Patrick , Radvanyi Laszlo 
Published: January 2023

Abstract
Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) is a powerful therapy for Stage IV melanoma patients who have failed first and second line therapies. TIL for adoptive transfer are expanded from melanoma surgical specimens with IL-2 followed by a “rapid expansion protocol” (REP), where the cells are activated with anti-CD3 antibody, PBMC feeder cells and IL-2 for two weeks, generating a final infusion product. The REP yields billions of highly-differentiated cytolytic CD8+ T cells that lose costimulatory molecules CD28 and CD27, and are hyporesponsive to further proliferative signals. We found “post-REP” CD8+ TIL express an alternative costimulatory molecule of the TNF-R family, 4-1BB. Our study tests the effects of an agonistic anti-4-1BB antibody added at the time of initiating the REP. We found adding an agonistic anti-4-1BB antibody increased CD8+ T-cell recovery, antigen specificity, BCL-2 gene expression and increased the frequency of CD28+ cells in the recovered CD8+ post-REP TIL. Adding anti-4-1BB improved post-REP TIL survival capacity in the absence of cytokine, with TIL continuing to divide even without added IL-2. Our findings suggest adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.
Tumor-derived mesenchymal stem cells promoted tumor development through CCR2-dependent recruitment of macrophages (66.40)
Authors: Ren Guangwen , Zhao Xin , Zhang Liying , Yuan Zeng-rong , Zheng Betty , Shao Changshun , Shi Yufang 
Published: January 2023

Abstract
Mesenchymal stem cells (MSCs) residing in tumor stroma have been recognized to originate from normal tissues, such as bone marrow. Although they present as a major component of tumor stroma, the exact role they play in tumor progression and how they distinguish from normal tissue MSCs are largely unknown. In this report, we characterized the function of mesenchymal stem cells in promoting tumor development. Compared to BM-MSCs or tissue-MSCs, lymphoma-derived MSCs (L-MSCs) exhibited a strong tumor-promoting effect in a tumor transplantation model. While L-MSCs did not differ from BM-MSCs in their ability to suppress the adaptive immunity, they induced more abundant accumulation of CD11b+Gr-1+ myeloid suppressor cells and F4/80+ macrophages in tumors. Depletion of macrophages, but not myeloid suppressor cells, completely ablated the effect of L-MSCs, indicating that the tumor-promoting effect of L-MSCs was mediated via the recruitment of macrophages. Furthermore, chemokines CCL-2, CCL-7 and CCL-12, all of which utilize CCR2 as a receptor, were found over-expressed in L-MSCs. Correspondingly, the tumor-promoting effect of L-MSCs was largely abolished in CCR2-/- mice. Collectively, these data demonstrated that L-MSCs contribute to tumor development through CCR2-dependent recruitment of macrophages to tumor sites. Our findings thus established a mechanistic link between two major types of tumor stromal cells, providing critical implications for the future cancer therapy.