bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic analysis of the role of mast cell- and basophil-derived TNF in severe sepsis (P4205)
Authors: Piliponsky Adrian , Lahiri Asha , Truong Phuong , Lilla Jennifer , Reber Laurent , Nedospasov Sergei , Galli Stephen 
Published: January 2023

Abstract
Mast cell-derived TNF can increase mortality in C57BL/6-KitW-sh/W-sh mice after severe cecal ligation and puncture (CLP). However, the mutation responsible for mast cell deficiency in these mice induces other abnormalities that may have influenced this finding. Therefore, we generated mice in which TNF production is specifically ablated in mast cells and basophils by crossing Cpa3-Cre mice with Tnffl/fl mice (Cpa3-Cre; Tnf fl/fl). Mast cells and basophils from Cpa3-Cre; Tnf fl/fl mice are impaired in their ability to produce TNF after stimulation by either IgE/antigen or LPS. Dermal leukocyte numbers in the ear pinnae of Cpa3-Cre; Tnf fl/fl mice were reduced at sites of IgE-dependent passive cutaneous anaphylaxis (PCA), suggesting that local release of mast cell- and/or basophil-derived TNF contributes to leukocyte recruitment after PCA. Serum TNF levels and intraperitoneal neutrophil numbers were significantly reduced after i.p. challenge with a sub-lethal dose of LPS, indicating that mast cells and/or basophils also contribute to systemic levels of TNF in this setting. Notably, Cpa3-Cre; Tnf fl/fl mice exhibited higher survival rates, reduced drops in body temperature, significantly lower numbers of intraperitoneal neutrophils, and decreased local and systemic TNF levels after severe CLP. Our findings in Cpa3-Cre; Tnf fl/fl mice provide strong evidence that mast cell- and/or basophil-derived TNF can contribute to increased mortality and inflammation in severe sepsis.
Autophagy regulates mycobacterial uptake by modulation of scavenger receptors (P4215)
Authors: Bonilla Diana , Bhattacharya Abhisek , Sha Youbao , Xu Yi , Xiang Qian , Gillard Baiba , Pownall Henry , Khan Arshad , Jagannath Chinnaswamy , Komatsu Masaaki , Eissa Tony 
Published: January 2023

Abstract
Autophagy and phagocytosis are highly conserved cellular functions involved in innate immunity. However, the nature of the interactions between these two critical processes remains essentially unknown. We evaluated the role of autophagy in regulating phagocytosis by measuring phagocytic activity in autophagy-deficient macrophages obtained from a myeloid-specific autophagy-related gene 7 knockout mice Atg7-/-. Importantly, in addition to increased mycobacterial growth, Atg7-/- macrophages exhibited higher bacterial uptake, when infected with Mycobacterium tuberculosis or M. tuberculosis var. bovis BCG. In addition, Atg7-/- mice, infected intranasally with BCG, showed increased bacterial loads and exacerbated inflammatory responses in lungs, compared to Atg7+/+ cells. Atg7-/- macrophages had increased surface expression of two class A scavenger receptors: macrophage receptor with collagenous structure MARCO and scavenger receptor-A. These findings were confirmed in Atg3-/-, Atg5-/- and Atg7-/- mouse embryonic fibroblasts. The increased surface expression of scavenger receptors was caused by increased activity of the transcription factor NF-E2-related factor 2 due to accumulation of p62 in autophagy-deficient macrophages. These new insights increase our understanding of host-pathogen relationship and suggest that therapeutic strategies against pathogens should be designed to include modulation of both phagocytosis and autophagy.
Adenosine binding A2B adenosine receptors is an essential danger signal for the initiation and development of the type 2 immune response to the intestinal parasite Heligmosomoides polygyrus (P4041)
Authors: Wu Wenhui , Patel Nirav , Mishra Pankaj , Hasko George , Gause William 
Published: January 2023

Abstract
A2B adenosine receptor (A2BAR) signaling modulates lymphocyte function in vitro and can control proinflammatory immune responses. To examine whether A2B receptors contribute to the in vivo protective type 2 immune response to helminthes, A2BAR-/- mice were infected with H. polygyrus (Hp). Worm burden and egg production were increased while serum IgE and IgG1 were reduced in A2BAR-/- mice compared to WT mice at day 14 after secondary Hp inoculation. Worms recovered from A2BAR-/- mice at day 11 after secondary inoculation exhibited higher protein and ATP levels than similarly inoculated WT mice, suggesting impaired host protective responses. Th2 cytokine expression and M2 macrophage markers were also inhibited in small intestines and in mesenteric lymph nodes (MLN) in A2BAR-/- mice during early luminal phase after secondary inoculation. After primary inoculation, increases in IL-33, and ILC2 cell activation and expression of IL-5 and IL-13 were inhibited in A2BAR-/- mice. IL-33 administration to Hp inoculated A2BAR-/- mice rescued ILC2 activation and cytokine expression. These studies indicate specific binding of adenosine to A2BAR, likely as a result of tissue damage, triggers the production of the alarmin IL-33 which then initiates the type 2 immune response. These results identify for the first time an essential danger associated molecular pattern required for the development of the helminth-induced type 2 immune response.
Withaferin A is effective in inhibiting the survival of B cell lymphomas and chronic lymphocytic leukemias (P2052)
Authors: Gachuki Beth , Oben Karine , Munniappan Latha , Alhakeem Sara , Rangnekar Vivek , Muthusamy Raj , Byrd John , Gupta Ramesh , Bondada Subbarao 
Published: January 2023

Abstract
Withaferin A is a withanolide from the ayurvedic medicinal plant, Withania Somnifera that has been shown to be a major contributor to the medicinal value of extracts from this plant. Withanolides, oxidation products of steroids, are known to have growth inhibitory effects on certain insect larvae. Withaferin A is the first withanolide to be isolated from the Aswagandha plant. It has been shown to have anticancer activity in breast and lung cancer models but its effects on hematological malignancies have not been studied in detail. Here we examined the effect of highly purified Withaferin A on the survival of diffuse large B cell lymphoma cell lines. Withaferin A strongly inhibited the survival of Ramos, Sudhl6, LY10 and murine A20luc and BKS2 B cell lymphoma cell lines, mainly by affecting cell cycle progression. It also reduced the survival of chronic lymphocytic leukemia cells (CLL) from the Emu-Tcl1 transgenic mice. We found that Withaferin A inhibited activation of Akt rapidly but of Src family kinase after 24 hours of incubation and also reduced the levels of pro-survival protein Bcl-x , in the murine CLL cells Withaferin A also reduced the levels of the pro-survival oncogene Tcl1 as well as the pro-apoptotic protein Par-4. We are currently investigating the mechanisms that underlie the opposite effects (reduction of both pro-survival and anti-survival proteins) and their relation to the anti-lymphoma/leukemia effects of Withaferin A.
Therapeutic potential in cancer: using adenoviral vaccine vector co-expressing tumorantigen and interleukin-2 (P2100)
Authors: Jensen Benjamin , Steffensen Maria , Christensen Jan , Holst Peter , Thomsen Allan 
Published: January 2023

Abstract
Cytokines role in the adaptive immune system has drawn significantly attention in recent years. An important finding is that T-cell quality and T-cell numbers tend to decline in cancer patients, partly as a consequence of inadequate amounts of the cytokine interleukin-2 (IL-2), due to a general tumor environmental down regulation of the T-cell activating molecules CD80/86. Thus, co-administration of cytokine(s), in combination with an antigen of interest, possesses the potential to modulate the immune response in a favorable direction. As a consequence, we have modified an adenoviral vaccine vector; designed it to co-express the glycoprotein (GP) from lymphocytic choriomeningitis virus (LCMV), alongside IL-2, in two disperse reading frames. Both components are exchangeable and expressed autonomously. Remarkably, adenoviral vaccine vectors have been postulated to be among the most potent and promising candidates for production of viral vaccines. The results exhibit increased tumor protection in wild type (WT) mice and impressive T-cell responses in CD80/86 knockout mice, which are otherwise known to have a diminished immune response, but most importantly, the described vaccine candidate enables CD80/86 knockout mice to restore tumor protection, undertake levels equivalent to WT mice. A concern regarding the use of IL-2 expressing vectors is the impact on the generation of long-term memory. However, we did not obtain any evidence pointing towards reduced CD8 T-cell memory.
α-Galactosylceramide analogues in cancer immunotherapeutics (P2102)
Authors: Sharma Shalu , Veerapen Natacha , Bhowruth Veemal , Zhang Lianjun , Donda Alena , Besra Gurdyal , Porcelli Steven 
Published: January 2023

Abstract
Natural Killer T cells (NKT) initiate potent anti-tumor responses upon activation by glycolipid antigens presented by CD1d, a non-classical MHC class I-like antigen presenting molecule. Anti-tumor responses are induced following NKT cell activation through their direct production of cytokines such as IFN-γ, and through a variety of indirect effects including dendritic cell maturation, the activation of NK cells and the cross-priming of tumor-specific CD8+ T cells. The glycolipid α-galactosylceramide (α-GC) is a CD1d ligand and potent NKT cell activator that enhances anti-tumor immune responses and decreases metastases in vivo. However, the response is short-lived, and causes liver toxicity and long term anergy of NKT cells. Here we report a novel class of chemically modified α-GCs, designated GCBs, that demonstrate unique binding properties to both mouse and human CD1d and enhanced NKT cell stimulation in vitro, as compared to previously described NKT cell activating ligands. A range of analyses have been carried out to characterize the structure and stability of GCB:CD1d complexes, revealing unique features that distinguish them from previously analyzed α-GC:CD1d complexes. Soluble GCB:CD1d complexes are currently being tested in vivo in mouse models for their ability to drive the sustained activation of NKT cells, which may provide a superior approach for development of improved cancer immunotherapies.
Reducing Fli1 levels in the MRL/lpr lupus prone mouse model impacts T cell activation and cytokine production (P4115)
Authors: Nowling Tamara , Richard Erin , Zhang John , Siskind Leah 
Published: January 2023

Abstract
The Ets factor Fli1 is implicated as a key modulator of lupus disease expression. Over-expressing Fli1 in healthy mice, results in the development of an autoimmune kidney disease similar to that observed in lupus. Lowering the global levels of Fli1 in two lupus mouse models significantly improved kidney disease and prolonged survival. The mechanisms by which Fli1 exerts this protective effect are not fully understood. We demonstrate that MRL/lpr lupus prone T cells expressing 50% less Fli1 (Fli1+/-) produce significantly less IL-4 and IL-13 and exhibit significantly reduced TCR-specific activation. Neuraminidase 1 (Neu1), a mediator of glycosphingolipid metabolism is required for TCR-specific activation and IL-4 production in T cells. Our results demonstrate that MRL/lpr Fli1+/- T cells express significantly reduced levels of Neu1 message and that Fli1 can regulate Neu1 promoter activity in T cells. Furthermore, MRL/lpr Fli1+/- T cells express significantly reduced levels of the glycosphingolipids lactosylceramide and glucosylceramide. Together, these results suggest that Fli1 may mediate lupus disease progression by modulating activation and cytokine expression of lupus T cells in part by regulating Neu1 expression and glycosphingolipid metabolism.
Affinity of T cells for chromogranin A remain constant in type I diabetic NOD mice. (P4119)
Authors: Hood Jennifer , Evavold Brian 
Published: January 2023

Abstract
In type 1 diabetes numerous autoantigens have been identified in the non-obese mouse model including insulin B 9-23, chromogranin A (ChgA29-42), and glutamic acid decarboxylase 65 (GAD65) to list a few, but the kinetics of antigen specificity during the progression from insulitis to overt diabetes is not known. A T cell clone, BDC2.5, isolated from a diabetic NOD was recently identified as being reactive to ChgA. The two-dimensional (2D) micropipette adhesion frequency assay was able to determine that BDC2.5 TCR transgenic cells specifically recognize ChgA29-42, albeit with 100-fold lower affinity compared to the highly stimulatory mimotope. As this TCR was originally derived from a diabetic NOD, we wanted to see if this highly sensitive assay could detect ChgA29-42 specific cells in the pancreatic lymph nodes (PLN) of diabetic NODs. ChgA29-42 specific CD4 T cells were detected in the PLN at a high frequency and their 2D effective affinity was not statistically different from the BDC2.5 affinity for ChgA29-42. Tracking both the frequency and affinity of antigen specific cells isolated from normoglycemic to overtly diabetic female NOD mice, we have determined that the frequency of antigen specific cells present in the PLN but not the overall affinity range changes for ChgA29-42 between these time points. Thus the micropipette adhesion frequency assay allows one to track the frequency and affinity of antigen specific T cells during the progression to autoimmune disease.
Characterization of the cytokine and transcription factor profile of CD4+ T cells from conditional LATY136F mice (P4133)
Authors: Swan Greg , Sullivan Sarah , Zhang Weiguo 
Published: January 2023

Abstract
LAT is an adaptor protein that is required for TCR signaling and T-cell activation. LATY136F mice have a point mutation at tyrosine 136 of LAT (Latm/m) whichprevents LAT from interacting with PLC-γ1 resulting in defective calcium flux and ERK activation upon TCR stimulation. Our lab created conditional LATY136F mice (LATf/m) in which the floxed allele can be deleted upon tamoxifen treatment. T cells from these mice undergo normal thymic development yet when thewildtype LAT allele is deleted in the periphery, develop a lymphoproliferative disease similar to LATY136F mice. Using these mice we were able to characterize transcription factor and cytokine production over the course of disease development in isolation from defective thymic development. We observed similar symptoms of disease to LATY136F mice includingsplenomegly and lymphadenopathy. Unlike CD4 cells from LATm/m mice, we observed, using immunostainingand qRT-PCR, elevated expression of Th1 cytokines and transcription factors during disease development that then declined by week 5. We also observed a stable population of cells that expressed both Th1 and Th2 cytokines. Finally we observed a transient decline in the pro-survival transcription factor BCL-2 and a steady decline of the pro-survival factor MCL-1. From this data we conclude that disease development in theperiphery of LATf/m mice is dynamic and differs from disease development in LATm/m mice.
Lateral distribution of the T cell receptor and cholesterol detected by high-resolution secondary ion mass spectrometry (P3370)
Authors: Wang Feng , Lozano Monica , Lillemeier Björn , Boxer Steven , Davis Mark 
Published: January 2023

Abstract
The engagement of the T cell antigen receptor (TCR) by a specific peptide-MHC ligand initiates a trans-membrane signaling that activates the T cell. An important event in T cell signaling is the recruitment of signaling proteins to the cell membrane and the assembly of a multi-protein (and lipid) signaling complex. Although it has been suggested that lipid/cholesterol composition plays an important role in the regulation of surface signaling complexes, direct visual evidence on lipid/cholesterol participation in the formation of these complexes is still lacking. Here, we characterize the clustering of TCR within T cell and model membranes by secondary ion mass spectrometry (NanoSIMS) with a spatial resolution of 50 nanometers or better. Quantitative information about the chemical composition of membrane complexes can be obtained through the use of orthogonal isotopic labeling of each signaling molecule. Analysis of nanoSIMS images shows the accumulation of TCRs into 60-150 nm clusters/islands within the membrane of an activating T cell and we further attempt to reveal the composition and lateral distribution of TCR and other membrane components, including cholesterol, within model membranes. Because these membrane structures may be shared with many other cell types, we believe the insights we gain in this system will be generalizable to other examples of cell surface receptor signaling.
Massively parallel phenotyping and clonotyping of single T cells (P3380)
Authors: Johnson David , Conant Carolyn , Withey Gary , Löhr Andrea , Hsu Andro , Meyer Everett 
Published: January 2023

Abstract
Flow cytometry captures T cell functionality but not individual T cell receptor clonal sequences. DNA sequencing characterizes the T cell clonotype repertoire, but lacks functional information. We report a method, T cell receptor-Effector Linkage Sequencing (TELS), which uses single-cell microfluidics and next-generation sequencing (NGS) to determine clonotype and phenotype for millions of T cells simultaneously. Single T cells are encapsulated in picoliter droplets. Overlap extension reverse transcriptase PCR amplifies T cell receptor beta (TCRb) and immune effector transcripts separately in early cycles. In later cycles, a linker DNA sequence drives synthesis of fusion products containing both TCRb and effector amplicons. After sequencing the fused products, we process the NGS data to determine clonotype-specific effector expression for each clonotype in the sample. We characterized single-cell expression levels for TCRb, Fas ligand (FasL), and interleukin 2 (IL-2) in four mouse T cell lines using qPCR. Using these cell lines spiked at 0.1% in murine peripheral blood, we produced TELS libraries with a set of multiplexed primers targeting the full TCRb repertoire, FasL, and IL-2. Across &gt;50 experiments, we accurately quantified (p&lt;2.2x10-16) T cell spike-in clonotype and phenotype. TELS could be used routinely for applications where knowing both T cell clonotype and function are helpful for discovery, such as transplantation, vaccine development, and cancer.
Techniques for thymic decellularization (P3392)
Authors: Falcon Jessica 
Published: January 2023

Abstract
Decellularization protocols for both whole organs and tissue parts have been developed and successful for many organs but not for the thymus. The decellularization process maintains the integrity of the organ by preserving the components of the extracellular matrix, including proteins such as collagen and fibronectin. This allows for an acellular, three dimensional biological scaffold of the desired organ which can then be repopulated with cells. Routine decellularization techniques published for other tissue such as the heart and the lung were inadequate for the thymus and ablated the extracellular matrix. Novel techniques were discovered. The aim of this project is to apply the idea of decellularization to the thymus, using thymic tissue procured from murine, porcine, and human models. Thymic tissue has been decellularized using two methods; SDS (sodium dodecyl sulfate) based protocols and by Deoxycholic Acid/Triton-X mixture protocols, and examined for remnant cells and extracellular proteins through histological staining and SEM imaging. We have confirmed complete decelluarization with murine thymic tissue and are advancing to porcine and human samples. We have identified varying decelluarization techniques that provide multiple degrees of integrity of the extra cellular matrix. These techniques may be used in tissue engineering strategies to repopulate a thymic construct for thymic reconstitution.
Fast and easy access to the overall B cell repertoire using the Blood B Booster® system (P3360)
Authors: Razanajaona-Doll Diane , Bertaux Ségolène , Clear Olivier , Laurant Stéphanie , Pin Jean-Jacques 
Published: January 2023

Abstract
Overall B repertoire analysis usually requires extensive molecular analysis of immunoglobulin genes. Determination of serum levels of specific antibodies by ELISA is restricted to “non steady-state” cases such as following vaccination, bacterial or viral infections. Dendritics has developed the Blood B Booster® system, a powerful B cell immortalization tool based on stimulation through CD40 and immortalization with Epstein-Barr virus. Briefly, the Blood B Booster® system is intended to generate B cell oligoclones representative of the overall B cell population. Here, using blood sample isolated from a healthy donor, we demonstrated that natural autoantibodies against various cytokines are detected at variable frequencies by ELISA in the supernatants of B cell oligoclones after 28 days of culture. Furthermore, we showed that Blood B Booster® is a suitable readout system to evaluate the effects of a given monoclonal antibody on B cell growth and/or Ig secretion. Finally, extension of the Blood B Booster® system to animal species allowed us to perform comparative studies between canine and human humoral responses against a candidate vaccine. In conclusion, analysis of the natural autoantibody repertoire, of the antibody responses against therapeutic agents (candidate vaccine, therapeutic mAbs) and human/animal comparative studies will be facilitated by the Blood B Booster® system which is shown here as an essential tool giving a fast and easy access to the overall B repertoire.
Simultaneous analysis of multiple fluorescent proteins and fluorochromes by a novel spectral flow cytometer (P3364)
Authors: Tomura Michio , Futamura Koji , Furuki Motohiro 
Published: January 2023

Abstract
We have been revealing immune system based of spatiotemporal regulation of immune cells in the entire body by using photoconvertible fluorescent protein (FP), Kaede and KikGR mice. Flow cytometric analysis of green and red signals of non-photoconverted and photoconverted Kaede or KikGR expressing cells with multiple fluorochrome-conjugated mAbs have been difficult and unable to increase color panels, because of the fluorescence signal overlap between FPs and fluorochromes. We have newly developed a spectral flow cytometer (FCM) based on a novel measurement principle. Unlike a polychromatic FCM, the spectral FCM with 32 channel linear array PMT detects the fluorescence derived from every fluorescent probe. The data of acquired spectra is analyzed with a unique algorithm. We detected spectral changes of KikGR color from green to red during photoconversion. We separated EGFP and Venus. One of the feature advantages of this instrument is that it can recognize spectral shape of each fluorescent probes and we could separate EGFP and FITC, their peaks of wavelength are almost same and only spectral shape of GFP is slightly broader than FITC. Finally, we successfully separated 11-colors including kikGR-green and -red simultaneously. Taken together, spectral FCM allow us to detection of FPs with multicolor fluorochromes, and thus, the system which uses KikGR mice with spectral FCM is a powerful tool to investigate spatiotemporal regulation of immune cells in the entire body.