bluuebunny/crossref_metadata_2025_split huggingface dataset:
Systems biology approach to determine critical T cell receptor signaling events for the induction of regulatory T cells (LYM8P.639)
Authors: Sheehan Robert , Hawse William , Morel Penelope , Faeder James 
Published: January 2023

Abstract
Antigen stimulation of the T cell receptor (TCR) in a naïve T cell triggers a complex network of signaling pathways that determine whether the cell becomes a tolerogenic T regulatory (Treg) or immunogenic T helper (TH) cell. We previously developed a Boolean model of T cell differentiation that predicted variation of TCR signal duration could be used to optimize the production of Tregs, a strategy that we confirmed in vitro. This model suggested that dynamics of PTEN, a lipid phosphatase that opposes the activation of Akt, might be predictive of cell fate. Subsequent experiments have demonstrated that several mechanisms for regulating PTEN, including posttranslational modification, proteasomal degradation, and transcriptional inhibition, play an important role in T cell fate determination. A more fine-grained computational model of T cell signaling, which considers site-specific phosphorylation of Akt and PTEN, negative regulation of mTORC2 by kinases downstream of Akt, and negative regulation of PTEN through several Akt-dependent and independent mechanisms, recapitulates the dose-dependent dynamics of PTEN and Akt activity. The net effect of these positive and negative feedback interactions is to create a strength- and time-dependent threshold for TCR stimulation to drive the sustained Akt activity required for the differentiation and proliferation of TH cells. The model also shows that sub-threshold TCR signals lead to transient Akt activation leading to a Treg phenotype.
An IL21 reporter mouse reveals a novel population of TFH precursors in young mice. (LYM8P.630)
Authors: Adkins Elisabeth , Wang Xulong , Sproule Thomas , Christianson Gregory , Park Giljun , Lane-Reticker Sarah Kate , Carter Gregory , Morse Herbert , Roopenian Derry 
Published: January 2023

Abstract
T follicular helper cells (TFH) are a specific subset of activated CD4+ T cells that localize to germinal centers (GC) and secrete Interleukin 21 (IL21). IL21 is vital for driving the proliferation of antigen-stimulated B cells within GC and their differentiation into memory B cells and antibody-secreting plasma cells. TFH and IL21 are crucial for normal humoral immunity, but their dysregulation has been implicated in immunodeficiencies and autoimmune diseases. However, the processes that result in the normal development of TFH and their expression of IL21 are still poorly understood. Here we use a novel IL21-venus fluorescent protein (VFP) reporter mouse to investigate the signals and pathways involved in the early differentiation of TFH and expression of IL21. Using this approach, we have identified a naturally occurring population of IL21-expressing CD44hi CD4+ T cells that are detected in the thymus and periphery of naïve C57BL/6J mice by 2 wks of age. RNAseq profiling and adoptive transfer experiments showed that these IL21-expressing cells are the earliest identified precursor (termed nascent TFH, nTFH) to the fully mature TFH state. nTFH display a highly diverse TCR repertoire, but restricting their TCR to a high affinity foreign antigen does not prevent their development. From this, we propose that nTFH arise by tonic self-antigenic stimulation and are primed for efficient differentiation to mature TFH in response to foreign or autoimmune antigen challenges.
Cryptococcus gattii infection dampens pulmonary inflammation by attenuating chemokine expression in the immunocompetent hosts (MPF6P.644)
Authors: Angkasekwinai Pornpimon , Sringkarin Nuntarat , Supasorn Oratai , Fungkrajai Madtika , Wang Yui-Hsi , Chayakulkeeree Methee , Ngamskulrungroj Popchai , Angkasekwinai Nasikarn , Pattanapanyasat Kovit 
Published: January 2023

Abstract
Cryptococcal infections are caused mainly by two fungal species; Cryptococcus neoformans and C. gattii. While C. neoformans primarily affects the immunocompromised hosts, C. gattii causes disease in immunocompetent individuals. Recent studies suggest that C. gattii is capable of infecting immunocompetent hosts because of its ability in dampening inflammatory cell recruitments. We hypothesizes that C. gattii dampens pulmonary inflammation by attenuating chemokine expression. Mice were infected with the highly virulent strains C. neoformans and C. gattii and then analyzed for kinetics of inflammatory cell infiltration and chemokine expression. Inflammatory cells and chemokine transcripts were found to be up-regulated and reached the peak levels at day 7 post-infection. Mice infected with C. gattii recruited much fewer myeloid dendritic cells, neutrophils, and CD4+ T helper cells into the lungs than those in C. neoformans-infected mice. Interestingly, the expression levels of Cxcl1, Cxcl2, Mcp1, Cxcl9, and Ip10 transcripts known to be important for recruiting neutrophil and Th1 cells were significantly higher in C. neoformans-infected mice than in C. gattii-infected mice, whereas the expression levels of Ccl20, Ccl11 and Ccl17 were comparable. Our data suggest that C. gattii infection dampens pulmonary inflammation by down-regulating the pulmonary chemokine expression, thus resulting in the inability to mount protective immunity in immunocompetent hosts.
Immunological and biochemical characterization of an immunogenic antigen (5G8) of Giardia lamblia (MPF6P.648)
Authors: Velazquez Carlos , Quintero Jael , Valdez Alejandra , Samaniego Brenda , Rascón Lucila , Calderon Boris , Rascón Raúl , Domínguez Víctor , Garibay Adriana , Astiazarán Humberto 
Published: January 2023

Abstract
The protozoan parasite G. lamblia is an intestinal parasite of humans, which the World Health Organization estimates to have infected 250 million people worldwide. Clinical manifestations of G. lamblia infections vary from the asymptomatic carrier state to severe diarrhea, abdominal pain, nausea, malabsorption and weight loss. Our knowledge about the immunogenic antigens of G. lamblia is limited. The purpose of this study was immunologically and biochemically characterize an immunogenic antigen (protein 5G8) of G. lamblia. We generated monoclonal antibodies (moAbs) spe­cific to the protein 5g8 of the parasite by using splenocytes derived from in­fected mice. All of these moAbs recognized a protein of 71 kDa by Western blotting. This protein was isolated from a G. lamblia trophozoite lysate by affinity chromatography. The purified 71 kDa protein was analyzed by mass spectrometry. This immunogenic antigen was expressed inside of the G.lamblia trofozoites (&gt;90% of total cell population) and only a small percentage (≈ 2-5%) of those trophozoites expressed high levels of the 5G8 protein on the cell surface. Trophozoite population expressing the 5G8 protein on the cell surface induced stronger antibody response that trophozoite population that does not express this protein on cell surface. The characterization of this antigen will provide a better understanding of the G. lamblia-host interaction, and will be important for developing effective control strategies against giardiasis.
CCR7+ dendritic cells are sufficient to recover a defective population of KLRG1+ T cells in the brain of chronically infected CCR7-/- mice (MPF7P.716)
Authors: Landrith Tyler , Noor Shahani , Wilson Emma 
Published: January 2023

Abstract
Toxoplasma gondii is a protozoan parasite that establishes a chronic infection in the brain. CCR7-/- mice are susceptible to challenge with normally avirulent strains of T. gondii, but if the mice are treated with sulfadiazine, a chronic infection is established. At this stage, equivalent numbers and proportions of parasite-specific T cells are seen in the brain of CCR7-/- and wild type mice suggesting normal priming is possible in the absence of CCR7. However, in contrast to their wild type counterparts, CCR7-/- mice fail to mount a protective response to rechallenge with a virulent strain, implying deficient effector mechanisms despite equivalent priming. Indeed, a profound defect is observed in the number and proportion of T cells expressing the effector marker KLRG1 in the brain of CCR7-/- mice. Here we present data showing that this defective effector population is restored upon transfer of CCR7+ dendritic cells at the chronic stage. We also present data on the role of transferred dendritic cells in survival upon rechallenge with the virulent RH strain. This data suggests a novel role for CCR7 during chronic T. gondii infection in guiding efficient dendritic cells interactions required to maintain effector populations of T cells in the brain.
Eosinophils support Th2 response and control parasite number in brain during murine neurocysticercosis (MPF7P.717)
Authors: Mishra Pramod , Li Qun , Munoz Luis , Mares Chris , Morris Elizabeth , Teale Judy , Cardona Astrid 
Published: January 2023

Abstract
Neurocysticercosis (NCC) is a common parasitic infection of the brain and leading cause of acquired epilepsy worldwide. NCC is caused by metacestode larvae of the tapeworm Taenia solium and characterized by an initial asymptomatic phase followed by a symptomatic phase. While the asymptomatic phase is attributed to the immunosuppressive capabilities of viable T. solium metacestodes, dying parasites induce inflammation including an eosinophil (EO) response causing pathology and symptoms. Previous studies in T. solium-infected pigs have shown that EOs are present around dying parasites. However, the role of EOs in killing of metacestode has not been verified. To determine the parasiticidal role of EOs in brain, we used a murine model of NCC in which wild type (WT) or EO deficient (ΔdblGATA) mice were infected intracranially. We found a strong EO response in WT NCC mice similar to porcine NCC. In comparison to WT NCC mice, EO deficient, ΔdblGATA NCC mice had an increased parasite burden. Analysis of infiltrates from infected brains revealed that ΔdblGATA mice had reduced leukocyte infiltration into the brain in comparison to WT mice. However, αβ T cell frequency was greater in ΔdblGATA mice brain. Further, higher αβ T cell frequency correlated with increased CD8+ T cell response and reduced CD4+ Th2 responses. Thus, our results show that EOs support Th2 response in brain microenvironment and play a critical role in controlling the parasite number in brain during murine NCC.
Inflammatory monocytes orchestrate innate antifungal immunity in the lung (MPF7P.701)
Authors: Espinosa Vanessa , Rivera Amariliz 
Published: January 2023

Abstract
Aspergillus fumigatus is a mold fungal pathogen and the most common cause of invasive aspergillosis (IA), a serious infection that develops in patients with compromised immune function. Although a variety of immune cells can help confer protection against IA, the mechanisms that govern immune cell cooperation culminating in the eradication of conidia and IA prevention are unclear. Using selective cell depletion strategies our recent studies have shown that, in addition to neutrophils, CCR2+ inflammatory monocytes (CCR2+Mo) and their derivative monocyte-derived dendritic cells (Mo-DCs) are required for the prevention of IA. In preliminary studies, we find that IA development in neutrophil-depleted animals is associated with diminished Mo-DC differentiation and lower conidiacidal activity in these cells. We hypothesize that neutrophil and CCR2+Mo functions are interdependent, continuously cross-regulating each other’s antifungal activities. We are currently investigating the mechanisms of CCR2+Mo and neutrophil cross-regulation, and characterizing their overall contributions to anti-fungal immunity.
MicroRNA-146a constrains intestinal immunity and increases susceptibility to DSS colitis (MUC8P.732)
Authors: Runtsch Marah , O'Connell Ryan 
Published: January 2023

Abstract
MicroRNA-146a (miR-146a) regulates multiple immunological processes via downregulation of proinflammatory pathways, and its loss or decreased expression often results in adverse chronic inflammatory phenotypes in mammals. Although miR-146a has been well studied in systemic compartments of the immune system, its role in intestinal immunity is not well understood. We have found that miR-146a is expressed in a variety of gut tissues, including the colon and small intestine. Further, we demonstrate that this miRNA represses a subset of intestinal barrier genes that include members of the Reg3 and Mucin families, adhesion molecules, IFN response genes, immunoregulatory enzymes, and cytokines, all within a network of immune-related signaling pathways that include NF-κB and Stat signaling. Moreover, miR-146a restricts the expansion of Tfh cells and GC B cells, which decreases the production of luminal IgA, in addition to reducing Th17 and Treg populations within the intestines. Consistent with an altered intestinal barrier, we observe shifts in communities of the fecal microbiota upon loss of miR-146a. Finally, we demonstrate that miR-146a-/- mice (which have a heightened barrier) are resistant to DSS-induced colitis, and find that miR-146a is elevated in colons of human IBD patients. Taken together, our data describe a role for miR-146a in constraining intestinal barrier function and find that this is consequential in the setting of intestinal diseases.
Isolation and ex vivo expansion of murine bone marrow-derived group 2 innate lymphoid cells (MUC8P.734)
Authors: Duerr Claudia , McCarthy Connor , Mindt Barbara , Fritz Jorg 
Published: January 2023

Abstract
Group 2 innate lymphoid cells (ILC2) are recently discovered lymphocyte subpopulations that have innate ability to produce large quantities of a specific set of cytokines regulating innate and adaptive immune responses. Importantly, deregulated ILC2 responses are implicated in detrimental type 2 immunopathologies such as allergies, asthma and atopic dermatitis, as well as fibrotic disease, both in rodents and humans. However, the progress in our understanding of ILC2 biology has been hampered by their scarcity at steady state as well as infected and inflamed tissues. Here, a simple, efficient and reliable method is presented to isolate murine ILC2 from the bone marrow by flow cytometric cell sorting and further cultivate them using a defined cytokine cocktail, yielding a 500-fold expansion. The resulting ILC2 constitute a pure cell population and show the same phenotypic and functional characteristics as ILC2 in vivo. Thus, this protocol enables a high yield of ILC2 from murine bone marrow and thereby provides sufficient ILC2 numbers for standard cell and molecular biology assays. An in-depth understanding of the cellular and molecular mechanisms of ILC2 is essential for the development of new strategies and drugs to fight unwanted type 2 immunopathologies and fibrotic disease.
Interleukin-22 directly regulates intestinal stem cell regeneration after tissue damage (MUC8P.722)
Authors: Hanash Alan , Lindemans Caroline , Mertelsmann Anna , van den Brink Marcel 
Published: January 2023

Abstract
Little is known about regulation of the intestinal stem cell (ISC) compartment after injury. We hypothesized that IL-22 could promote intestinal regeneration by either acting on ISCs or by acting on Paneth cells (PCs) making up the ISC niche. Utilizing an in vitro regeneration model, we found that organoids derived from murine small intestine (SI) and large intestine (LI) crypts demonstrated substantially increased size after culture with IL-22. Innate lymphoid cell co-culture also increased organoid size in an IL-22-dependent fashion. IL-22 expanded the Lgr5+ ISC pool within SI organoids, activated STAT3 phosphorylation in Lgr5+ cells, and increased size of organoids cultured from purified SI ISCs. Using a murine allogeneic bone marrow transplant (BMT) model, we found that IL-22 administration led to reduced intestinal graft vs. host disease pathology, increased maintenance of Lgr5+ ISCs, and significantly greater ISC proliferation. ISC protection post-BMT was not due to niche augmentation, as PC numbers, PC-derived growth factors (EGF, Wnt3), and stroma-derived growth factors (R-spondin3) were all unchanged after IL-22 administration. IL-22 receptor (IL-22R) staining further indicated a direct effect of IL-22 on ISCs rather than the niche, as PCs were found to express very little IL-22R and demonstrated no evidence of STAT3 phosphorylation upon in vitro culture. In summary, we found that IL-22 regulates epithelial regeneration after tissue damage by acting directly on ISCs.
Foxp3+ regulatory T cells restrain pathogenic RORγt+ group 3 ILC-mediated colitis (MUC8P.728)
Authors: Joyce-Shaikh Barbara 
Published: January 2023

Abstract
IL-10 receptor beta (IL-10Rb) is a common receptor chain for both IL-10 and IL-22. Since IL-10 is a protective cytokine that restrains inflammatory responses and IL-22 signaling is required for epithelial cell-dependent tissue repair, targeted deletion of Il10rb should greatly exacerbate inflammatory bowel disease (IBD). Unexpectedly, we found that Il10rb-/- mice are completely resistant to development of spontaneous colitis. In contrast, Il10-/- mice develop IBD by 12 weeks of age and harbor a large number of IL-22-producing RORgt+ innate lymphoid cells (ILCs). Using a CD45RBhi T cell transfer model, we found that induction of intestinal inflammation requires the presence of gut resident RORgt+ ILCs. The IL-22-producing ILCs are essential to perpetuate the disease process initiated by the T cells. Furthermore, Foxp3+ Tregs directly inhibit ILC expression of IL-23R and other tissue injury sensors, thereby suppressing innate cell-driven colitis. Our study has uncovered a novel immune network that maintains intestinal homeostasis.
Bacillus subtilis exopolysaccharide suppresses mast cell responses (MUC9P.754)
Authors: Swartzendruber Julie , Knight Katherine 
Published: January 2023

Abstract
Commensal bacteria are being studied for their probiotic, or beneficial effects in a number of diseases. Probiotics have been proposed as a therapy for food allergy but the mechanism and efficacy of how probiotics may be preventing disease are not understood. We previously showed that we can limit diarrheal disease caused by the enteric pathogen Citrobacter rodentium using the probiotic Bacillus subtilis. In addition we isolated the protective molecule, exopolysaccharide (EPS) and shown EPS alone is sufficient for protection of disease. Food allergy and the anaphylactic response following allergen exposure are mediated by mast cell degranulation. We have evidence that EPS can suppress mast cell-dependent degranulation and anaphylaxis. Therefore, we hypothesize that EPS protects against anaphylaxis through the suppression of mast cell activation. Identifying the mechanism by which EPS prevents anaphylaxis will provide the first safe and effective therapeutic use of a probiotic for the treatment of food allergy.
Mechanism by which commensal exopolysaccharides limit inflammation (MUC9P.744)
Authors: Paynich Mallory , Jones Sara , Knight Katherine 
Published: January 2023

Abstract
Trillions of bacteria live within the gastrointestinal tract and are critical for maintaining intestinal homeostasis; however, the mechanisms utilized by specific bacterial molecules to contribute to homeostasis are not well understood. We utilize a mouse model in which a single dose of the probiotic, B. subtilis, protects mice from acute colitis induced by the enteric pathogen C. rodentium. Our goal is to elucidate the mechanism by which B. subtilis prevents inflammation. We identified exopolysaccharides (EPS) to be the active molecule of B. subtilis, and a single dose of EPS protects mice from disease. EPS specifically binds F4/80+CD11b+ peritoneal macrophages (MΦ). Following EPS treatment, these cells increase expression of CD206+Arg1+YM-1+IL-4Rα+ and inhibit T cell activation and proliferation in vitro, indicative of M2 MΦ. CD4+ T cells drive much of the inflammation associated with C. rodentium infection, and we hypothesized that EPS inhibits CD4+ T cell responses in vivo. Accordingly, we measured levels of IFNγ (Th1), IL-17 (Th17), and IL-13 (Th2) in splenic T cells following EPS treatment and found decreased levels of these cytokines. Similarly, mesenteric lymph node from mice orally gavaged with B. subtilis spores produced decreased levels of T cell cytokines in an EPS dependent manner. We hypothesize that EPS-induced M2 MΦ mediate inhibition of CD4+ T cell responses in vivo and further, that these cells mediate protection from acute colitis induced by C. rodentium.
Generation of antigen-specific monoclonal antibodies by in vitro immunization (TECH2P.904)
Authors: Heilmann Katja , Holzlöhner Pamela 
Published: January 2023

Abstract
Monoclonal antibodies are one of the most important tools in biomedicine but the generation by hybridoma technology is very time consuming and laborious. The adaptation of the process to an in vitro approach is therefore a smart alternative to reduce the disadvantages of hybridoma technology. In this study a protocol for in vitro immunization was developed which enables hematopoietic stem cells to differentiate into naïve dendritic cells. These cells were specifically activated with the antigen of interest and cocultivated with naïve B and T lymphocytes in order to generate antigen specific B lymphocytes. After 4 days the cells were fused with myeloma cells and antigen specific hybridomas were selected. The produced antibody was purified and characterised in comparison to a conventional monoclonal antibody reacting against the same antigen. Further this protocol could be adapted to human cells and the generation of antigen specific hybridoma cells secreting human IgG molecules could be established. The developed protocols allowed a generation of antigen specific hybridoma cells of murine and human origin within 3 weeks. Therefore it seems that the immunization in vitro is useful for avoiding the disadvantages of conventional hybridoma technology.
Measuring autophagic flux by assessing LC3, p62 and LAMP1 co-localization using imaging flow cytometry (TECH2P.908)
Authors: Pugsley Haley , Friend Sherree , Kong Raymond , Vaidyanathan Shobana , Morrissey Phil 
Published: January 2023

Abstract
Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome; during starvation it is a survival mechanism that reallocates nutrients from unnecessary processes to more vital processes in the cell. In autophagy, cytoplasmic LC3 protein is processed and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to cause the breakdown of the autophagosome vesicle and its contents. The ubiquitin-associated protein p62 which binds to LC3 can also be used to measure autophagy. p62 is an important marker for the induction of autophagy, clearance of protein aggregates and the inhibition of autophagy. In addition, p62 positive cytoplasmic inclusions have been found in several neurodegenerative diseases. Immunofluorescence microscopy has been used to visually identify LC3 puntca, p62 and/or lysosomes on a per-cell basis; however, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, we employed the ImageStreamX Mark II imaging cytometry platform. We collected large numbers of cell images to assess autophagy and co-localization of LC3, p62 and the lysosomal marker LAMP1 in an objective, quantitative, and statistically robust manner. Defects in autophagic flux have been linked to immune-mediated diseases; we believe this technique may bring insight to these diseases.