bluuebunny/crossref_metadata_2025 huggingface dataset:
The dietary component, curcumin, inhibits the proliferation, survival and function of bone-marrow derived mast cells (P6032)
Authors: Mathias Clinton , Shah Sagar , Statham Rebecca , Kinney Shannon
Published: January 2023

IgE antibodies and mast cells play critical roles in the establishment of allergic responses. Curcumin, the active ingredient of the naturally occurring curry spice turmeric, has anti-allergic properties, both in vitro and in vivo, and thus may have the capacity to modulate mucosal mast cell activation and function during allergic responses. Here, we show that curcumin inhibits the proliferation, survival and function of bone-marrow derived mast cells (BMMCs). BMMCs were grown in the presence of IL-3+SCF or monomeric IgE for 6 days. Co-culture with curcumin completely inhibited the proliferation and long-term survival of BMMCs, suggesting that curcumin has suppressive effects on mast cell expansion. Similarly, curcumin also suppressed the proliferation of peritoneal mast cells. Flow cytometric analysis of the BMMCs demonstrated that curcumin-treated BMMCs were undergoing apoptosis, as indicated by expression of Annexin V and propidium iodide. Intracellular cytokine staining further suggested that these BMMCs produced lower levels of TNF-α. Finally, curcumin-treated BMMCs, activated in the presence of antigen, exhibited reduced β-hexosaminidase activity in comparison with vehicle-treated controls. Our data demonstrate that dietary components such as curcumin can regulate the expansion, activation and function of mast cells, and thus may affect the outcome of mast-cell-dependent allergic responses in vivo.
Quantifying the unanticipated diversity of the human NK cell repertoire (P1456)
Authors: Strauss-Albee Dara , Horowitz Amir , Dogan Ozge , Mackey Sally , Swan Gary , Dekker Cornelia , Davis Mark , Parham Peter , Blish Catherine
Published: January 2023

Like their T and B lymphocyte counterparts, natural killer (NK) cells exist in small and specialized subpopulations. However, while T and B cell repertoire diversity is generated primarily through DNA rearrangement to produce a single antigen-specific receptor, the diversity of the NK cell repertoire is determined by the expression of a spectrum of activating and inhibitory receptors. To evaluate the human NK cell repertoire on a single-cell basis, we simultaneously measured more than 25 NK cell receptors in the peripheral blood of 22 healthy individuals via mass cytometry. We used Boolean gating to classify cellular phenotypes. Based on the Simpson index, an ecological measure designed to quantify population diversity, we found that total NK cell repertoire diversity was approximately normally distributed across individuals. Using rarefaction curves and non-parametric species estimators, we calculate the expansiveness of the NK cell repertoire at a minimum of 100,000 unique phenotypes. Furthermore, within an individual, the ex vivo addition of the homeostatic cytokine IL-15 biased the repertoire toward highly common and uncommon phenotypes, increasing the total population richness but decreasing the overall diversity. These data show, for the first time, that the receptor-based NK cell repertoire is exquisitely diverse. They further suggest an optimal baseline diversity that is highly sensitive to optimization by exogenous factors.
Transgenic mice expressing functional CD8αβ without an active CD8αα implies the contribution of CD8αα in the biological function of CD8αα+TCRαβ+ iIELs (P1466)
Authors: Kang Yoon-Joong , Chang Hsiu-Ching
Published: January 2023

To understand the functional significance of CD8αα complexes in vivo, we generated Tg mice carrying a variant CD8αβ (CD8αm3β) capable of forming inactive CD8αα/pMHCI complexes. These mice show a sub-optimal thymic differentiation with reduced populations of CD8 single-positive thymocytes. Tg CD8 T cells exhibit a compromised developmental capacity when competing with CD8 T cells from B6 mice in the adoptive bone marrow transferred host. However, once emigrated to the peripheral lymphoid organs, these CD8 T cells exhibit normal effector function against viral infection. Furthermore, TCRαβ+ T cell populations expressing CD8αβ+ or CD8αα+ reduced to about 50% in our Tg mice compared to normal B6 mice, and none of these CD8αα+TCRαβ+ T cells were stained by TL tetramer. Furthermore, this mice shows reduced infmmatory responses in DSS-induced colitis model. The findings obtained from our Tg animal models are going to be important to dissect the distinct functions of CD8αα molecule.
The microbiota-derived signal indole regulates T-cell fate by reciprocal control of Th17 and Treg development (P3178)
Authors: Katepalli Madhu , Mueller Carrie , Steinmeyer Shelby , Jayaraman Arul , Alaniz Robert
Published: January 2023

The presence of a normal microbiota and immune regulation in the gut limits the onset of several inflammatory disorders. However, the specific microbiota mechanisms or chemical effectors that promote homeostasis are largely unknown. Previously, we reported that indole, a microbiota-derived (not host derived) tryptophan-metabolite, attenuates indicators of inflammation in intestinal epithelial cells. In addition, we show that indole treatment rescues mice from colitis in vivo. Based on our results, we hypothesized that indole influences mucosal CD4 T-cell function. Because Foxp3+ regulatory T cells (Tregs) and pro-inflammatory Th17 cells are key players in the balance of gut homeostasis, we tested the impact of indole on Treg and Th17 development. Here, we reveal that indole regulates Treg/Th17 lineage fate by dramatically augmenting TGF-β-induced Treg expansion, function, and stability. Interestingly, indole also promoted Treg Foxp3 expression independent of TGF-β or IL-2 suggesting a novel mechanism for the induction of Foxp3 expression in CD4 T cells. In a reciprocal fashion, indole inhibited Th17 development via STAT3 and RORgt pathways and decreased IL-17 production. These data reveal a novel mechanistic paradigm on how the microbiota influence mucosal immunobiology and establish the metabolite indole as a potent regulator of Treg and Th17 cell balance. Moreover, our results suggest microbiota-derived metabolites offer a rich pool of potent immunomodulatory compounds.
A role for microbiota metabolites in generation of mucosal dendritic cells (P3160)
Authors: Mueller Carrie , Katepalli Madhu , Steinmeyer Shelby , Jayaraman Arul , Alaniz Robert
Published: January 2023

The gut mucosa normally exhibits tolerance towards the commensal microbiota by active suppression of inflammation. When homeostasis is disrupted, inflammatory bowel diseases may develop, during which microbiota dysbiosis may also occur. Resulting chronic inflammation induces loss of intestinal epithelial integrity, gastrointestinal (GI) distress, and colon cancer risk. We hypothesize that under normal conditions, the microbiota produce beneficial compounds that promote homeostasis. Previously, we reported that indole exerts immunomodulatory and anti-inflammatory effects on intestinal epithelial cells (IECs). We propose that indole, an abundant, freely diffusible, and strictly microbiota-derived molecule in the GI tract, conditions IECs and immune cells for optimal gut functions. Here, we report that indole potently modulates dendritic cell (DC) responses, which are central regulators of gut homeostasis. Specifically, indole-conditioned DCs produce decreased pro-inflammatory cytokines after microbial activation, and indole strongly synergizes with TGF-β for this effect. In addition, indole regulates the expression of the mucosal DC markers CCR9, B220, and aldh1a2. Furthermore, indole-treatment rescues host inflammation in a murine model of colitis. Given its availability and immunomodulatory properties, we suggest indole is a member of a novel class of microbiota compounds that regulate gut immune cells and may be a potential treatment of inflammatory bowel disease.
Gut immunity and microbiome: from defense to metabolism (P3168)
Authors: Fu Yang-Xin , Upadhyay Vaibhav
Published: January 2023

We have demonstrated that lymphotoxin (LT) from innate lymphoid cells (ILC) is essential for innate IL-22 production against Citrobacter rodentium infection (CR). By using a conditional gene targeting approach, we demonstrate that an LT-driven positive feedback loop controls IL-22 production by ROR + ILCs via LTβR signaling in DCs. Intriguingly, LT-controlled immune responses also control gut flora that is required for more efficient energy saving and fat accumulation in diet-induced obesity (DIO) and type II diabetes. The ability to transfer obesity phenotype through the transplantation of microbiota to germ free mice and through co-housing of wild type and lymphotoxin deficient mice confirm the essential role of host responses in diet-induce immunity for obesity and diabetes. Furthermore, HFD promotes host immune responses that reshape gut flora. Sequencing of cecal DNA and subsequent metagenomic analysis revealed gene profiling which adapts to the obese state and contributes to weight gain. This study has paved a way to explore whether and which innate or adaptive respond for DIO in LT dependent fashion and which microbiota are essential for DIO in LT dependent way. This opens new avenue to study how immune system regulates gut flora for DIO and set new model to identify the nature of obese microbiome.
Macrolides suppress key features of experimental steroid-sensitive and steroid-resistant asthma (P6229)
Authors: Hansbro Philip , Horvat Jay , Essilfie Ama-Tawiah , Kim Richard , Mayall Jemma , Starkey Malcolm , Foster Paul
Published: January 2023

Steroid-resistant asthma is an important clinical problem and effective therapies are urgently required. Macrolides have been shown to be efficacious in treating steroid-resistant forms of asthma. However, how these immunomodulatory antibiotics induce their effects is not known. Using mouse models of Chlamydia and Haemophilus lung infection and ovalbumin-induced allergic airway disease (AAD) we have previously shown that both infections induce airways hyperresponsiveness (AHR) and neutrophilic inflammation in AAD that is resistant to steroid treatment. In the current study the effects of clarithromycin versus amoxicillin on immune responses in Th2-associated, steroid-sensitive eosinophilic and infection-induced, Th1/Th17-associated, steroid-resistant neutrophilic AAD was assessed. We show that clarithromycin, but not amoxicillin, treatment reduces AHR and inflammation in both steroid-sensitive and steroid-resistant AAD. Suppression of these features of disease was associated with a reduction in a number of Th1, Th2 and Th17-associated immune factors. Significantly, clarithromycin treatment reduced Th2-associated factors in steroid-sensitive AAD and Th1/Th17-associated factors in infection-induced, steroid-resistant AAD. These findings suggest that macrolide treatment can have broad anti-inflammatory effects on immune responses in the asthmatic lung and that the specific immune responses that are suppressed may be dependent on the responses that predominate during disease.
Study of the inverse correlation between the level of T regulatory cells and IgE during a type 1 hypersensitivity reaction in the equine model. (P6235)
Authors: Behrens Nicole , Gershwin Laurel
Published: January 2023

Recently there has been an increase in allergic responses to vaccination in horses. Since many viral vaccines share contaminating non-target proteins that can act as an antigen to elicit IgE, we compared T regulatory cell % and IgE levels pre and post vaccination. We hypothesize: high IgE responding horses to BSA have low levels of specific T regulatory cells. Furthermore high IgE responders produce IL-4, IL-10 and TGF-β, which stimulate a T helper 2 response and inhibit a T helper 1 response; a strong IFN-γ response, which stimulates a T helper 1 response and inhibits a T helper 2 response, occurs in non IgE responders. Sixteen horses were evaluated pre vaccination, and 7 days post for T regulatory cell production and specific IgE. Peripheral blood mononuclear cells isolated pre and post vaccination analyzed for expression of CD4 and CD25, and intracellular Foxp3 with multicolor flow cytometry. IgE was evaluated by ELISA. A T regulatory cell recall response was examined by in vitro lymphocyte stimulation one-month post vaccination to re-stimulate T regulatory cells. Supernatant was analyzed for cytokine levels. Levels of IL-4, were higher in horses with high IgE as compared to horses with low IgE; while IFN-γ was higher in horses with low IgE responses as compared to horses with high IgE responses. In conclusion horses with high IL-4, and low IFN-γ have less T cell down-regulation and produce a T helper 2 response leading to high IgE production and allergic reactions.
An experimental model for steroid-refractory immune response in severe asthma (P6216)
Authors: Raundhal Mahesh , Morse-Zinger Christina , Oriss Timothy , Khare Anupriya , Krishnamoorthy Nandini , Wenzel Sally , Ray Prabir , Ray Anuradha
Published: January 2023

Asthma is a disease of the airways caused by a dysregulated immune response. Although for the most part, asthma can be controlled by inhaled corticosteroids (CS), severe asthma (SA) is difficult to control by either inhaled or systemic CS. It is becoming increasingly apparent that the most severe and poorly controlled form of SA displays mixed neutrophilic/eosinophilic airway inflammation, often displaying neutrophil dominance. The lack of animal models for SA has limited our understanding of mechanisms governing steroid refractoriness in SA as well as the development of alternatives to CS therapy. Our analysis of human asthma samples has shown that unlike patients with milder disease, SA patients consistently have a mixed Th1/Th17 immune profile in their airways with residual Th2 responses despite high dose CS therapy. With the association of viral and bacterial infections with asthma exacerbations, we hypothesized that the combination of an allergen with agents that mimic infection would induce a SA phenotype in mice. We would present a murine model of SA in which the induced immune response resembles that in severe asthmatics. As in humans, this immune response elicits mixed granulocytic airway inflammation and airway hyperresponsiveness that is poorly responsive to CS. This model would allow us to understand interactions between innate and adaptive immune responses that induce the SA phenotype. Additionally, the model would be amenable to testing novel therapies for SA.
NRROS negatively regulates the production of reactive oxygen species in phagocytes during host defense and autoimmunity (P1339)
Authors: Noubade Rajkumar , Wong Kit , Ota Neko , Rutz Sascha , Valdez Patricia , Peng Ivan , Sebrell Andrew , Caplazi Patrick , DeVoss Jason , Soriano Robert , Modrusan Zora , Hackney Jason , Sai Tao , Ouyang Wenjun
Published: January 2023

Reactive oxygen species (ROS) produced by phagocytes are essential for host defense against bacterial and fungal infections. Individuals with defects in the ROS production machinery develop chronic granulomatous disease. On the other hand, ROS can cause collateral tissue damage during inflammatory processes and therefore needs to be tightly controlled. Here we describe a novel protein that limits ROS generation by phagocytes upon inflammatory stimuli. We named this protein as Negative Regulator of ROS (NRROS). NRROS is a previously uncharacterized leucine rich repeat containing protein expressed mainly in myeloid cells. NRROS controls protein expression and/or stability of NOX2 and p22phox, the membrane-bound subunits of the NADPH oxidase complex. NRROS deficient cells produce increased ROS upon inflammatory challenges. In vivo, NRROS deficient mice show higher capacity to control invading bacteria such as Escherichia coli and Listeria monocytogenes. Conversely, these mice develop severe experimental autoimmune encephalomyelitis, accompanied with significant mortality, due to oxidative tissue damage in the central nervous system. Interestingly, NRROS expression is differentially regulated by inflammatory stimuli. Tissue necrosis factor-α but not interferon-γ suppresses NRROS expression in macrophages. NRROS, thus, provides a hitherto undefined mechanism for ROS regulation; one that enables phagocytes to control invading pathogens while minimizing collateral tissue damage.
Involvement of DNA-PKcs in the pro-inflammtory cytokine and type I IFN response to CpG-ODN in dendritic cells is mediated by its interaction with TRAF6 (P1359)
Authors: Chu Wen-Ming , Ma Chi , Muranyi Marianna , Chu Catherine , Zhang Jianhua
Published: January 2023

CpG-ODN stimulates dendritic cells (DCs) to produce cytokines, which are important for autoimmune disorder development and vaccine strategy for cancer. CpG-ODN activates the TLR9/TRAF6 cascade leading to IKK-NF-κB and IKK-IRF7 activation, which is critical for production of pro-inflammatory cytokines and the type I IFNs, respectively. However, whether other molecules are involved in activation of CpG-ODN signaling is still unclear. Here we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is involved in this activation process. DNA-PKcs-deficient DCs exhibited a defect in the IL-6 and IL-12 response to CpG-ODN in a dose- and time- dependent manner. Also, loss of DNA-PKcs impaired the IFNa response to CpG-ODN and blockade of IFNR signaling further reduced the IL-6 and IL-12 response to CpG-ODN in DNA-PKcs deficient DCs. Additionally, loss of DNA-PKcs impaired phosphorylation of IKKa, IκBα, NF-κB, JNK, IRF7 and STAT1 in response to CpG-ODN. Interestingly, TLR9- and DNA-PKcs/TLR9- deficient DCs showed almost no IL-6 and IL-12 response to CpG-ODN, but they exhibited about 2/3 to 3/4 defect in the IFNa production compared to WT controls. Finally, CpG-ODN bound DNA-PKcs in the absence of TLR9, whereas it induced DNA-PKcs’s association with TRAF6 in a TLR9-dependent manner. Our data suggest that DNA-PKcs is a player in CpG-ODN signaling and may explain why DNA-PKcs is implicated in the pathogenic process of autoimmune disease.
Impact of altered gravity on the ROS-signalling in macrophages (P1293)
Authors: Brungs Sonja , Kolanus Waldemar , Hemmersbach Ruth
Published: January 2023

Astronauts suffer from a higher susceptibility to infections during spaceflights. Different stressors could lead to a diminished immune defense. Macrophages present the first line of defense of the innate immune system. Their main challenge is the recognition, engulfment and destruction of pathogenic bacteria, viruses and dead cells. The killing of bacteria is accomplished by the production of Reactive Oxygen Species (ROS) during oxidative burst. We investigated the effects of altered gravity on the pattern recognition and ROS production of macrophages. Our results show that real microgravity (parabolic flight), simulated microgravity (2D fast-rotating clinostat) as well as hypergravity (centrifuge) alter the production of ROS after stimulation of cell surface receptors Dectin, TLR2/6, complement and Fcγ. We also find that phosphorylation of the tyrosine kinase Syk, an essential link between pattern recognition, cytoskeleton and ROS production, is sensitive to microgravity. We can conclude that both microgravity and hypergravity cause very fast changes in ROS production, characterising the oxidative burst as a gravisensitive processes. Altered gravity might have impact on the interaction of different pattern recognition signalling pathways, which are crucial for the bacteria clearance in terms of ROS production. Therefore, the study of ROS signalling under altered gravity is not only of interest for humans in space, but also for human health on Earth.
Catecholamines promote an M2 macrophage activation phenotype (P1299)
Authors: Grailer Jamison , Haggadone Mikel , Ward Peter
Published: January 2023

Based on their function, activated macrophages are categorized as having M1 or M2 phenotypes. M1 macrophages are pro-inflammatory and mediate host defenses against pathogens. M2 macrophages promote immunosuppression and wound healing. Here, we report significant alterations in activated macrophage phenotypes by catecholamines. Epinephrine and norepinephrine dramatically reduced TNF production by LPS-stimulated mouse macrophages in vitro, with IC50s of 5.5 and 700 nM, respectively. Co-treatment of macrophages with LPS and either catecholamine promoted an anti-inflammatory phenotype, including increased IL-10 mRNA and protein production. Interestingly, in LPS-stimulated macrophages, co-presence of either catecholamine reduced iNOS (a marker for M1 macrophages) mRNA by 75-81%, while Arginase (M2 marker) mRNA expression was enhanced by 8.3 to 13.5-fold. Therefore, catecholamines promoted an M2-like macrophage activation phenotype. This effect was determined to be mediated through the beta-2 adrenergic receptor and the phosphoinositol 3-kinase pathway. Catecholamines modulated both MyD88-dependent and MyD88-independent signaling through several different TLRs. In rodent models of acute lung injury, co-administration of a beta-2 adrenergic receptor agonist reduced inflammatory mediator production and prevented injury. These data have significant clinical implications for diseases ranging from allergy and asthma to sepsis.
Transcription factor Oct1 (Pou2f1) and its coactivator OCA-B (Pou2af1/OBF-1) are critical for CD4 memory T cell formation and function in vivo and in vitro (P1132)
Authors: Tantin Dean , Williams Matthew , Shakya Arvind
Published: January 2023

Stable epigenetic changes occurring during T cell activation are crucial for the generation of immunological memory. Our prior work indicates that a widely expressed transcription factor, Oct1, is required to generate a poised gene expression state at the Il2 locus in resting but previously activated primary CD4 T cells. A known Oct1 coactivator, OCA-B, is not expressed in naïve CD4 T cells or within 6 hr after activation, but is expressed 48 hr after T cell activation. We find that OCA-B is also required to generate a poised gene expression state at Il2. Using in vivo pathogen infection models together with Oct1 and OCA-B loss of function alleles, we find that both Oct1 and OCA-B are critical for the generation of functional CD4 memory T cells. The mechanism underlying this phenotype involves Oct1 and OCA-B recruitment of a specific histone lysine demethylase, KDM3A/Jmjd1a, to maintain chromatin at target loci in a configuration that allows target genes to be rapidly and strongly induced upon antigen re-encounter.
Optimal CD8 T cell integrin function and differentiation, but not proliferation, are dependent on ADAP (P1138)
Authors: Fiege Jessica , Shimizu Yoji
Published: January 2023

Efficient T cell activation through the TCR is dependent on stable integrin-mediated adhesion to an antigen-presenting cell (APC) and intracellular signaling that promotes T cell expansion and differentiation. ADAP is a multifunctional scaffold that coordinates the formation of signaling complexes that promote TCR-mediated activation of LFA-1, NFκB and JNK. CD4 T cells lacking ADAP exhibit defects in integrin-dependent adhesion and T cell proliferation in vitro and in vivo. We demonstrate similar defects in integrin-mediated contacts between CD8 T cells and APCs. Unlike CD4 T cells, we do not observe any impairment in proliferation or clonal expansion of ADAP-deficient CD8 T cells following antigen challenge. In addition, experiments examining the T cell response to Listeria monocytogenes identify a previously unappreciated role for ADAP in CD8 effector T cell differentiation. These findings support a role for ADAP in regulating CD8 T cell differentiation during an immune response and highlight inherent differences between CD4 and CD8 T cells during T cell activation.
Early growth response genes -2 and -3 are required for naïve T cell activation and primary response against virus infection (P1150)
Authors: Wang Ping , Miao Tizong , Symonds Alistair , Li Suling
Published: January 2023

T cells robustly respond to virus infection. Egr-2 and -3 are induced in both naïve and tolerant T cells (PMC2652694, PMID15585857, PMID15834410). The deficiency of Egr-2 and -3 specifically in B and T cells results in autoimmunity (PMID23021953). It is however unknown whether they also play roles in protective immmunoresponses. We have now shown that Egr-2 and -3 are required for naïve T cell activation in response to virus infection. Defect in Egr-2 and -3 restrains the IL-2 production and the expansion of viral specific CD4 and CD8 T cells, leading to the development of chronic infection despite active phenotypes and excessive production of IFNg. The induced Egr-2 and -3 in naïve T cells are essential for the maintenance of optimal AP-1 activation and IL-2 production leading to the rapid expansion of viral specific T cells. In addition to the proliferation of viral specific T cells, Egr-2 and -3 control the production of inflammatory cytokines and the activation phenotypes of viral specific T cells at early stages of infection to limit inflammatory pathology and build a pool of responding T cells for a robust anti-viral effect with minimum immunopathology. The functions of Egr-2 and -3 are limited in naïve T cells in primary immunoresponses and are not involved in the development of the memory pool and secondary responses. In conclusion, in addition to the maintenance of immunohomeostasis, Egr-2 and -3 are important to primary immune responses against virus infection.
Human Fc receptor-like 5 interacts with key signaling proteins in B cells (P1116)
Authors: Franco Andrea , Damdinsuren Bazarragchaa , Dement-Brown Jessica , Li Huifang , Tolnay Mate
Published: January 2023

B cell response is modulated by an array of co-receptors which transduce activating and/or inhibitory signals. The balance of these stimuli will determine cell function and fate. Human Fc receptor-like 5 (FCRL5) is a transmembrane protein containing immunoglobulin superfamily domains and is expressed by distinct subsets of B cells. The functional role of FCRL5 is poorly understood. However FCRL5 has been reported to be upregulated in several B cell tumors, including hairy cell leukemia, mantle cell lymphoma and multiple myeloma. The cytoplasmic tail of FCRL5 has two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and one predicted imunoreceptor tyrosine-based activation motif (ITAM). Both ITIMs were shown to interact with the protein phosphatase SHP-1 upon co-ligation of the B cell antigen receptor (BCR) and FCRL5, inhibiting calcium mobilization and tyrosine phosphorylation. Using Rec-1 FCRL5-positive cells and Daudi cells which have been transfected to stably express FCRL5, we investigated signaling components and pathways engaged upon FCRL5 stimulation. We found that FCRL5 forms protein complexes with phospholipase C (PLC) gamma 2 and CD19. CD19 is a well established positive regulator of BCR signaling, whereas PLC gamma 2 is critical for calcium flux in B cells. Therefore our findings could be part of the mechanism by which FCRL5 regulates B cell activation through signal transduction and calcium mobilization.
Effects of maternal silver acetate exposure on immune biomarkers in a rodent model (P1030)
Authors: Babu Uma , Balan Kannan , Bigley Elmer , Pereira Marion , Black Thomas , Olejnik Nicholas , Keltner Zachary , Scott Michael , Sprnado Robert
Published: January 2023

The bacteriocidal effects of silver mixtures are well characterized and as a consequence they are used worldwide in many food contact substances. However, the long-term effect of silver ion exposure on the developing immune system has not been well studied. In this study, 50 d old male and female rats were exposed to 0.4, 4 or 40 mg/Kg body weight silver acetate (AgAc) in drinking water for 10 wk prior to mating and during the 3 week mating period. Sperm positive females remained within their dose groups and were exposed to AgAc during gestation and lactation. Splenic and thymic lymphocyte subsets from postnatal (PN) 4 and 25 d old pups were assessed by flow cytometry for changes in immune biomarker profile. Functional indices included natural killer (NK) activity and mitogen-induced lymphocyte proliferation. Spleens from PN 4 d pups had lower percentages of CD8+ lymphocytes in 4 and 40 AgAc groups; lower CD3 in 40 AgAc group, and reduced Con A response in all 3 AgAc groups. In PN 25 d pups, splenocytes from 40 mg AgAc fed rats had a higher B cell population. However, Con A and LPS mediated lymphocytic responses were significantly reduced in this group. Further, splenic NK activity was higher in PN 25 d old pups exposed to 0.4 AgAc diet. In conclusion, maternal exposure to AgAc had a significant impact on rat immune system development especially in the early lactation period.
SNARE protein requirements for cytotoxic T lymphocyte-mediated cell killing (P1032)
Authors: Giraudo Claudio , Spessott Waldo , McCormick Margaret
Published: January 2023

Cytotoxic T Lymphocytes (CTLs) and Natural Killer (NK) cells selectively kill virally infected or cancer cells by releasing the content of lytic granules at the contact area with target cells called immunological synapse (IS). SNAREs mediate the maturation and exocytosis of “fully-armed” lytic granules at the IS. Deregulation of SNARE-mediated fusion events, such as in Familial Hemophagocytic Lymphohystiocytosis-4 and -5, in which Syntaxin11 and Syntaxin Binding Protein-2 are mutated, respectively; severely impaired lytic granule release and cytotoxicity of CTL and NK-cells. We have identified cognate SNAREs that specifically interact with Syntaxin11 to perform its function during CTL activation and lytic granule release. Biochemical experiments showed that these interacting proteins can form stable SNARE complexes and confirmed the specificity of these interactions. Stimulated Emission Depletion (STED) Superresolution microscopy studies revealed two pools of Syntaxin 11 in CTLs, one localized to a M6PR-containing vesicular compartment, and one localized at the plasma membrane. We have precisely visualized the localization patterns Sytanxin11-interacting partners and other membrane trafficking proteins involved in this process. These results support the conclusion that the identified Syntaxin11-containing SNARE complex plays a crucial role during CTL-mediated cytotoxicity and FHL pathophysiology.
Expression of redox-regulating molecules rather than mitochondrial biogenesis correlate with susceptibility to apoptotic stimuli in human CD62L T cell subsets (P1034)
Authors: Kesarwani Pravin , Al-Khami Amir , Kaur Navtej , Fang Quan , Naga Osama , Beeson Gyda , Johnson Christina , Mayer Elizabeth , Beeson Craig , Nishimura Michael , Mehrotra Shikhar
Published: January 2023

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells) and exhibit impaired in vivo persistence and anti-tumor efficacy, compared to T cells with central memory-like phenotype (TCM cells). However, the intrinsic and metabolic factors that attribute to this differential outcome of T cell subsets are not well understood. Here we show that CD62Lhi TCM-like T cells exhibited increased expression of cell surface sulphydryl groups (-SH; thiols), compared to TEM phenotype like CD62Llo TEM-like T cells. This increase in thiols coincided with an up-regulation of the redox-regulating anti-oxidant proteins catalase, SOD1, NRF2 and inversely correlated to the generation of ROS, RNS, extent of T cell proliferation and glycolytic enzymes. Further, we observed that T cells pretreated with rapamycin, a drug that favors the generation of a CD62Lhi TCM-like phenotype of T cells and renders reduced susceptibility to apoptosis, also up-regulated thiol expression and level of anti-oxidative genes. Conversely, thiol donor L-NAC pretreatment reduced mTOR activation. However, both rapamycin and L-NAC treated T cells showed a decrease in oxygen consumption rate, key glycolytic enzymes and expression of PGC1α, a key regulator of mitochondrial biogenesis. These data suggest that modulation of anti-oxidant capacity, mitochondrial metabolism and glycolytic pathway of T cell subsets could increase persistence and improve immunotherapy.
Elucidating immunological synapse organisation: a CAZ-protein study (P1036)
Authors: Maier-Peuschel Monika , Pattu Varsha , Krause Elmar , Rettig Jens
Published: January 2023

The highly adaptive and sophisticated network of our immune system ensures a potent defense against a variety of possible threats. As a part of this machinery, cytotoxic T lymphocytes (CTLs), upon antigen recognition, are inducing programmed cell death by forming an immunological synapse (IS) between CTL and target cell, followed by release of granzyme B and perforin from lytic granules into the synaptic cleft. Whereas some mechanisms underlying the fusion of lytic granules with the plasma membrane have already been determined, temporal and spatial organization of these vesicles right before their fusion at the IS still remains elusive. From chemical synapses in the hippocampus it is known, that vesicle fusion occurs only at the active zone which is being formed by an exclusive number of CAZ (cytomatrix of the active zone) proteins like the 420 kDa protein bassoon. Our qRT-PCR data from primary human and murine CTLs show that CAZ proteins are not only expressed, but are specifically upregulated in activated CTLs, suggesting a possible role during the killing process for example as scaffolding proteins. As specific target killing is a process of paramount importance for survival, it seems likely that vesicle fusion at the IS is precisely organized as well. We present data from primary CTLs derived from CAZ-deficient mice that investigate a possible contribution of CAZ proteins in the organisation and function of the IS.
Protective function of FoxP3+ regulatory T cells in experimental necrotizing enterocolitis (P1010)
Authors: Tran Dat , Dingle Bridgette , Liu Yuying , Fatheree Nicole , Rhoads J Marc
Published: January 2023

Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3+ regulatory T cells (Tregs) are central to gut homeostasis. While Tregs are reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their function in regulating experimental NEC induction. Utilizing a rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC (p=0.002). Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35% (p<0.05). The Tregs suppressed the upregulation of costimulatory molecule CD80 on dendritic cells and inhibited the trafficking of effector cells from leaving the MLN. These findings suggest that there is a developmental delay in Tregs which contributes to the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the induction of NEC by regulating DCs and modulating effector cell trafficking. Treg immunotherapy has a therapeutic potential in the prevention of NEC.
Use of a recombinant miRNA expressing influenza A virus to modulate SOCS1 expression (P6166)
Authors: Stambas John , Jenkins Kristie , Tizard Mark , Izzard Leonard
Published: January 2023

The impact of influenza infection is a major public health concern. Significant morbidity and mortality is associated with infection especially in susceptible populations such as the elderly. Efforts to improve vaccine efficacy and long-term immunological memory are currently areas of significant research interest. miRNAs are naturally occurring 19 to 25 nucleotide (nt) long single stranded RNAs that bind to mRNA and cause inhibition of translation. It is predicted that over 30% of all human genes are targeted by microRNAs (miRNAs), including many immune related genes. For this reason, the artificial use of miRNAs for RNA interference (RNAi) is a rapidly emerging field. However, key limitations exist within the field; most notably delivery of miRNAs to the desired target cell populations in vivo. The suppressors of cytokine signaling (SOCS) proteins are important in the regulation of cytokine production, with SOCS1 and SOCS3 having key roles in innate immunity. miR-155 directly targets SOCS1 and is implicated in the proliferation of various T-cell subsets. This study used a reverse engineered influenza A virus to overexpress miR-155 in infected VERO cells. Using a dual luciferase assay we were able to show a successful reduction in SOCS1 expression. Our long-term goal is to use this viral miRNA delivery system to manipulate the immune system, with the aim of enhancing the efficacy and potency of vaccine induced immune responses.
Generation of memory CD4+ T cells after influenza virus infection requires PSGL-1 and selectin binding activity (P6148)
Authors: Tinoco Roberto , Nguyen Jennifer , Lin Melissa , Bradley Linda
Published: January 2023

CD4+ memory T cells have a critical role in immunity to new influenza viruses. In particular, lung-resident memory CD4+ T cells are essential for production of innate inflammatory cytokines that promote early virus control. However, the contribution of adhesion molecules in regulating CD4+ T cell memory remains unclear. We assessed the role of PSGL-1, a ligand for the selectin family of adhesion molecules that is highly upregulated on CD4+ T cells during infection. In adoptive transfer studies, we found that PSGL-1 KO CD4+ T cells had increased accumulation in the lungs. However, compared to WT cells, few PSGL-1 KO cells persisted into the memory phase and these cells failed to re-expand upon secondary challenge. When PSGL-1 KO mice were re-challenged with a high dose influenza infection, early virus levels in the lungs were increased and KO mice had prolonged morbibity. To address whether functional selectin binding activity was necessary for the development of memory CD4+ T cell responses we used mice deficient in two α-(1,3)-fucosyltransferases, IV and VII (FuT KO) that generate biologically active PSGL-1. FuT KO CD4+ T cells did not display impaired primary responses in the lymph node but showed altered accumulation in the lungs. Like PSGL-1 KO CD4+ T cells, FuT KO cells failed to re-expand upon secondary challenge. Our findings demonstrate a critical role for PSGL-1 and functionally active selectin ligand generation in the development of effective memory CD4+ T cells.
Myeloid derived suppressor cells as therapy for experimental myasthenia gravis (P5229)
Authors: Lin Feng , Li Yan , Qian Shiguan , Fung John , Kusner Linda , Kaminski Henry , Lu Lina
Published: January 2023

We recently demonstrated that hepatic stellate cells (HSC) induce myeloid derived suppressor cells (MDSC) differentiation from myeloid progenitors. In this report, we found that adoptive transfer of the MDSC was effective in reversing disease progression in experimental autoimmune myasthenia gravis (EAMG), a T cell-dependent, and B cell-mediated model for myasthenia gravis (MG). In accordance with ameliorated disease severity, the MDSC-treated EAMG mice showed suppressed AchR-specific T cell responses, decreased levels of serum anti-AChR IgGs and reduced complement activation at the neuromuscular junctions. We also discovered that the MDSC inhibited the proliferation of, and antibody production from B cells activated by anti-IgM or anti-CD40 antibodies in vitro, and inhibited the production of antibodies against a T cell-independent antigen in vivo, indicating that in addition to their T cell inhibitory activities, MDSC also directly inhibit B cells. These results demonstrated that HSC-induced MDSC concurrently suppress T- and B- cell autoimmunity, leading to effective treatment of established EAMG. These data also suggest that the MDSC could be further developed as a novel approach for MG treatment, and even more broadly, for other diseases in which T and B cells are involved in the pathogenesis.
Myeloid resident cells in the islets of Langerhans are key players in presentation of islet antigens and islet trophic function: identification of their phenotype and ontogeny (P5232)
Authors: Calderon Boris , Unanue Emil
Published: January 2023

The entry of autoreactive T cells to their target organ is important in autoimmunity since this initiates the inflammatory process. Islets of Langerhans contain a monocyte/macrophage-derived cell with important features and properties. This islet myeloid cell plays a central role in the interaction with diabetogenic T cells. We have studied in-depth the phenotype of the islet myeloid cells in the NOD and non-diabetic mouse strains and find 2 myeloid subsets: one subset (~85%), expressing MHC-II, CD11c, CD11b, F4/80 and LysM; and a minor subset, (~15%) expressing MHC-II, CD11c, CD11b, CD103, BTLA and LysM. Islet myeloid cells are stable in numbers and decrease 30% 7 days after whole body irradiation. Studies testing eGFP bone marrow transplantation has shown that islet myeloid cells can be replaced as early as 2 weeks post-transplant. Finally, we consider that in addition to their role of antigen presentation, the islet myeloid cell has a different and perhaps a more important role in islet physiology, which is to maintain the health of the islet. When a genetic mutation affects the presence of myeloid cells in islets (CSF-1-deficient mice), islets are reduced in size and show an altered glucose homeostasis. Our findings highlight the importance of the islet myeloid cell as central in presentation of islet antigen and trophic function of the islet. Their phenotype, ontogeny and trophic identification, will bring insights into diagnostics and point of interventions in diabetes.
A novel regulatory pathway for autoimmune disease: binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T cell tolerance (P5219)
Authors: Vandenbark Arthur , Meza-Romero Roberto , Benedek Gil , Andrew Shayne , Yu Xiaolin , Mooney Jeffrey , Burrows Gregory
Published: January 2023

Treatment with partial MHC class II-β1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. New studies revealed a novel regulatory pathway that involves RTL binding to CD11b+ monocytes through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized DR-α1/CD74 interaction that inhibited CD74 expression on the cell surface and blocked binding and activity of its ligand, macrophage migration inhibitory factor (MIF), resulting in reduced severity of clinical and histological EAE. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance. Furthermore, the natural anti-inflammatory activity and universal expression of the DR-α1 domain supports its possible application as a new therapeutic agent suitable for treatment of a wide variety of human inflammatory conditions.
Small molecule blockers reveal an important role for Orai1-NFAT-orphan receptor pathway in Th17 differentiation (P5169)
Authors: Kim Kyun-Do , Srikanth Sonal , Tan Yossan-Var , Yee Ma-Khin , Jew Marcus , Damoiseaux Robert , Jung Michael E , Shimizu Saki , An Dong Sung , Waschek James A , Ribalet Bernard , Gwack Yousang
Published: January 2023

Orai1 is the pore subunit of the Calcium Release Activated Channels (CRAC) that activate downstream calcineurin-NFAT pathway. Loss of function mutation in Orai1 causes lethality due to severe defects in immune system, which can be rescued by bone marrow transplantation. Using a high throughput chemical library screening, we have identified a class of small molecule blockers that inhibit Orai1. One of the small molecules, compound 5D blocked the activity of a constitutively active mutant of Orai1, suggesting a direct block of ion permeation via Orai1. Primary murine Th17 cells showed highest sensitivity to block by this compound. Treatment with compound 5D not only suppressed cytokine production, but also inhibited differentiation of Th17 cells by suppression of the retinoic-acid-receptor-related orphan receptors RORα and RORγt. Exogenous expression of RORα, RORγt or a constitutive active mutant of NFAT (CA-NFAT) rescued the blocking effect, identifying the Orai1-NFAT- ROR signaling pathway to be important for Th17 differentiation. The defects in cytokine production and differentiation were recapitulated in Th17 cells from Orai1-deficient mice suggesting Orai1 as a specific target of the blockers. In vivo administration of Orai1 blockers effectively delayed the onset and reduced the severity of autoimmune disease in mice. These data indicate that derivatives of compound 5D can be used as chemical templates for development of therapeutic agents to alleviate autoimmune diseases.
A novel CMKLR1 small molecule antagonist suppresses experimental autoimmune demyelinating disease (P5179)
Authors: Zabel Brian , Zhang Jian , Lewen Susanna , Burke Thomas , Carlson Jeffrey , Zoudilova Maria , Sobel Raymond , Butcher Eugene , Graham Kareem
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) is a widely studied mouse model that shares many clinical and histological features with multiple sclerosis (MS). Chemokine-like receptor-1 (CMKLR1) is a chemoattractant receptor that is expressed by key effector leukocytes in EAE and MS, including macrophages, subsets of dendritic cells, and natural killer cells. We previously showed that clinical and histological EAE is less severe in CMKLR1-deficient (CMKLR1 KO) mice compared with wild-type. In this study, we sought to identify small molecular CMKLR1 inhibitors that recapitulate the CMKLR1 KO phenotype in EAE. We identified α-naphthoyl ethyltrimethylammonium iodide (α-NETA) as a selective CMKLR1 small molecule antagonist that inhibits chemerin-stimulated β-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1+ cell migration. Structure-activity relationship studies indicated that the trimethylamine region is critical for CMKLR1 potency. Prophylactic dosing with α-NETA (i.e. prior to the onset of clinical signs) significantly delayed the onset of EAE induced in C57BL/6 mice by active immunization with myelin oligodendrocyte glycoprotein peptide 35-55. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides proof-of-concept data that pharmaceutical targeting of CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.