bluuebunny/crossref_metadata_2025 huggingface dataset:
PTEN regulates natural killer cell trafficking and effector functions (IRM7P.484)
Authors: Leong Jeff , Schneider Stephanie , Sullivan Ryan , Fehniger Todd
Published: January 2023

Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the PI3 kinase pathway, members of which are essential for regulating natural killer (NK) cell signaling and activation. To evaluate whether PTEN was required for normal NK cell functions, we utilized a mouse model of NK specific PTEN deficiency (Ncr1iCre knockin x PTENflox). PTEN deletion resulted in significant loss of NK cells in the bone marrow and other lymphoid tissues, but markedly increased numbers within the peripheral blood and lung. Surprisingly, we observed near equivalent NK cell maturation within peripheral organs regardless of PTEN expression, suggesting that PTEN operates to re-distribute NK cells during development. Similarly, despite impaired migration to chemoattractants in vitro, PTEN-deficient NK cells egress more efficiently from the bone marrow and preferentially reside in sinusoidal compartments, but are specifically retained in the lung and peripheral vasculature. Given the inappropriate localization in the absence of PTEN, we further evaluated whether cytotoxic functions were altered. While PTEN deficient NK cells have enhanced cytotoxicity against co-localized RMAS tumors, they are unable to migrate to distal tumor sites. These data suggest that PTEN is required for the trafficking of NK cells during both homeostasis and in the presence of malignancy, during which it represses cytotoxic functions.
Indoleamine 2,3-dioxygenase inhibits B cell immune response to T independent antigens (IRM8P.710)
Authors: Shinde Rahul , Chaudhary Kapil , Shimoda Michiko , Pacholczyk Gabriela , McGaha Tracy
Published: January 2023

T cell independent (TI) antibody responses are crucial for humoral immunity to viruses and encapsulated bacteria. Here we report a novel mechanism where the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) 1 regulates the B cell response to TI antigens. Immunization with NP-Ficoll led to rapid splenic induction of IDO, primarily in the extra-follicular space. When IDO1KO mice were immunized there was a significant increase in humoral responses with increased formation of extra-follicular IgM and IgG3 foci, antibody secreting cells (ASCs), and increased antibody titers. This was not associated with an alteration in affinity maturation suggesting the primary impact of IDO1 deficiency was increased B cell proliferation and plasma cell formation. IDO1 did not affect immune responses to protein antigens as immunization with NP-OVA elicited similar antibody titers regardless of IDO1 function. In addition, adoptive transfer of IDO1 deficient B cells to µMT-/- (B cell deficient) mice was sufficient to replicate increased TI responses observed in IDOKO mice. Moreover, in vitro LPS rapidly induced IDO1 in MACS-purified B cells and IDO deficient B cells display enhanced LPS and CpG-driven proliferation associated with increased production of IgM, IgG3, and IL10. Thus, our results demonstrate a novel role of IDO in suppressing T cell independent antibody response that provides insight into the understanding of B cell immune responses in autoimmunity and vaccine biology.
Novel ubiquitination-like modifications that regulate recombination of immunoglobulin loci gene segments leading to antibody diversification (IRM8P.711)
Authors: Basu Uttiya , Sun Jianbo , Keim Celia , Grinstein Veronika
Published: January 2023

Programmed DNA mutagenesis events in the immunglobulin (Ig) loci of B cells utilize the common and conserved mechanism of ubiquitination of proteins prior to proteasomal degradation to generate the required antigen-receptor diversity. Recombinase proteins RAG1 and RAG2, required for V(D)J recombination, and activation-induced cytidine deaminase (AID), an essential mutator protein for catalyzing somatic hypermutation (SHM) and class switch recombination (CSR), are regulated by various ubiquitination events affecting protein stability and activity. AID associates with transcribing RNA polII and non-coding RNA processing complex RNA exosome to introduce DNA lesions in the IgH locus that are required for CSR and SHM. Failure in regulating AID’s DNA mutagenic activity is considered as a primary cause for B cell lymphomagenesis, especially Diffuse Large B cell Lymphoma (DLBCL). Here, we present our recent observations that demonstrate that the AID/RNA exosome/RNA polymerase II complex is regulated by ubiquitination and neddylation modifications. Using proteomics based experiments we have identified AID associated protein factors that promote ubiquitin-like modification of AID/RNA polII/RNA exosome complex. Using various biochemical assays, knockdown experiments and genetically mutated mouse models, we demonstrate the ubiquitin-like modification of AID-protein complex is important for regulating AID activity during CSR and overall maintenance of B cell genomic integrity.
Mouse models for immunology research available from the Jackson Laboratory repository. (IRM9P.718)
Authors: Valenzuela Alicia , Lutz Cathleen , Rockwood Stephen , Sasner Michael , Donahue Leah
Published: January 2023

The Jackson Laboratory Repository serves as a centralized facility for the development, rederivation, distribution and cryopreservation of mouse models of human biology and disease. Hundreds of strains are added annually to one of the largest collections of characterized mouse strains available to the biomedical research community. This poster presents new knockout (KO) strains carrying genes associated with T cells and antigen (Ag) presentation. Lcp2 (SLP-76)-null mice lack T cells and γδ-TCR+ cells. Psmb8 (LMP-7) KO mice exhibit impaired Ag presentation and reduced T cells. In Clec9a (DNGR-1) KO/GFP knock-in (KI) mice, cross-priming of CD8+ T cells against dead-cell-associated antigens is altered. Nlrp3-/- (cryopyrin) mice are useful for studying role of the inflammasome in immune responses. New floxed strains include Cxcl12, Csf1r and a floxed-stop ubiquitous Myc-driven human CD2 KI. Cxcl12 functions in lymphoid progenitor and hematopoietic SC maintenance. The cytokine, Csf1r, is associated with macrophage function. Other additions include: a tamoxifen-inducible KI of EGFP/cre/ERT into the Foxp3 locus, a transcription factor required for development and function of Treg cells; a Notch reporter with GFP expression directed to the Notch pathway; an RORgamma-driven cre transgene that directs expression to αβ T cells, immature thymocytes and lymphoid tissue inducer cells. To have your strain considered for inclusion in the Repository please visit www.jax.org/donate-a-mouse.
Cyclic dinucleotides mediate gap junction-dependent spread of DNA-induced IRF3 activation. (IRM9P.729)
Authors: King Kevin , Patel Suraj , Yarmush Martin , Weissleder Ralph
Published: January 2023

Cell death and sterile inflammation are central to the pathogenesis of human diseases such as myocardial infarction and acute liver failure. Since patients often present after injury has begun, a primary therapeutic goal is to limit the extent and duration of injury. Therefore, we investigated the molecular mechanisms by which innate immune responses to dying cells are spread. When a cell dies, it releases danger signals such as DNA, which are sensed by neighboring cells and spread by undefined mechanisms, ultimately defining the extent and duration of injury. To study this process, we created stable monoclonal fluorescent reporter cell lines for key transcriptional regulators of danger signaling (IRF3 and NFkB), exposed them to various danger signals, and monitored their spatiotemporal activation by quantitative fluorescence microscopy. We found IRF3 to be strongly activated not only in cells sensing DNA, but also in their unexposed neighbors. Using pharmacologic inhibitors and genetically modified co-cultures, we found that spread of IRF3 activity is contact-dependent and requires gap junctions. We also provide evidence that the recently described cyclic dinucleotide cGAMP is a gap junction permeable mediator of IRF3 activation spread. In vivo experiments show that genetic or pharmacologic modulation of gap junction communication is protective against acute organ injury, demonstrating the importance of injury spread, and its promise as a therapeutic target.
Endogenous HMGB1 prevents nuclear catastrophe and proinflammatory nucleosomal release (IRM9P.719)
Authors: Tang Daolin , Kang Rui , Lotze Michael , Zeh Herbert
Published: January 2023

The mechanisms by which tissue injury alerts the immune system and drives a systemic inflammatory response syndrome remain unknown. Here we identify a high mobility group box 1 (HMGB1)-mediated nuclear regulatory mechanism for the control of the inflammatory response during tissue injury. We show that conditional knockout of HMGB1 within the pancreas, while benign without provocation, rendered mice dramatically more susceptible to experimental acute severe pancreatitis, as manifested by accelerated tissue injury and animal lethality. Deficiency of endogenous pancreatic HMGB1 led to exaggeration of L-arginine-induced nuclear catastrophe and nucleosome release, which then recruited and activated inflammatory cells with subsequent HMGB1 release locally and into the circulation. Serum levels of tissue enzymes (e.g., amylase, lactate dehydrogenase, and pancreatic myeloperoxidase) and pro-inflammatory cytokines were significantly higher in conditional pancreas-specific HMGB1 knockout mice compared to their wild-type control littermates. Moreover, neutralizing extracellular histone and HMGB1 conferred protection against acute pancreatitis in conditional pancreas-specific HMGB1 knockout mice. Thus, intracellular HMGB1 may serve as a previously underappreciated negative regulator of inflammation, shedding light on the role of the innate immune response in infection and tissue damage.
A steroid-mimicking nanomaterial that mediates inhibition of human lung mast cell responses (IRM9P.725)
Authors: Dellinger Anthony , Zhou Zhiguo , Kepley Christopher
Published: January 2023

Water-soluble fullerenes can be engineered to regulate activation of mast cells (MC) and control MC-driven diseases in vivo. To further understand their anti-inflammatory mechanisms a C70-based fullerene conjugated to four myo-inositol molecules (C70-I) was examined in vitro for its effects on the signaling pathways leading to mediator release from human lung MC. The C70-I fullerene stabilize MC and act synergistically with long-acting β2-adrenergic receptor agonists (LABA) to enhance inhibition of MC mediator release through FcεRI-simulation. The inhibition was paralleled by the upregulation of dual-specificity phosphatase one (DUSP1) gene and protein levels. Concomitantly, increases in MAPK were blunted in C70-I treated cells. The increase in DUSP1 expression was due to the ability of C70-I to prevent the ubiquitination and degradation of DUSP1. These findings identify a mechanism of how fullerenes inhibit inflammatory mediator release from MC and suggest they could potentially be an alternative therapy for steroid resistant asthmatics.
Immunomodulation of volatile anesthetics (IRM9P.726)
Authors: Stollings Lindsay , Lu Binfeng , Xu Yan
Published: January 2023

Introduction: The impact of anesthetic exposure on the immune system is relatively unknown at both mechanistic and clinical levels. In this in vitro study, we employ immunological assays and understanding of immune cell function to evaluate the effect of volatile anesthetic (VA) exposure on bone marrow dendritic cells (BMDCs). BMDCc play a critical role in antigen presentation and link the innate and adaptive arms of the immune response. Hypothesis: We believe that VA exposure may modulate immune responses, specifically BMDC function. Methods: Bone marrow cells were harvested from C57BL/6 mice and cultured for six days to generate BMDCs. They were stimulated with low and high dose lipopolysaccharide (LPS). Simultaneously, the cells were exposed to 1.5% isoflurane and incubated for 5 hours. Upon completion of VA exposure, the BMDCs were lysed with Trizol, RNA extracted, and reverse transcript PCR used to generate cDNA. RT-qPCR was performed to evaluate gene expression of common BMDC cytokines. Results: BMDC exposure to isoflurane resulted in decreased gene expression of IL-10, IL-6, and IL-1β. RT-qPCR data indicated 2-4 fold decreases in gene expression of these cytokines. Conclusions: BMDCs exposed to VA in vitro displayed decreased gene expression of inflammatory cytokines. Significance: Better understanding of immunomodulation by anesthetics is critical for optimizing patient outcomes and improved perioperative management.
Quantitative phosphoproteomics reveals novel Vav1-dependent pathways in T cell receptor signaling (IRM10P.736)
Authors: Helou Ynes , Salomon Arthur
Published: January 2023

Vav1, a Rac/Rho guanine nucleotide exchange factor, is a critical component of the T cell receptor (TCR) signaling cascade and is rapidly tyrosine phosphorylated in response to T cell activation. Previous studies using Vav1 knockout models have determined its importance in TCR-dependent signaling. However, a wide-scale characterization of Vav1-dependent phosphorylation events is still lacking. Using quantitative mass spectrometry, a phosphoproteomic analysis of 668 tyrosine phosphorylation sites was performed on Vav1-deficient T cells across four time points of TCR stimulation. Compared to controls, cells lacking Vav1 expression showed statistically significant decreases in phosphorylation on actin cytoskeletal signaling proteins, supporting the role of Vav1 in cytoskeletal reorganization. Interestingly, loss of Vav1 led to perturbations in phosphorylation on signaling molecules proximal to the TCR. Furthermore, proteins implicated in negative feedback pathways, including CBL and PAG, showed altered phosphorylation profiles. Effects further downstream of the TCR were also observed in Vav1-deficient cells, notably, hyperphosphorylation of inhibitory sites on various cyclin dependent kinases, suggesting cell cycle arrest. This data demonstrates novel signaling networks regulated by Vav1 and support the pleiotropic functions of Vav1 downstream of the TCR.
Ca2+-TAGLN2 polymerizes G-actin in a mechanism crucial for T cell activation (IRM10P.738)
Authors: Jun Chang Duk
Published: January 2023

Actin dynamics is required for controlling T-cell activation and effector function. We report that TAGLN2, an actin-binding protein predominantly expressed in immunogenic cells—including CD8+ T cells, induces extensive actin polymerization under low-salt or high-Ca2+ conditions (where spontaneous nucleation and actin polymerization do not normally occur) and inhibits cofilin-induced depolymerization. Mutation of the Ca2+-binding motif results in impaired actin polymerization in vitro and T-cell function in vivo. TAGLN2 localizes to the outer actin ring in the immunological synapse; knockout of TAGLN2 (TAGLN2-/-) leads to diminished F-actin content and impaired actin ring formation in synaptic T cells. These cells demonstrate reduced PLC-γ phosphorylation and calcium signaling after T-cell receptor stimulation, suggesting a positive feedback calcium loop in this pathway. Furthermore, the cytotoxic function of TAGLN2-/- CD8+ T cells in targeting cells is inefficient. Thus, a novel mechanism of F-actin assembly mediated by Ca2+-TAGLN2 binding positively might affect T-cell functions.
Studying the role of CAML in T cell biology (IRM10P.741)
Authors: Chan Siaw Li , Lindquist Lonn , Bram Richard
Published: January 2023

Effective T cell function protects us from pathogens as well as cancer. However when T cell activation is dysfunctional, it has detrimental effects. If T cell activation cannot be shut down, it may cause autoimmunity. Therefore, it is crucial for us to study how T cell activation is regulated. Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed endoplasmic reticulum protein. Using a transgenic conditional knock-out mouse model to delete CAML, we previously demonstrated that CAML is required for early embryonic development and thymopoiesis. Additionally, cells lacking CAML do not respond to EGFR stimulation due to a receptor recycling defect. However, the role of CAML in mature, peripheral T cells is not well understood. Here we provide preliminary data showing that loss of CAML in peripheral T cells leads to defective T cell activation and proliferation upon TCR stimulation, and inducing cell death through Fas ligand engagement. We hypothesize that CAML regulates components that are required to sustain optimum TCR signaling and function. T cell responsiveness to TCR stimulation is critical for T cells to deliver effector functions during antigen recognition and activation. Therefore understanding the regulation of TCR signaling by CAML will provide insight into novel mechanisms of immune regulation. These studies may provide potential treatments of immunodeficiency, autoimmunity or other inflammation associated diseases.
Optineurin and NEMO: a potential novel interplay to fine tune NF-κB activation upon TCR engagement. (IRM10P.742)
Authors: Montecalvo Angela , Kane Lawrence
Published: January 2023

Optineurin (OPTN) is a homolog of NF-κB essential-modulator (NEMO), the regulatory subunit of the IκB kinase (IKK) complex. Dysregulation of OPTN has been linked to several neurodegenerative diseases. Although OPTN shares with NEMO a high degree of homology, OPTN is not part of the same high-molecular weight complex containing IKKα and IKKβ. Although, OPTN cannot substitute for NEMO during (LPS)-induced NF-κB activation, OPTN negatively regulates TNF-α induced NF-κB activation by competing with NEMO for ubiquitinated proteins. We have investigated the potential role of OPTN during TCR induced NF-κB activation. OPTN overexpression down-regulates TCR-induced NF-κB activation in a dose-dependent manner. Furthermore we observed a transient loss of OPTN after stimulation of T cell lines or primary human or murine T cells. We further determined that the transient loss of OPTN upon TCR stimulation does not seem to be due to proteosomal degradation, suggesting a potential involvement of lysosomal degradation pathway. The present study provides new information regarding the role of OPTN in regulating NF-κB during TCR stimulation, thus revealing the possible importance of OPTN during inflammation and/or autoimmune diseases.
Impaired transitional B cell maturation in Flt3-ligand deficient mice. (LYM6P.775)
Authors: Medina Kay , Dolence Joseph , Gwin Kimberly , Hsu Fan-Chi , Shapiro Virginia
Published: January 2023

The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.
The fate of B cells in HIV infection - shifting the balance from plasma cell towards regulatory B cell (Breg) differentiation (LYM6P.776)
Authors: Kaltenmeier Christof , Gawanbacht Ali , Lindner Stefanie , Beyer Thamara , Härter Georg , Grüner Beate , Bommer Martin , Kirchhoff Frank , Schrezenmeier Hubert , Jahrsdörfer Bernd
Published: January 2023

Certain regulatory lymphocyte populations suppress T cell expansion in a granzyme B (GrB)-dependent manner. Recently, we found that human B cells also contribute to immune regulation by interleukin (IL-)21-dependent expression of GrB. GrB-expressing Bregs are induced by incompletely activated CD4+ T cells, which express high levels of IL-21, but only low CD40 ligand. In contrast, fully activated CD4+ T cells strongly induce plasma cell differentiation. Here we demonstrate that up to 60% of B cells from HIV-infected patients with high viremia exhibit a regulatory (Breg) phenotype with expression of GrB and potent suppression of T cell proliferation by GrB-dependent degradation of the T cell receptor zeta-chain (TCR-zeta). These results are supported by additional findings showing that T cells in HIV-infected individuals produce high levels of IL-21, while expressing only low levels of CD40L and TCR-zeta. Importantly, soluble CD40L multimers can restore the differentiation of Bregs derived from HIV-infected individuals into plasma cells. Our results suggest that incompletely activated CD4+ T cells in HIV patients lead to a misrouting of B cell differentiation into Bregs at the cost of fully functional plasma cells. Apart from a disturbed humoral immune response these B cells may further aggravate the immune status of HIV patients. The use of CD40L multimers may disrupt the defective B cell-T cell interactions and may prove beneficial for future HIV vaccination approaches.
Generation of IgA plasma cells during a secondary response in the absence of IgA memory B cells (LYM6P.800)
Authors: Pape Kathryn , Taylor Justin , Xiong Huizhong , Lafaille Juan , Jenkins Marc
Published: January 2023

IgA antibodies are important for protection against infection at mucosal surfaces. Despite this importance in protective immunity, the differentiation of IgA B cells is not well understood. To address this issue, an antigen-based enrichment method was used to track polyclonal B cells specific for the model antigen R-Phycoerythrin (PE) during the course of a T-dependent immune response. While 10-40% of the PE-specific short lived plasma cells expressed IgA, less than 0.05% of the PE-specific germinal center and memory B cells expressed IgA. Despite the lack of IgA memory B cells, secondary antigen challenge resulted in the generation of large numbers of PE-specific IgA plasma cells that appeared at times prior to the appearance of IgA plasma cells during the primary response. Since IgA memory B cells were infrequently detected, we hypothesize that IgA plasma cells generated during the secondary challenge are the result of a secondary switch from memory B cells expressing other isotypes. We are currently examining the role that individual isotypes of memory B cells play in the generation of IgA plasma cells by looking for IgG switch remnants in the PE-specific IgA plasma cells generated after secondary challenge, and by testing the ability of purified isotypes of memory B cells to produce IgA plasma cells after transfer and challenge of recipient mice.
Hypoxia, metabolism, and long-lived plasma cells (LYM6P.765)
Authors: Minges Wols Heather , Aguilera Alexya , Witte Pamela
Published: January 2023

Evidence from multiple laboratories agrees that long-lived humoral immunity derives from long-lived plasma cells (LLPC) that reside in the bone marrow (BM), and existing literature makes a compelling case explaining that short-lived PC die due to high metabolic stress. However, the mechanism by which LLPC escape the same fate as SLPC is unexplored. We tested the hypothesis that glycolysis is used by BM LLPC, which might be triggered by hypoxic areas of the bone marrow. Hypoxyprobe™-1 (PIM) was used as an indicator of BM LLPC residing in areas of low O2 tension. PIM positive stain in PC suggests that most BM LLPC reside in hypoxic niches. In addition, data obtained from both ELISPOT and 24h ELISA suggest that Ig secretion from BM cultured in a hypoxia chamber (3% O2) is slightly enhanced in hypoxic versus normoxic conditions. Since BM PC appear to reside in hypoxic niches, we assessed whether glycolysis is the predominant metabolic strategy employed by PC. Using the MitoTracker probe that fluoresces upon oxidation in respiring mitochondria, we found BM PC in both MitoTracker-hi and -lo populations, suggesting PC may differ in their metabolic strategies, using aerobic respiration and glycolysis, respectively. BM LLPC in both Mitotracker subsets are functional as determined by ELISA and ELISPOT. Future studies will assess the role of the plasma cell “niche” in promoting and sustaining a metabolic phenotype, which likely governs long-term immunity.
CD14+CD16+ monocytes play distinct role in Plasmodium vivax malaria (MPF3P.817)
Authors: Antonelli Lis , Leoratti Fabiana , Costa Pedro , Rocha Bruno , Diniz Suelen , Tada Mauro , Pereira Dhelio , Golenbock Douglas , Gonçalves Ricardo , Gazzinelli Ricardo
Published: January 2023

During malaria, the Plasmodium triggers high levels of cytokines, which both help the immune response controlling the parasite and contribute to the symptoms observed during the diseases. Our data show that monocytes are the main source of cytokines upon P. vivax infection, suggesting their important role during disease. While it is recognized that monocytes are heterogeneous, the physiological relevance of this is not yet completely understood. The different monocyte subsets seem to reflect developmental stages with distinct roles. Our goal is to define the role of monocyte during P. vivax infection. Our data show that higher levels of cytokines, accompanied by increased frequencies of monocytes in P. vivax-infected patients. The infection triggers the expression of activation markers, adhesion molecules and chemokine receptors on monocytes. Moreover, these cells can be distinguished into three monocyte subsets based on the expression of CD14 and CD16. Each of these subsets display an distinct profile, suggesting that they distinctly act during P. vivax infection to induce inflammation, control the infection and modulate immune response. Importantly, CD14+CD16+ monocytes displayed higher phagocytic activity and killing potential than their other counterparts. Identification of mechanisms that regulate monocyte responses during malaria will provide important information on the development of therapeutic strategies that are targeted to modify their particular cell subsets
Regulation of Helicobacter Pylori replication by macrophage PPARγ (MPF3P.823)
Authors: Kronsteiner-Dobramysl Barbara , Bassaganya-Riera Josep , Viladomiu Monica , Philipson Casandra , Schiff Eric , Hontecillas-Magarzo Raquel
Published: January 2023

Helicobacter pylori infects 50% of the human population and in about 15% of colonized individuals it can cause gastritis and gastric malignancies. Although previously considered an exclusively extracellular bacterium, H. pylori has been found inside gastric epithelial cells, macrophages and dendritic cells. Data from our mouse model show significant depletion of H. pylori from the stomach when macrophages acquired a pro-inflammatory phenotype due to the lack of the transcription factor peroxisome proliferator activated receptor gamma (PPARγ). To dissect the underlying mechanism, we polarized WT and PPARγ-deficient BM-derived macrophages towards an M1 phenotype prior to bacterial co-culture. Proliferation of H. pylori was almost completely abrogated in PPARγ-deficient M1 and the bacteria were cleared 6 hours earlier compared to WT M1, WT M0 and PPARγ-deficient M0. In line with the importance of an M1 like phenotype, gene expression results obtained from a time-course experiment showed higher iNOS expression in cells lacking PPARγ and increased NFκB activity upon bacterial challenge of cells treated with a PPARγ inhibitor. The importance of iNOS in bacterial clearance was confirmed by inhibition of iNOS resulting in increased re-isolation in both WT and PPARγ-deficient macrophages. Together our data suggest that PPARγ might be a critical factor in the outcome of H. pylori infection and thus represents a promising target for new therapeutic interventions.
CD8+ T cell activation in pathological giardiasis (MUC4P.837)
Authors: Keselman Aleksander , Singer Steven
Published: January 2023

Infection with the protozoan Giardia duodenalis is the most common intestinal parasitic disease in the United States. Clinical giardiasis presents with diarrhea and malabsorption of nutrients. One major pathological hallmark of giardiasis is the reduction of intestinal digestive enzymes, such as disaccharidases. Our lab and others in the field have demonstrated that CD8+ T cells are required for Giardia-induced disaccharidase deficiency but are not involved in protective immunity. In light of these observations, we have devoted our efforts to understanding how G. duodenalis infection activates intestinal CD8+ T cells to drive pathology. Analysis of surface marker expression on intestinal CD8+ T cells revealed an increased CD44hiCD69hi population following infection within the duodenal lamina propria. These activated CD8+ T cells produced IFNγ and TNFα. Furthermore, antibiotic treatment limits both CD8+ T cell activation and intestinal disaccharidase reduction in infected mice. Because the microbiota effects the development of these CD8+ T cell responses, we are currently determining if Giardia-activated CD8+ T cells are mounting a specific response to parasite antigen or are acting as bystanders. Further characterization of these intestinal CD8+ T cell responses to G. duodenalis infection will expand our understanding of pathogenesis and help identify novel therapeutic targets for the clinical setting.
COP9 regulates intestinal microbial ecology and susceptibility to colitis (MUC4P.839)
Authors: Wang Baomei , Deng Zhong-bin , Dryden Gerald , Miller Donald , Wei Ning , Zhang Huang-Ge
Published: January 2023

Inflammatory Bowel Disease (IBD) has been considered as the dysfunctional interplay between intestinal microbiota, epithelium and the immune system. Located at the heart of this complex interaction, intestinal epithelial cells (IECs) play a crucial role in coordinating commensal bacterial-dependent intestinal homeostasis. Separated from commensal bacteria by mucus layer, the mechanism that IECs confer a stable microbiota remains incompletely understood. Here, we reported that mice with an IEC-specific deletion of one subunit of COP9 signalosome, which regulates the ubiquitin-proteasome system, exhibited extreme susceptibility to dextran sulfate sodium (DSS) induced colitis. Deficiency of one subunit of COP9 resulted in dysfunction of paneth cell and colonic enterocyte, which led to impaired AMP (antimicrobial peptide) production, shifted composition of intestinal microbiota and skewed mucosal immune cells towards a proinflammatory phenotype. Deep sequencing and quantitative PCR analysis of luminal microbiota from proximal colon revealed a significant reduction of the relative abundance of Lactobacillus and increase of segmented filamentous bacteria. Correspondingly, the Th1/Th17 ratio is reversed in colonic lamina propria. Moreover, the susceptibility to colitis was alleviated by continued broad-spectrum antibiotic treatment. Collectively, these data suggest that COP9 signalosome in IEC regulates intestinal homeostasis by modulating the composition of microbiota.
Chronic morphine in the context of HIV infection, induce gut microbiome dysbiosis and lead to persistent GALT inflammation. (MUC4P.843)
Authors: Banerjee Santanu , Sindberg Gregory , Wang Fuyuan , Sharma Umakant , Johnson Timothy , Ramakrishnan Sundaram , Roy Sabita
Published: January 2023

Clinical observations show a strong correlation between microbial translocation and HIV disease progression. Chronic morphine, on its own, is known to induce bacterial translocation, impair immune activation and pathogenic clearance during infection and results in long-term persistent inflammation in the affected organs. Lack of response to therapy, and non-AIDS related comorbidities have been linked to immune activation as a consequence of microbial translocation. The gut microbiota plays a significant role in maintaining gut homeostasis and barrier integrity. Loss of microbes, which are part of the normal flora in healthy hosts, increase the susceptibility to a more virulent composition, that can contribute to barrier disruption and microbial translocation. In this study, we show that morphine induces bacterial translocation across gut mucosa, which is exacerbated with HIV infection. We also show HIV and morphine mediated gut microbiome changes and its influence on the immune system leading to persistent inflammation. Finally, we show that these co-morbidities preferentially favor class firmicutes over others and also increase adherence and alter virulence factors to tweak the host immune system to bring about the said effects.
Metabolomics reveals the impact of vitamin D on the microbiome (MUC4P.856)
Authors: Bora Stephanie , Ooi Jot Hui , Smith Philip , Patterson Andrew , Cantorna Margherita
Published: January 2023

Dysbiosis of the microbiome is an important factor in susceptibility to diseases such as inflammatory bowel disease (IBD). Vitamin D has known immunoregulatory roles and decreased levels of vitamin D are linked to IBD. Mice lacking the enzyme 1-α-hydroxylase (Cyp27B1 KO) cannot metabolize 25(OH)D3 into bio-active 1,25(OH)2D3. These mice are more susceptible to experimental colitis compared to WT controls. Here we wanted to determine the effect of vitamin D3 on the urinary metabolome, since microbial metabolites can be detected in urine. Urine was collected from WT, WT+20,000IU vitamin D3, and WT and Cyp27B1 KO littermates. Urine samples were profiled by ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and multivariate data analysis tools. WT mice supplemented with vitamin D and WT and Cyp27B1 KO mice displayed changes in microbial metabolites compared to unsupplemented mice. Metabolites associated with the gut microbiota: p-cresol sulfate, indoxyl sulfate, xanthurenic acid, and betaine were different as a function of vitamin D3 supplementation or Cyp27B1 expression. Alterations in the urinary metabolome suggest a role for vitamin D3 in regulating the microbiota. Metabolite analysis will aid our understanding of the mechanisms by which vitamin D3 affects host health and the microbiota, and future work will address how changes in the microbiota affect susceptibility to experimental IBD.
Formyl-peptide receptor 2-expressing cells in Peyer’s patch are closely associated with the regulation of antigen-specific mucosal immune induction (MUC4P.832)
Authors: Jang Yong-Suk , Kim Sae-Hae , Lee Ha-Yan , Park Jisang , Lee Kyung-Yeol
Published: January 2023

It is known that formyl peptide receptor-2 (FPR-2) acts as innate immune sensor through recognition of peptides derived from pathogens such as H. pylori and HIV, mitochondrial peptides, amyloidogenic peptide including serum amyloid A, and inflammatory response-associated peptides such as LL-37. However, the role of FPR-2-expressing cells in mucosal immune inductive area, Peyer’s patch (PP), is still unraveled yet. In this study, we identified the expression of FPR-2 in both M cells located in follicle-associated epithelium of PP and follicular dendritic cells (FDCs) localized in germinal center. In addition, the level of Cxcl13 and TGF-β gene transcripts in FDCs, which are closely associated with recruitment and differentiation of IgA-secreting B cells was substantially increased by treatment of cathelicidin LL-37, one of FPR-2 ligands. Moreover, enhanced immune induction was verified in vivo mouse system through monitoring the antigen-specific mucosal immunity after oral administration of LL-37-conjugated antigen. Treatment of LL-37 has also shown the immune modulatory effects such as recruitment of CD11c+ cells and IL-17-secreting cells together with activation of FPR-2-expressing cells. Collectively, this study suggests that FPR-2-expressing cells are associated with regulation of mucosal immune induction and LL-37 could be used as a modulator for the cells.
S-nitrosothiols (SNO) imported through LAT1 (L-type amino acid transporter type 1) ameliorates phagocytosis in alveolar macrophages and neutrophils (MUC5P.868)
Authors: Brahmajothi Mulugu , Mason Stanely , Auten Richard
Published: January 2023

Phagocytosis is a receptor mediated process where cytoskeletal rearrangement occurs to internalize an organism or particles. Macrophages and neutrophils have this potential as they form the first line of defense in any immune response. During lung inflammation, neutrophils and monocytes are freshly recruited; infiltrating neutrophils outnumber other cells but are short lived. As a result, more focus is on macrophages. Our study was the first to describe LAT1 expression in macrophages. We wanted to see the expression of LAT1 and its effects on SNO uptake in neutrophils. We administered LPS (50µg/kg) intratracheally to induce inflammation in juvenile rats. After 24hrs, BALF was collected by intratracheal aspiration, cells were isolated and enumerated. LAT1 expression was observed in neutrophils, monocytes, red blood cells and macrophages. When these cells were exposed to 100 µm of CysNO, neutrophils and macrophages exhibited remarkable phagosomes, rapidly engulfed particles and other senescing neutrophils. These properties were not observed when L-Leu or JPH 203 (competitor and inhibitor) were used. We observed neutrophils cannibalizing senescing neutrophils and neutrophil clearance by macrophages. This was a preliminary observation and further studies are underway. We propose that s-nitrosylation is involved in post translational modifications of some key signaling and adaptor proteins that may be of therapeutic significance in immune response.
Fully immunomagnetic isolation of untouched purified lymphocyte populations directly from whole blood without the need for red blood cell sedimentation, hypotonic lysis or density gradient centrifugation in just 25 minutes. (TECH2P.871)
Authors: Kokaji Andy , MacDonald G. , Sun C. , Kellerman Drew , Le Fevre Tim , Leung Nathan , Ng Victoria , Tabatabaei-Zavareh Nooshin , McQueen Karina , Fairhurst Maureen , Thomas Terry , Eaves Allen
Published: January 2023

Human whole blood is made up of 93-96% red blood cells (RBCs), 4-7% platelets and 0.1-0.2% leukocytes consisting mainly of granulocytes, lymphocytes and monocytes. Due to the low frequency of lymphocyte subsets within whole blood, their isolation typically requires a pre-processing step such as Lymphoprep™ or hypotonic lysis to deplete RBCs prior to cell isolation. We have therefore developed a new, fully immunomagnetic method for the negative selection of untouched cells directly from whole blood, without the need for any pre-processing steps. The EasySep™ Direct procedure involves labeling RBCs, platelets and unwanted leukocytes present in human whole blood with antibody complexes and magnetic particles. The magnetically labelled unwanted cells are separated from the untouched desired cells using an EasySep™ magnet by simply pouring or pipetting the desired cells into a new tube. In just 25 minutes, purities of 92-99% total lymphocytes, 93-98% T cells, 87-97% CD4+ T cells, 75-91% CD8+ T cells, 91-99% B cells, 87-98% naïve B cells and 65-84% NK cells can be achieved with little to no residual RBC contamination. The isolation of cells directly from whole blood will enable rapid access to highly purified cells immediately ready for downstream functional assays with minimal sample handling.
Nontargeted urinary metabolite profiling of a chronic mouse model of multiple sclerosis (THER3P.884)
Authors: Giri Shailendra , Laila Poisson , Suhail Hamid , Singh Jaspreet , Deshpande Mandar , Datta Indrani , Denic Aleksandar , Rodriguez Moses , Rattan Ramandeep , Mangalam Ashutosh
Published: January 2023

In search of metabolites signature as biomarkers during EAE disease, we profiled urine from B6 EAE using global untargeted metabolomics. Evaluation of metabolomic profiling of urine from EAE and healthy B6 group by using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found that 132 out of 322 (41%) metabolites were differentially altered (P<0.05) indicating robust alteration in the urine metabolomics profile during disease. Among the perturbed metabolites, 11 were up regulated in EAE urine whereas 121 were down regulated. We conducted pathway analysis of the biochemical pathways of the KEGG and considered both concerted changes in metabolite intensity within the pathway (GlobalTest) and alterations of high impact, and found that a number of pathways were significantly altered including glyoxylate and dicarboxylate, phenylanine metabolism, porphyrin & chlorophyll metabolism, primary bile acid biosynthesis, cysteine & methione metabolism, taurine and hypotaurine metabolism, glycine, serine & threonine metabolism, and beta-alanine metabolism. Alteration in these pathways during EAE disease suggesting that perturbation of certain central metabolites could have impact on multiple metabolic pathways. While some of these metabolite changes could easily be developed as biomarkers, the key to translating metabolomics into therapeutics would require figuring out the central altered metabolic pathway(s), once studied in detail.
GM-CSF-expanded CD1dhiCD5+ B cells suppress experimental autoimmune myasthenia gravis (THER3P.886)
Authors: Sheng Jian Rong , Quan Songhua , Soliven Betty
Published: January 2023

IL-10-competent subset within CD1dhiCD5+ B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. Whether B10 cells can prevent or suppress the development of experimental autoimmune myasthenia gravis (EAMG) has not been studied. In this study, we investigated whether low dose granulocyte macrophage-colony stimulating factor (GM-CSF) can expand B10 cells in vivo, and whether adoptive transfer of CD1dhiCD5+ B cells would prevent or suppress EAMG. We found that treatment of EAMG mice with low-dose GM-CSF increased the proportion of CD1dhiCD5+ B cells and B10 cells. In vitro co-culture studies revealed that CD1dhiCD5+ B cells altered T cell cytokine profile but did not directly inhibit T cell proliferation. On the other hand, CD1dhiCD5+ B cells inhibited B cell proliferation and its autoantibody production in an IL-10-dependent manner. Adoptive transfer of CD1dhiCD5+ B cells to mice could prevent disease and suppress ongoing EAMG. This was associated with downregulation of mature dendritic cell markers and expansion of regulatory T cells resulting in the suppression of acetylcholine receptor (AChR)-specific T cell and B cell responses. Thus, our data have provided significant insights into the mechanisms underlying the tolerogenic effects of B10 cells in EAMG. These observations suggest that in vivo or in vitro expansion of CD1dhiCD5+ B cells or B10 cells may represent an effective strategy in the treatment of human myasthenia gravis.
Profile of circulatory metabolites in chronic mouse model of multiple sclerosis using untargeted global metabolomics (THER3P.883)
Authors: Giri Shailendra , Laila Poisson , Singh Jaspreet , Suhail Hamid , Deshpande Mandar , Datta Indrani , Rodriguez Moses , Rattan Ramandeep , Mangalam Ashutosh
Published: January 2023

Multiple sclerosis is a chronic inflammatory and demyelinating disease of the CNS. Although, MS is well characterized in terms of the role played by immune cells, cytokines and CNS pathology, nothing is known about the metabolic alterations that occur during the disease process in circulation. Here, we profiled the plasma metabolites of B6 with EAE, a known model of chronic MS using untargeted global metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, a total of 326 metabolites were identified, with significant changes observed in 105 metabolites (13 up- and 92 down-regulated), that mapped to lipid and amino acid pathways, followed by the peptide, xenobiotic, carbohydrate, nucleotide and energy pathways (p<0.05). To understand the functional role of these alterations, the KEGG metabolic library was analyzed using Metaboanalyst. The top nine most significant pathways in terms of GlobeTest p-value and impact are 1) fatty acid biosynthesis, 2) lysine degradation, 3) histidine metabolism, 4) beta-alanine metabolism, 5) pentose phosphate pathway, 6) arachidonic acid metabolism, 7) linoleic acid, 8) D-arginine and ornithine. Overall, these metabolic changes could be exploited as biomarkers for EAE/MS disease and to design new treatment paradigms where metabolic interventions could be combined with present and experimental therapeutics to achieve better treatment.
Ex vivo-induction of tumor-specific effector and memory T cells for adoptive T cell therapies using antigen-loaded DCs (TUM4P.910)
Authors: Hansen Morten , Wieckowski Eva , Kalinski Pawel
Published: January 2023

Adoptive transfer of ex vivo expanded tumor infiltrating lymphocytes (TILs), or blood-derived T cells genetically-modified to express specific T cell receptors have been used in adoptive cell therapies (ACT) of melanoma and hematologic malignancies. However, wider application of ACT has been limited by poor T cell infiltration of many cancers, terminal differentiation and limited proliferative and functional capacity of TILs from many lesions, or lack of defined tumor-rejection antigens. Here we show that these limitations can be circumvented by in vitro sensitization (IVS) of blood lymphocytes, using antigen-loaded dendritic cells (DCs) to generate CTLs specific against multiple tumor-specific epitopes. Type-1-polarized DCs (aDC1s) consistently induce high frequencies of functional tumor-specific CTLs from PBMCs of healthy donors and cancer patients. Such IVS-induced CTLs can be further expanded up to 1000-fold, in a single cycle of activation with anti-CD3, irradiated feeder cells and low-dose IL-2. Unexpectedly, presence of aDC1 supernatants during the rapid expansion phase improved not only the functional activity of the expanded CTLs, but also the frequencies of tumor-specific cells, their CD62L and IL-7R expression, and ability to survive and perform killer function in the absence of exogenous IL-2. The current data show a new strategy of generating high numbers of functional CTLs for ACT, with several important advantages compared to high-dose-IL-2 expanded TILs.
Regulation of cell death and antitumor immunity by a natural triterpenoid in malignant lymphoma (TUM4P.930)
Authors: Haque Azizul , Radwan Faisal , God Jason , Hossain Azim , Elvington Michelle , Tomlinson Stephen
Published: January 2023

B-cell lymphoma is a leading cause of hematological malignancies, accounting for almost 10% of new cancer-related deaths in the United States. In this study, we report that Ganoderic acid A (GA-A), a natural triterpenoid from the medicinal mushroom Ganoderma lucidum, induces anti-proliferative activity and apoptotic cell death in human B-cell lymphoma lines and primary B-cell tumors. GA-A treatment induced caspase-dependent apoptotic cell death characterized by a dose-dependent increase in active caspases 9 and 3, up-regulation of pro-apoptotic Bim and Bax proteins, and a subsequent loss of mitochondrial membrane potential with release of cytochrome c. In addition to GA-A’s anti-growth activity, we show that lower doses of GA-A enhance HLA class II-mediated antigen presentation and CD4+ T cell recognition of lymphoma in vitro. The therapeutic relevance of GA-A treatment was tested in vivo using the EL4 syngeneic mouse model of metastatic lymphoma. GA-A-treatment significantly prolonged survival of EL4 challenged mice and decreased tumor metastasis to the liver, an outcome accompanied by a marked reduction of myeloid-derived suppressor cells (MDSC) in the host. Thus, GA-A not only selectively induces apoptosis in tumor cells, but also enhances cell-mediated immune responses by reducing MDSC and elevating Ag presentation and T cell recognition. The demonstrated therapeutic benefit indicates that GA-A is a candidate for future drug design for the treatment of lymphoma.