bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel TIR domain containing adapter, BCAP, links Toll-like receptors to PI3K/AKT activation and regulates inflammation (P1203)
Authors: Pasare Chandrashekhar 
Published: January 2023

Abstract
Stimulation of TLRs leads to recruitment of several adaptor proteins leading to activation of NF-κB, MAP kinases, and IRFs, culminating in an inflammatory response. Interaction between TLRs and all known adaptor proteins depends on heterotypic interactions between respective Toll-Interleukin-1 receptor (TIR) homology domains. In addition to activation of NF-κB, MAP kinases, and IRFs, TLR signaling also leads to activation of phosphoinositide 3-kinase (PI3K). Activation of PI3K regulates the TLR induced inflammatory response, however, the mechanism of TLR mediated PI3K activation is unclear. Using in silico analysis, we identified an uncharacterized cryptic TIR domain at the amino-terminus of B cell adaptor for PI3K (BCAP, also known as Pik3ap1). We found that BCAP associated with MyD88 and TIRAP and attenuated TLR mediated activation of NF-κB. Both of these functions were dependent on BCAP’s TIR domain. Mechanistically, BCAP was phosphorylated upon TLR activation and the PI3K p85 subunit was recruited to BCAP upon TLR stimulation. BCAP deficient macrophages were severely limited in activation of PI3K/AKT and secreted increased pro-inflammatory cytokines upon TLR stimulation. In vivo studies revealed BCAP deficient mice to be highly susceptible to inflammatory colitis, as evidenced by increased weight loss, as well as enhanced tissue destruction. Additionally BCAP has a major role in wound healing and tissue repair.Together, our results demonstrate that BCAP is a TIR domain containing TLR signaling adaptor crucial for connecting TLRs to PI3K/AKT activation and regulation of inflammatory responses both in vitro and in vivo.
IRAK4 kinase activity is not required for induction of endotoxin tolerance but significantly contributes to TLR2-mediated tolerance (P1224)
Authors: Medvedev Andrei , Xiong Yanbao , Pennini Meghan , Vogel Stefanie 
Published: January 2023

Abstract
Prior exposure to LPS induces “endotoxin tolerance” that reprograms TLR4 responses to subsequent LPS challenge by altering expression of inflammatory mediators. Endotoxin tolerance limits the excessive cytokine storm and prevent tissue damage during sepsis, but renders the host immunocompromised and susceptible to secondary infections. IRAK4 acts as a kinase and an adapter, activating subsets of divergent signaling pathways. In this study, we addressed mechanistically the role of IRAK4 kinase activity in the induction of TLR4 and TLR2 tolerance using macrophages from wild-type (WT) vs. IRAK4 kinase-dead knock-in (IRAK4KDKI) mice. Pretreatment of WT and IRAK4KDKI macrophages with LPS similarly inhibited LPS-induced degradation of IRAK1 and IκB-α, MAPK phosphorylation, induction of proinflammatory cytokines, and up-regulated expression of negative regulators of TLR signaling, A20 and IRAK-M. Prior exposure of WT macrophages with Pam3Cys, a TLR2-TLR1 agonist, ablated p38 and JNK phosphorylation in response to subsequent challenges with Pam3Cys or LPS. In contrast, IRAK4KDKI macrophages exhibited attenuated TLR2-elicited homo- and heterotolerance at the level p38 and JNK activation. Thus, IRAK4 kinase activity is not required for the induction of endotoxin tolerance, but significantly contributes to TLR2-elicited homo- and heterotolerance.
Inhibition of TLR4 signaling by TRAM-derived decoy peptides in vitro and in vivo (P1232)
Authors: Toshchakov Vladimir , Piao Wenji , Vogel Stefanie 
Published: January 2023

Abstract
Toll/IL-1R domain- (TIR-) containing adapter inducing IFN-β- (TRIF-) related adapter molecule (TRAM) enables recruitment of TRIF to activated TLR4 and thereby mediates the TLR4-driven induction of TRIF-dependent cytokines. A library of cell-permeating decoy peptides derived from TRAM TIR domain has been screened for the ability of individual peptides to inhibit TLR4 signaling in primary macrophages. Peptides derived from TRAM TIR BB loop (TM4) and C helix (TM6) inhibited the LPS-induced activation of MyD88- and TRIF-dependent cytokines, as well as MAPK activation. TM4 and TM6 did not block macrophage activation induced by TLR2, TLR9, or RIG-I-like receptor agonists. Both TM4 and TM6 blocked co-immunoprecipitation of TRAM and TLR4 ectopically expressed in HEK293T cells. Both peptides also blocked the LPS-induced recruitment of MyD88 to TLR4 in primary murine macrophages. In vivo examination of TRAM-derived peptides demonstrated that all peptides that were inhibitory in vitro, profoundly suppressed systemic inflammatory response elicited in mice by a sublethal LPS dose, and protected against a lethal LPS challenge. Both intraperitoneal and intravenous administration of a select peptide effectively mitigated the cytokine production induced by an intraperitoneal LPS injection. This research identifies novel TLR inhibitors effective in vitro and in vivo and validates the approach taken in this study as a rational way for development of signaling inhibitors and therapeutics leads.
Differences in both Toll-like receptor 4 and MD-2 account for the host-specific recognition of Bordetella pertussis lipid A (P1240)
Authors: Maeshima Nina , Hajjar Adeline , Fernandez Rachel 
Published: January 2023

Abstract
The lipid A portion of lipopolysaccharide (LPS) activates innate immunity through the Toll-like receptor 4 (TLR4)/MD-2 complex on host cells. Variation in lipid A has significant consequences for TLR4 activation and thus may be a means by which pathogenic bacteria modulate host immunity. We have previously seen that strain BP338 of Bordetella pertussis modifies its lipid A by the addition of glucosamine moieties which promote TLR4 activation in human macrophages. In the absence of glucosamine modification, TLR4 activation is attenuated. This effect is host-specific: glucosamine modification does not affect mouse TLR4 activation. We hypothesized that the species-specific effect is due to differences in TLR4, and used inter-species chimeric receptors to test this hypothesis. We found that the middle 330 amino acids of TLR4 were sufficient to generate species-specific responses to B. pertussis lipid A, dependent on co-expression of the matching species of MD-2. Based on the published crystal structure of TLR4/MD-2 in complex with E. coli LPS, we narrowed our search to the interface where TLR4/MD-2 complexes dimerize for activation, and used site-directed mutagenesis to identify specific amino acid residues in TLR4 and MD-2 that promote activation in response to B. pertussis lipid A. Taken together, we have demonstrated that charged amino acids in TLR4 and MD-2 contribute to host-specific immune responses to lipid A variants from B. pertussis.
Processing of human TLR7 by furin-like proprotein convertases is required for its accumulation and activity in endosomes. (P1244)
Authors: Hipp Madeleine , Gileadi Uzi , Sheperd Dawn , Reis e Sousa Caetano , Cerundolo Vincenzo 
Published: January 2023

Abstract
Toll-like receptor 7 (TLR7) triggers antiviral immune responses by recognizing viral single-stranded RNA in endosomes. Although this mechanism underscores the ability of TLR7 to distinguish between self and not self RNA, the biosynthetic pathway of TLR7 remains unclear. Here, we show for the first time that human TLR7 is proteolytically processed and that the C-terminal fragment selectively accumulates in endocytic compartments, where it is functionally active. Human TLR7 processing depends on the action of furin-like proprotein convertases and occurs at neutral pH. Knockdown of Furin and mutation of a furin-like recognition site within TLR7 inhibits processing of the receptor. Furthermore pro-inflammatory stimuli such as PMA and IFN-γ enhance processing of and signalling through TLR7, consistent with an observed upregulation of Furin. This mechanism might serve as a positive feedback loop generating active TLR7 after infection. As self-RNA can under certain conditions activate TLR7 and trigger autoimmunity, our results identify furin-like proprotein convertases as a possible target to attenuate TLR7-dependent autoimmunity and other immune pathologies.
Neuritin promotes the expansion and persistence of regulatory T cells in vitro and in vivo. (P1051)
Authors: Barbi Joseph , Yu Hong , Pardoll Drew , Pan Fan 
Published: January 2023

Abstract
Regulatory T cells (Tregs) represent a major force for immune control and the prevention of excessive or self-directed responses. These cells are marked by expression of the transcription factor Foxp3 and their ability to suppress leukocyte activation. Deficits in Treg function or numbers result in immune mediated pathologies, reflecting their importance as a layer of immune regulation. However the factors and mechanisms responsible for maintaining these cells are incompletely understood. Here, we report that a GPI-anchored molecule known as neuritin is highly and uniquely expressed by Treg and this molecule potently drives expansion and persistence of the ‘Treg-pool’. Transgenic expression of neuritin by Tregs enhanced their division in vitro while treatment with anti-neuritin antibody stunted Treg proliferation. Further suggesting a role in maintaining the Treg population, neuritin transgenic mice displayed expedited recovery in the EAE model of autoimmune disease accompanied by elevated frequencies of Foxp3+ Tregs. Conversely, antibody mediated neuritin blockade prolonged disease and stunted proliferation of Foxp3+ cells during EAE. Further dissection of neuritin activity revealed the involvement of paradoxically enhanced IL-2 production by this Treg specific factor. In all these findings strongly implicate neuritin as a Treg derived factor responsible for the persistence of these cells and their control of immune responses.
Low dose intradermal infection with Trypanosoma congolense leads to expansion of regulatory T cells and enhanced susceptibility to reinfection (P1055)
Authors: Onyilagha Chukwunonso , Kuriakose Shiby , Singh Rani , Okwor Ifeoma , Uzonna Jude 
Published: January 2023

Abstract
BALB/c mice are highly susceptible to experimental T. congolense infection. Interestingly, a recent report showed that these mice are relatively resistant to primary intradermal low dose infection. Paradoxically, repeated low dose intradermal infections predispose to enhanced susceptibility to an otherwise non-infectious dose challenge. Here, we explored the mechanisms responsible for this low-dose-induced susceptibility to subsequent low dose reinfection. We found that akin to intraperitoneal infection, low dose intradermal infection leads to production of IL-10, IL-6, IL-12, TGF-β and IFN-γ by spleen and draining lymph node cells. Interestingly, despite the absence of parasitemia, low dose intradermal infection led to expansion of CD4+CD24+Foxp3+ cells (Tregs) in both the spleens and lymph nodes draining the infection site. Depletion of Tregs by anti-CD25 mAb treatment during primary or before reinfection following repeated low dose infection completely abolished the low dose-induced enhanced susceptibility to rechallenge infection. In addition, Tregs depletion was associated with dramatic reduction in serum levels of TGF-β and IL-10. Collectively, these findings show that low dose intradermal infection leads to rapid expansion of Tregs and these cells mediate enhanced susceptibility to subsequent infection.
T cell activation threshold regulated by E3 ubiquitin ligase Cbl-b determines fate of inducible regulatory T cells (P1025)
Authors: Zhao Yixia , Qiao Guilin , Yang Tianlun , Zhang Jian 
Published: January 2023

Abstract
E3 ubiquitin ligase Cbl-b is critical for establishing the threshold for T cell activation, and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of inducible CD4+CD25+ regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by CD4+CD25- T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is down-regulated in vitro. Using TCR transgenic Rag-/- mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.
Reprogramming tumor microenvironment in cancer immunotherapy (P4274)
Authors: Kalinski Pawel , Muthuswamy Ravikumar , Obermajer Natasa , Zeh Herbert , Zureikat Amer , Edwards Robert , Bartlett David 
Published: January 2023

Abstract
Type-1 immune effector cells (CTLs, Th1 and NK cells) express high levels of CXCR3 and CCR5 and depend on the relevant chemokine ligands (CXCL9, CXCL10 and CCL5) for entry into tumor microenvironments. We observed that local production of prostaglandin (PG)E2 suppresses the induction of immune effector cell and selectively inhibits the production of the effector cell-attracting chemokines within peritoneal tumors (ovarian and colorectal cancers and different forms of peritoneal carcinomatosis). COX2 is also the key factor driving the MDC/CCL22- and SDF1/CXCL12-mediated accumulation of Tregs and myeloid-derived suppressor cells (MDSCs) in tumor tissues, orchestrating local inhibition of CTL function. COX2 blockers antagonized the accumulation and suppressive function of MDSCs and proved to be a necessary (but not sufficient) element of strategies aimed at restoring local immunosurveillance. While TLR-ligands used as stand-alone vaccine adjuvants can amplify the PGE2-driven suppressive events, their combination with COX2 inhibitors and IFNα allows to selectively induce effector cell-attracting chemokines within tumors (bu tnot marginal tissues) and suppress the MDSC- and Treg-attracting chemokines (CXCL12 and CCL22). Our current trials evaluate the effectiveness of tumor-selective conditioning of tumor microenvironments to promote the entry of spontaneously-arising and vaccination-induced CTLs into tumors and counteracting local immunosuppression.
Transcriptomic studies of Mycobacterium avium subsp. paratuberculosis infected monocyte-derived macrophages from individual dairy cows (P6095)
Authors: Switzenberg Jessica , Kabara Edward , Sommer Sandra , Coussens Paul 
Published: January 2023

Abstract
Johne’s disease in cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic wasting disease characterized by granulomatous enteritis, persistent diarrhea, and death. Following ingestion, MAP is phagocytosed by intestinal macrophages, eliciting an early pro-inflammatory Th1-like response. However, an ineffective Th2-like response becomes predominate during a long subclinical phase, allowing MAP to persist within host cells. Previously, our group has shown that infected macrophages are defective in response to CD40 ligand stimulation with reduced expression of IL-12p40 and iNOS. Infected macrophages also exhibit increased expression of IL-10 and decreased apoptosis relative to control cells. A core set of 93 genes exhibit altered expression during early infection by various strains of MAP, as assessed by microarray studies. These genes provide candidates that may be involved in efficiency of infection and intracellular survival of MAP. Indeed, susceptibility and infection rate of macrophages from individual cows are highly variable, possibly due to genetic differences between individuals. We present initial studies using RNA-seq to further analyze transcriptome changes in cultured bovine monocyte-derived macrophages (MDMs) from different cows that have also been genotyped. Measurements of percent infectivity and spontaneous apoptosis are also utilized to help determine additional candidate genes that may be involved in MAP survival in host cells.
From viral genome to specific peptide epitopes: methods for identifying porcine T cell epitopes based on in silico predictions, in vitro identification and ex vivo verification (P6098)
Authors: Pedersen Lasse , Rasmussen Michael , Harndah Mikkel , Patch Jared , Nielsen Morten , Buus Soren , Golde William , Jungersen Gregers 
Published: January 2023

Abstract
The affinity with which major histocompatibility complex (MHC) class I molecules bind peptides is instrumental to presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). We analyzed three swine leukocyte antigen (SLA) molecules for complete nonamer peptide-based binding matrices in order to predict likely candidates for peptide-SLA binding. These results were combined with binding predictions generated by the algorithm, NetMHCpan (http://www.cbs.dtu.dk/services/NetMHCpan/) in order to select peptide candidates for in vitro analysis. The correlation between high affinity and high stability was determined using luminescence oxygen channeling (LOCI) and scintillation proximity assay (SPA) for peptides bound by the commonly occurring SLA molecules, SLA-1*0401, SLA-2*0401, and SLA-3*0401. Using these tools, peptides bound by SLA with high affinity and stability were identified from a library of 10.000 peptides. T cell epitopes were identified using peptide-SLA complexes assembled into fluorescent tetramers to stain swine influenza specific CTLs derived from immunized animals and MHC-defined pigs vaccinated against foot-and-mouth disease virus. These results demonstrate the broad applicability of methods originally developed for analysis of human leukocyte antigen (HLA) presentation of peptides. The methods presented provide a timely and cost-effective approach to CTL epitope discovery that can be applied to diseases of swine and of other mammalian species of interest.
B cell immunogenetics in the T cell receptor repertoire generation of shark (P6069)
Authors: Criscitiello Michael , Eubanks Jeannine , Chen Patricia , Castro Caitlin , Wallace Andrea , Ohta Yuko , Flajnik Martin 
Published: January 2023

Abstract
Rearranged immunoglobulin (Ig) variable (V) regions undergo somatic hypermutation (SHM) in activated B cells during a T-dependent humoral immune response, allowing affinity maturation. TCR loci were believed not to undergo SHM, until the demonstration of SHM at the TCRγ locus of sandbar sharks. Our study in the nurse shark corroborates this seminal finding, as we have found SHM of TCRγ loci in spleen; we further show that TCRγ V regions are highly mutated in clones isolated from the spiral valve (shark intestine), suggesting a role for T cell SHM in mucosal immunity. Surprisingly, we also found SHM to occur extensively at shark TCRα loci. Unlike B cell SHM that follows antigen exposure in sharks and most other vertebrates, SHM at TCRα loci occurs during thymic development. TCRα SHM is supported with isolation of clusters of mutated clones having the same CDR3 rearrangement.We suggest that such SHM, in addition to TCRα receptor editing, permits developing T cells with evolving receptors to scan cortical epithelial cells for positive selection on self-peptide/self MHC. Additionlly, we have found that shark TCRδ can employ variable gene segments of IgM and IgW in rearrangements, a situation that may be similar to the IgHV use by TCRδ noted in higher vertebrates. Future studies must further study the lineage commitment of shark B and T cells and the receptor plasticity of these lineages in all vertebrates.
Coral as a model to study intrinsic and innate immune responses to herpes-like viruses (P6081)
Authors: Garrison Keith , Daisa Nicole 
Published: January 2023

Abstract
Porites corals have been shown to produce herpes-like viruses when triggered by environmental stressors. We intend to use coral as a model species to study the immune responses to herpes-like viruses in an invertebrate species. The long-term goals of our study include measuring virus production in multiple coral species, determining if virus production increases under stress, and identifying possible immune genes in corals expressed in response to virus production. One frag of Porites and one of Acropora were grown in separate tanks, each containing artificial seawater and live rock. Stabilization of the corals was achieved through the use of timed overhead lighting, regular feeding, weekly testing of the water for calcium and dKH levels, and periodic addition of KH and magnesium. Two DNA extractions were done on each coral sample and used for PCR. Four genes are initially being targeted: ND5, a control; Spondin2, a putative immune gene; Hsp16, a stress gene; and Thymidylate synthase, produced by the herpes-like virus identified in Porites. Standard PCR has been effective at amplifying these sequences from genomic DNA. After optimization of PCR conditions and product sequencing, qPCR protocols will be developed to quantify gene expression. Future experiments will test the utility of measuring immune dysfunction as a leading indicator of coral reef stress.
Influenza virus infection elicits antibodies and T cells to host cell antigens previously identified as tumor associated antigens: a new view of cancer immunosurveillance (P2131)
Authors: Iheagwara Uzoma , Finn Olivera 
Published: January 2023

Abstract
We are testing the hypothesis that febrile viral infections or other acute inflammatory events cause transient aberrant expression of many self-proteins and induce immune responses and immune memory that participates in cancer immunosurveillance by targeting some of the same molecules aberrantly expressed by malignant cells. We previously used infection of mice with two different strains of influenza virus and profiled post-infection anti-tumor IgG responses against mouse tumor 3LL with the 2-D gel technique DIGE. We identified over a 60 molecules recognized by post-infection IgG and selected for further study GAPDH, Histone H4, Malate Dehydrogenase (MDH), Annexin A2, and HSP90, all previously reported as tumor associated antigens. To determine if the infection induced CTL, we selected peptides MDH251-260, GAPDH300-310, Annexin A2184-191, and H487-95 based on their MHC I binding affinity. We infected C57BL/6 mice with influenza virus, harvested spleens five days after secondary infection, purified T cells, and stained with Dimer-X reagent loaded with individual peptides. We detected GAPDH, H4, MDH, and Annexin A2 specific CD8 T cells showing that in addition to antibodies, aberrant expression of these molecules on flu infected tissues elicits CD8 T cells. Studies are in progress to examine the ability of this immune response to control tumor growth.
Conversion of CD11bhighCD27high natural killer cells into myeloid-derived suppressor cells in the tumor environment is inhibited by interleukin-2 (P2143)
Authors: Park Young-Jun , Song Boyeong , Kang Chang-Yuil 
Published: January 2023

Abstract
NK cells are an important arm of tumor immunity. The sustained numbers and functions of NK cells are barriers against tumor formation and produce a good prognosis in tumor patients. In this study, we demonstrated a potential mechanism of NK cell reduction in implanted tumor models. The CD11bhiCD27hi NK cells from the tumor-bearing mice were converted into MDSCs, indicating the cause of the NK cell reduction. IL-2 inhibited the conversion of the NK cells, and the IL-2 treatment in vivo preserved the NK cell phenotype and may have therapeutic effects on the implanted tumor. Our findings contribute to understanding the mechanisms by which tumors subvert the immune system and are also noteworthy that our data suggested the possibility of conversion between lymphoid (NK cells) and myeloid cells (MDSCs) which are derived from distinct progenitor cells.