bluuebunny/crossref_metadata_2025 huggingface dataset:
The RiboPuroMycylation method reveals surprising protein synthesis in bone marrow T cells within a day of vaccinia virus infection (P3385)
Authors: Seedhom Mina , David Alexandre , Bennink Jack , Yewdell Jonathan
Published: January 2023

Protein translation is an essential and intricately regulated cellular process. We recently described the RiboPuroMycylation Method (RPM) to measure protein translation in cultured cells. RPM is based on ribosome-catalyzed addition of puromycin to nascent chains followed by detection with a monoclonal antibody specific for puromycin in fixed and permeabilized cells. To apply RPM in vivo, we administered puromycin intravenously and measured puromycin staining via flow cytometry. We were able to detect a robust signal that was blocked by ribosome targeting antibiotics that prevent nascent chain puromycylation. After infection with either vaccinia virus (VACV) or influenza virus, we find profound increases in protein translation in activated T and B cells. In mice with adoptively transferred OT-1 T cells we find that translation of individual cells peaks 3 days after a foot pad infection, i.e. several days before T cell numbers peak. One day after an intra-peritoneal VACV infection, almost 40% of the CD4+ and CD8+ T cells in the bone marrow (BM) have increased protein translation as compared to only 3% of the T cells in the spleen. Using transgenic OT-1 T cells and VACV with or without SIINFEKL, we find that the increased protein translation in the spleen and BM is antigen-dependent. Taken together, these findings illustrate the ability of in vivo RPM to provide novel information regarding the participation of translational regulation in workings of the immune system.
A robust and sensitive flow cytometric method for multiplex RNA in-situ hybridization analysis of blood cells using oligo probes and branched DNA based signal amplification (P3356)
Authors: Lai Chunfai , Levinson Shauna , Lin Audrey , Pache Jared , Goltsev Yury , Nolan Garry , Bers George , Nguyen Quan
Published: January 2023

We present here a novel method for in-situ hybridization (ISH) detection of multiple RNA targets in single cells using standard flow cytometer. This method is based on the use of oligo pairs as probes and bDNA signal amplification for highly sensitive and specific ISH detection of RNA. The oligo pairs are designed to hybridize to specific target sequences and can be readily synthesized. The oligo probes/RNA target complex is detected after signal is generated by bDNA signal amplification. The method is compatible with immunostaining, so simultaneous staining of protein and RNA in single cells is feasible. We demonstrated the co-staining of RNA and surface marker proteins in PBMC, B cells, T cells and leukemic cell lines of U937, K562, Jurkat and M1. In addition, the flow cytometric analysis of cytokine gene induction by lipopolysaccharides/R848 in PBMC was shown by staining with antibody for CD14 protein and probes for RNAs encoding interleukin-1 beta, -6, -8 and tumor necrosis factor-alpha. Taken together, this novel RNA ISH flow cytometry assay offers an invaluable tool for studying immune response, stem cell biology and infectious diseases.
IL-17 mediates the protective actions of PPAR γ blockade during enteroaggregative Escherichia coli infection (P3238)
Authors: Philipson Casandra , Bassaganya-Riera Josep , Hontecillas Raquel
Published: January 2023

Enteroaggregative Escherichia coli (EAEC) is an etiologic agent of enterocolitis and persistent diarrhea worldwide, especially in malnourished children and immunosuppressed individuals. Expression of IL-17, which is produced by a number of immune and epithelial cells, increases during EAEC infection. We previously demonstrated that blocking peroxisome proliferator-activated receptor gamma (PPARγ) enhances mucosal Th17 responses that contribute to bacterial clearance in EAEC-infected mice. Here we investigate whether IL-17 mediates the effects of PPARγ blockade in the gut. We show that increasing doses of PPARγ antagonist GW9662 (0.5, 1, or 2µM) directly correlated to increased IL-17 expression in the colon. C57BL/6 wild type mice were challenged with EAEC strain JM221 on day 0 and administered anti IL-17 neutralizing antibody (50μg) i.p. on days 0, 2, and 4 of the study. Anti (α)-IL17 antibody-mediated neutralization of IL-17 abrogated the beneficial effects of PPARγ blockade on weight loss and fecal EAEC burden. In addition, infiltration of CD11c+ dendritic cells and CD4+ T cells into the colonic lamina propria, as well as colonic antimicrobial peptide expression were significantly reduced in mice treated with αIL-17 and GW9662 when compared to mice receiving GW9662 alone. These findings suggest that the beneficial effects from PPARγ blockade in disease activity and bacterial burden during EAEC infection are largely mediated through an IL-17-dependent mechanism.
Annexin 1 in microparticles promotes intestinal mucosal wound repair during inflammation. (P3264)
Authors: Nusrat Asma , Leoni Giovanna , Neumann Philipp-Alexander , Alam Ashfaqul , Lambeth David , Hilgarth Roland , Kusters Dennis , Reutelingsperger Chris , Perretti Mauro , Parkos Charles , Neish Andrew
Published: January 2023

Inflammation-induced epithelial injury results in compromised mucosal barrier function. In response to injury, epithelial cells migrate as a sheet to cover denuded surfaces. We have recently reported that a calcium dependent phospholipid binding protein Annexin A1 (AnxA1) is a pro-resolving mediator that signals via epithelial formyl peptide receptors (FPRs) to promote intestinal mucosal wound repair. Analysis of signaling pathways downstream of epithelial FPRs identified intestinal epithelial NADPH oxidase-1 (Nox1) and Rac1 as key elements responsible for generation of reactive oxygen species (ROS). We determined that ROS-induced oxidative modification of phosphatases, in turn, activate proteins involved in controlling focal cell matrix adhesions and epithelial cell migration. Exogenous AnxA1 administration was observed to promote intestinal mucosal wound closure in WT mice but not in mice lacking intestinal epithelial Nox1 (Nox1-/-IEC). Analysis of AnxA1 released from the epithelial cells revealed that it is shed in membranes that have properties of microparticles (<1 µm) as an N-terminal cleaved product. Incubation of wounded epithelial monolayers with AnxA1containing microparticles promoted wound closure. These findings define a mechanism by which intracellular AnxA1 is cleaved and released in epithelial-derived microparticles during inflammation which can then signal in a paracrine fashion via FPR and Nox1 to promote intestinal mucosal wound repair.
Toll-like receptor signaling regulates mucosal barrier function and antigen acquisition in the small intestine (P3266)
Authors: Miller Mark , Xu Jack , McDole Jeremiah , McDonald Keely , Knoop Kathryn , Newberry Rodney
Published: January 2023

The mucosal immune system must efficiently recognize foreign antigen during intestinal infection and yet avoid triggering inappropriate inflammatory responses to commensal microbes and food antigens. In vivo two-photon imaging revealed that low-molecular weight material can cross the epithelium of the small intestine during goblet cell secretion, a phenomenon termed goblet cell-associated antigen passages (GAPs). GAPs are abundant in the steady-state and selectively deliver foreign antigen to CD103+ lamina propria dendritic cells with tolerogenic potential. However, under pathological conditions, goblet cell secretion and epithelial permeability are dramatically altered. The cellular mechanisms that regulate barrier function in response to luminal microbial products are poorly understood, but have important ramifications for intestinal pathogenesis and oral vaccine efficacy. We found that Toll-like receptor (TLR) ligands introduced acutely into the intestines of germ free mice modulated GAP formation in vivo. Furthermore, mice deficient in TLRs that signal through TRIF, lacked GAPs and had reduced epithelial permeability. We propose that a subset of villus epithelial cells expressing TLR3/TLR4 and containing high levels of serotonin (5HT) can induce GAP formation in the small intestine by secreting 5HT in response to TLR ligands.
Delineating residential macrophage heterogeneity in lung disease (P3282)
Authors: Duan Mubing , Li Waichu , Steinfort Daniel , Irving Louis , Anderson Gary , Hibbs Margaret
Published: January 2023

In healthy lungs, alveolar macrophages comprise >95% of the immune cells in the alveolar airspaces where they regulate local lung surfactant production and act as the primary sentinels of respiratory pathogens. Interestingly, increased AMΦ numbers are observed in many animal models of chronic lung diseases and also clinically in patients with COPD. Combining flow cytometry and chimeric mice studies, we observed that residential AMΦs form two distinct subpopulations (by either remaining Mac-1neg/low or turning Mac-1pos) during acute inflammation and in chronic inflammatory lung disease subsequent to SHIP-1 deletion. SHIP-1-/- mice which spontaneously develop COPD-like lung disease maintain both Mac-1neg/low and Mac-1pos AMΦ subpopulations. This additional Mac-1pos subpopulation highly expresses MMP-12 and tracks with the induction of lung disease using SHIP-1-/- chimeric mice. Altogether, our results suggest that residential AMΦ heterogeneity is a component of acute lung inflammation and chronic inflammatory lung disease. Consequently, whether residential AMΦ heterogeneity may be a novel hallmark of inflammation-driven human lung diseases has been our final area of study. Overall, our studies of both animal models and patient samples may allow us to better understand the role of AMΦ heterogeneity in lung homeostasis and disease.
Oxidation resistance 1 attenuated lupus nephritis by suppressing oxidative stress and inflammation in kidney (P5119)
Authors: Li Quanzhen , Li Yajuan , Yan Mei , Raman Indu , Mohan Chandra
Published: January 2023

Oxidation resistance 1 (Oxr1) was identified to be up-regulated in mouse kidney upon anti-GBM antibody challenge and may play a role in anti-GBM disease and lupus nephritis. Since Oxr1 has been shown to confer resistance against oxidative stress by detoxify reactive oxygen species (ROS), we speculate that Oxr1 may have protective effect against oxidative stress induced renal damage. Thus, we utilized mesenchymal stem cells (MSCs) as vehicles to deliver Oxr1 into the injured kidneys of lupus nephritis model mice and investigated the influence of Oxr1 on glomerulonephritis. Human OXR1 gene was transfected into mouse bone marrow-derived MSCs via lentiviral vector pCDH to establish a stable hOXR1-MSC cell line. 106 hOXR1-MSCs were injected (i.v.) into B6.Sle1.Sle2.Sle3 lupus mice and nephritis phenotype was observed for 6 months. Compared with vector-MSCs treated mice, the hOXR1-MSCs administrated mice showed significantly decreased blood urea nitrogen (BUN), proteinuria and ameliorated renal pathology. hOXR1-MSCs transplantation significantly reduced macrophage and T lymphocyte infiltration by inhibiting the expression of CCL2, CCL7, IL-1β, IL-6 and NFκB. hOXR1-MSCs prevented hydrogen peroxide induced oxidative stress and its implantation reduced NO in mouse serum and urine and increased catalase and GPX1 to inhibit tubular cell apoptosis. In conclusion, OXR1 exerted protective effect on nephritis by suppressing inflammation and oxidative stress.
Oxidative damage and transmethylation micronutrient effects on the T cell epigenome in lupus (P5141)
Authors: Ray Donna , Richardson Bruce
Published: January 2023

The development of human lupus is attributed in part to T cell DNA demethylation. The mechanisms causing T cell DNA demethylation in lupus patients are poorly understood. DNA methylation is dependent on the methyl donor S-adenosylmethionine (SAM) and DNA methyltransferase (Dnmt) activity, and is inhibited by S-adenosylhomocysteine (SAH). Several dietary nutrients affect SAM levels including folate and methionine. Additionally, Dnmt levels are low in lupus patients due to defective ERK pathway signaling, caused by oxidative stress resulting in PKCδ nitration. We hypothesized that T cells subjected to oxidative stress will overexpress genes normally suppressed by DNA methylation due to low Dnmt levels, and the overexpression will increase in the presence of low folate and methionine levels. We treated PHA-stimulated human T cells with the oxidant peroxynitrite, cultured the cells in media containing high or low folate or methionine levels, and compared the effects on expression of the methylation sensitive genes CD70 and KIR. Peroxynitrite treated T cells cultured in lower nutrient concentrations overexpressed CD70 and KIR compared to those cultured in higher concentrations and/or no oxidant. These results suggest that low nutrient levels may increase T cell epigenetic changes caused by oxidation. Since several environmental insults resulting in oxidative stress are associated with lupus, dietary supplementation may mitigate the damage.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Effects of colorectal cancer liver metastases on intrahepatic B cells (P2093)
Authors: Thorn Mitchell , Burga Rachel , Ngyen Cang , Licata Lauren , Espat N. , Katz Steven
Published: January 2023

Background: We and others have found that liver metastases (LM) mediate tolerogenic effects. B cells make up a significant proportion of liver lymphocytes but their function in the normal liver and in the setting of LM is poorly defined. Therefore, we attempted to define the features of HBC in the setting of LM. Methods: We have used a murine model of CEA+ colorectal LM to study the effects of malignancy on HBC. Tumor cells were injected into the portal circulation of C57Bl/6 mice to induce LM, and HBC were analyzed after two weeks. Results: 50% of mice with LM secreted anti-CEA antibodies, suggesting that the tumor is recognized by B cells in our model. HBC comprised 45±10% of lymphocytes in normal livers, and their frequency was reduced in mice with LM (25±3%, p=0.03). A significant reduction in the frequency of IgM+IgDlo transitional B cells, precursors of mature B cells, was observed in mice with LM (23±2% and 8±4% in normal and metastatic livers respectively, p=0.02). HBC exhibited significant down-regulation of two molecules associated with T cell activation: MHCII (7.5±3.5%, down from 31±5% in normal livers, p=0.03) and CD80 (0.9±0.4%, down from 9.6±1.4% in normal livers, p=0.02). Conclusion: The ability of HBC to mount an immune response may be compromised in the setting of LM as evidenced by reduction of HBC number and diminished HBC expression of MHCII and CD80. Further research is needed to elucidate the function of hepatic B cells during liver metastasis.
Reducing agents such as ascorbic acid or N-acetylcysteine support the chemotactic and proliferation-inducing effect of S100A4 on mesenchymal stromal cells (P2165)
Authors: Grassl Stefan , Yildiz Tatjana , Jahrsdörfer Bern , Schrezenmeier Hubert , Lotfi Ramin
Published: January 2023

Chronic inflammatory response is found in most advanced solid tumors with subsequent necrosis associated with release of various damage-associated molecular patterns (DAMPs) including S100A4. Mesenchymal stromal cells (MSCs) seem to interfere with the anti-tumor-immune response resulting in a poor prognosis for cancer patients, given the immunosuppressive capacity of these cells. In previous experiments we already showed that S100A4 induces MSC proliferation with an optimum at 100ng/ml. We now tested higher S100A4 concentrations and observed a bimodal effect with a second optimum achieved when using more than 2000ng S100A4/ml. Furthermore, stimulation of MSCs with graded concentrations of S100A4 inhibited dose-dependently the proliferation of lymphocytes. Based on our previous observations that S100A4 activity is enhanced in the presence of uric acid, we now tested further reducing agents like ascorbic acid, dithiothreitole, and N-acetylcysteine and could demonstrate increased chemotactic activity of S100A4 on MSCs when mentioned antioxidants were present, while they had minimal - if any - effect by themselves. Considering that MSCs respond to necrosis-associated factors, we conclude that creating an oxidative environment could be one possible strategy to inactivate not only S100A4 but also further oxidation-sensitive DAMPs like HMGB1, which contribute to the immunosuppressive nature of tumor microenvironment.
Gap junction intercellular communication enhances NK cell activation and cytotoxic capacity (P2185)
Authors: Salazar-Onfray Flavio , Tittarelli Andres , Mendoza-Naranjo Ariadna , López Mercedes , Chambers Benedict , Lundqvist Andreas , Guerrero Israel
Published: January 2023

Gap junctions (GJ) mediate intercellular communications between adjacent cells. Previously, we showed that connexin 43 (Cx43) was accumulated between DCs mediating antigen transfer and recruited to the immunological synapse during T cell priming. Moreover, this intercellular crosstalk contributed to T cell activation, intracellular Ca2+ responses, and cytokine release. However, the role of Cx43-GJ in NK cell activation and NK cell cytotoxicity against tumor cells, have not been elucidated. Here, we evaluated GJ role in the NK-DC interaction and in NK cell cytotoxicity against tumors. Cx43 expression was quantified on NK-DCs and in NK-tumor cell co-cultures using CX43 Abs. GJIC was evaluated by Calcein transfer and GJ inhibitors. Cytotoxicity, Granzyme-B activity and Ca2+ signaling was evaluated by flow cytometry. We found that NK cells form functional GJ with DC and tumor cells. The polarization of Cx43 increases in the NK-DCs synapsis and between NK and melanoma or K562 cells. The blocking of Cx43 using an analogue peptide or antisense RNA inhibited Ca2+ influx, Granzyme-B activity and NK cytotoxicity. NK-DC communication was bidirectional, although blocking of GJIC inhibited NK cell activation but not DC function. Moreover, Cx43-blocking inhibited tumor lysis by NK cells. Thus, our data suggest that GJ play an important role in the NK cell activation and anti-tumor effector function.
Tumor infiltrating lymphocytes derived from metastatic melanoma during intralesional immunostimulatory therapy exhibit a phenotype reflecting tumor progression (P2193)
Authors: Sieling Peter , Faries Mark , Morton Donald , Lee Delphine
Published: January 2023

Intralesional immunostimulatory therapy is a rational approach for in transit metastatic melanoma, as lesions are accessible and provide a source of matched tumor antigen to facilitate local and systemic immune responses. Moreover, the lesions are accessible for evaluation of the local immune response at the site of disease. To examine the immunological status at the site of disease during the course of intralesional therapy, T cells were extracted from lesions, cultured to preserve their in vivo phenotype, and expanded for functional analysis. T cells isolated from the skin lesions two weeks after initiation of intralesional therapy produced elevated Th1 cytokines relative to pre-therapy lesions. In contrast, T cells isolated at nine weeks produced decreased Th1 cytokines, but elevated Th2 cytokines, soluble IL-2Ra and IL-6, mediators associated with tumor progression. Expression of skin homing receptor cutaneous lymphocyte antigen (CLA) on lesion derived T cells correlated with Th1 cytokine production. Strikingly, lesion-derived Th1 cytokines and CLA expression were inversely correlated with tumor size during the course of therapy, whereas the phenotype of T cells derived from the blood did not change dramatically over the course of the therapy. Our studies indicate that T cells isolated from the site of disease in melanoma provide insight into the clinical state of the patient and suggest that they are critical regulators in the progression of tumor in malignant melanoma.
OX40 engagement promotes potent tumoricidial CD4+ T cells (P2199)
Authors: Hirschhorn-Cymerman Daniel , Budhu Sadna , Kitano Shigehisa , Merghoub Taha , Wolchok Jedd
Published: January 2023

Several immune-based strategies are currently used to treat melanoma in the clinic; however, very few result in a complete response. CD4+ T cells are potent mediators of anti-tumor immunity and adoptive transfer of specific CD4+ T cells leads to tumor regression. OX40, a costimulatory molecule expressed primarily on activated CD4+ T cells, promotes and enhances anti-tumor immunity with limited success on large tumors in mice. Here we show that OX40 engagement induces a novel CD4+ T cell population characterized by the expression of the master regulator eomesodermin that leads to both terminal differentiation and central memory phenotype, with concomitant secretion of Th1 and Th2 cytokines. This subpopulation of CD4+ T cells eradicates very advanced melanomas in mice, and an analogous population of human tumor-specific CD4+ T cells can kill melanoma in an in vitro system. Interestingly, the combination therapy effectively eliminates tumor escape variants. Our results show that these uniquely programmed effector CD4+ T cells have a distinctive phenotype with increased tumoricidal capability and support the use of immune modulation in reprogramming the phenotype of CD4+ T cells
Tumor growth and anti-tumor immunity depend upon extent of metabolic energy devoted to thermoregulation in laboratory mice (P2099)
Authors: Kokolus Kathleen , Capitano Maegan , Lee Chen-Ting , Eng Jason , Hylander Bonnie , Waight Jeremy , Gordon Christopher , Abrams Scott , Repasky Elizabeth
Published: January 2023

Laboratory mice are housed under relatively cool conditions and even though their core temperature remains normal, mice are cold stressed and exhibit significant increases in metabolic heat production. We compared tumor growth in mice housed at a standard ambient temperature (22oC) and at a thermoneutral temperature (30oC) which reduces metabolic effort needed for maintaining core temperature. We found significantly reduced tumor incidence and growth rates in animals housed at thermoneutral vs standard temperature using four different syngeneic tumor models, including a metastasis model. Because this difference was not seen in SCID or NUDE mice or in mice depleted of CD8+ T cells, we suspected that a prominent result of metabolic cold stress is diminished adaptive immunity. Immunohistochemistry and flow cytometry confirmed the presence of more CD8+ T cells (including antigen-specific cells) within the tumor microenvironment and spleens of animals at thermoneutral vs standard temperature. Conversely, animals at standard temperature had significantly increased infiltration of immunosuppressive cell subsets including GR-1+CD11b+ myeloid derived suppressor cells and Foxp3+ cells. These data reveal a fundamental relationship between energy utilized for thermoregulation and CD8+ T cell-dependent immunity and strongly suggest that immunological data collected from tumor bearing mice housed at standard temperature could be significantly biased by chronic cold stress.
ERK mitogen-activated protein kinase regulates Th17 cell production of IL-10 and the pathogenesis of colitis (P3197)
Authors: Liu Houpu , Yao Suxia , Qin Hongwei , Elson Charles , Cong Yingzi
Published: January 2023

Distinct from proinflamamtory Th17 cells which produce high levels of IL-17 and IL-22, and mediate the pathogenesis of inflammatory bowel disease, regulatory Th17 cells have recently been identified, which produce low levels of IL-17 but high level of IL-10. However, the factors and signaling pathways regulating Th17 production of IL-10 are still largely unclear. In this report, we investigated the role of ERK MAPK signal pathway, which is activated by both TGF-β and IL-6, in Th17 cell differentiation and IL-10 production. We demonstrated that blockade of ERK activation by both ERK inhibitor or siRNA specific for ERK activity inhibited Th17 cell development while upregulating Treg cells under Th17 polarization conditions. Inhibition of ERK decreased IL-6 induction of RORγt but enhanced TGF-β induction of Foxp3. Importantly, blockade of ERK activation promoted Th17 cell IL-10 production. ERK inhibitor-treated Th17 cells function differently from proinflamamtory Th17 cells, in that ERK inhibitor-treated Th17 cells possessed suppressive function in vitro as they inhibited naïve T cell proliferation and IFNγ production at the levels comparable to that of Tregs, and had a decreased potency to induce colitis in vivo. Collectively, our data demonstrated that the ERK pathway regulated Th17 differentiation and IL-10 production, and thus interfering with the ERK pathway could provide a therapeutic treatment for inflammatory bowel diseases and other Th17-related autoimmune diseases.