bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency of the leukocyte integrin inhibitor, Del-1, exacerbates experimental autoimmune encephalomyelitis through increased Th17 cell infiltration. (P5198)
Authors: Lim Jong-Hyung , Choi Eun Young , Samus Maryna , Lee Seung Hwan , Kim Hyesoon , Hajishengallis George , Chavakis Triantafyllos
Published: January 2023

Del-1 is an endogenous endothelial-derived inhibitor of LFA-1-mediated leukocyte adhesion (Choi et al. Science 2008). As Del-1 has high expression in brain, we assessed here its role in neuroinflammation in experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis. We found decreased Del-1 expression in the inflamed spinal cord of mice subjected to the EAE protocol. Moreover, Del-1-deficiency in mice resulted in increased EAE severity, associated with increased inflammatory cell recruitment to the inflamed spinal cord. Immunohistochemistry showed that demyelination and axonal damage of the spinal cord was higher due to Del-1-deficiency. Recently, we proved that Del-1 suppressed IL-17-triggered inflammation (Eskan et al., Nat Immunol, 2012). Consistent with this finding, we found that the infiltration of IL-17 producing CD4+ T cells (Th17 cells) was significantly increased at the onset of the disease. This resulted in enhanced infiltration of CD45highCD11b+ monocytes/macrophages that was up to the effector phase of EAE. Subsequent studies revealed that IL17R-/- deficiency reversed the aggravated EAE phenotype of Del-1-deficiency, accompanied by a decrease of leukocyte infiltration into the spinal cord. Our results suggest that Del-1 deficiency exacerbates EAE disease severity by elevating Th17 cell infiltration to the inflamed spinal cord, which implies that Del-1 could be used as a therapeutic approach for neuro-inflammatory disorders.
Identification of novel cell surface markers on mouse and human TH17 cells (P6354)
Authors: Bonnevier Jody , Guerra Alberto , Goetz Christine
Published: January 2023

TH17 cells are a subset of CD4+ T helper cells that protect against extracellular bacteria, but may also be involved in autoimmune disease progression. Consequently, basic and clinical research on TH17 cells has become a prominent and active area of research in immunology. The TH17 subset is defined by the ability to produce cytokines, such as IL-17 and IL-22, and also by the expression of cell surface markers, such as the IL-23 receptor. Much research effort in TH17 cell biology is the search for surface molecules that can be used to consistently characterize TH17 cells in different immune environments, and ultimately lead to potential therapies for autoimmunity. To date, only a few surface molecules have been found that are exclusively expressed by TH17 cells, making the use of surface markers for this cell type a challenge. Our objective was to identify new surface markers to distinguish TH17 cells from other subsets of helper CD4+ T cells. In order to accomplish this goal, we used a high-throughput flow cytometer to screen over 700 surface antibodies on mouse and human TH17 cells. We identified 26 novel surface markers in human TH17 cells that also coexpress IL-22, IL-17, and RORgT, confirming these markers were on functional TH17 cells. Interestingly, 4 of these markers (BTLA, CD200, CD99, and IL-18Ra) were also identified in mouse TH17 cells. Further study of these markers will enable the development of therapies for autoimmunity and cancer research.
Simultaneous multicolor flow cytometric detection and analysis of IL-17A and IFN-γ mRNA and protein levels expressed in human T lymphocytes (P6364)
Authors: Wu Xiaowei , Martin Jody , Park Emily , Ernst David , Berry Jody , Sepulveda Homero , Rohrer Jurg
Published: January 2023

The ongoing development of multiparameter flow cytometers, fluorescent dyes and specific antibodies for surface and intracellular markers provides valuable tools to analyze and define cellular and molecular mechanisms that underlie complex biological systems. These tools have contributed significantly to both basic and clinical research applications including immune system monitoring, drug screening, and diagnostic tests. Recently-developed techniques and reagents allow for the specific, amplified fluorescent detection of different RNA transcript levels expressed in individual cells. In this study, we evaluated the use of multicolor flow cytometry for the simultaneous analysis of differentially coexpressed mRNA and protein levels in primary cells. Using a human Th17-like cell model system, we demonstrate that in vitro-differentiated Th17-like cells can be simultaneously probed for their differentially expressed levels of IL-17A and IFN-γ proteins as well as IL-17A and IFN-γ mRNAs and IL-23 receptor mRNA. These results suggest that numerous potential new mRNA biomarkers can now be defined and used along with conventional immunofluorescent staining of protein biomarkers, to more fully characterize the nature of cells that function in complex biological systems. Simultaneous multicolor flow cytometric analysis of mRNA and protein in individual cells constitutes an important new toolset that can provide novel information from basic and clinical research investigations.
Natural killer cell impairment is associated with liver fibrotic progression in chronic HBV infection (P3361)
Authors: Zhang Zheng , Zhao Juanjuan , Li Yonggang , Wang Fu-Sheng
Published: January 2023

Natural killer (NK) cells have been implicated in inducing fibrosis remission in mice model; however, their roles in fibrotic progression remain to be elucidated in liver fibrosis (LC) patients. We comprehensively characterized peripheral and intrahepatic NK cells in 50 chronic hepatitis B (CHB) patients and 68 HBV-associated LC patients as well as 35 healthy subjects (HC). Both peripheral and intrahepatic NK cell frequencies in LC and CHB patients were significantly decreased, and they expressed lower levels of TRAIL, activation marker, activation receptors and granzyme and perforin and higher levels of inhibitory receptors in LC patients than those from CHB patients, thus displaying the decreased CD107a and interferon (IFN)-γ production and reduced killing capacity against hepatic stallete cells (HSCs). In vitro, HSCs-derived TGF-β could suppress NK cytolytic activity, and blockade of TGF-β significantly enhanced NK cell-derived CD107a and IFN-γ production. The immunohistochemical staining showed that NKp46-positive cells were more enriched in the α-SMA-negative area in livers from LC patients. Finally, NK cell cytolytic activity was also correlated negatively with liver fibrosis scores in HBV infected patients, which is further confirmed by the longitudinal follow-up of LC patients. Our findings may facilitate the rational development of immunotherapeutic strategies to enhance NK activity while limiting or abolishing liver fibrosis in chronic HBV infection.
Exploiting RNA aptamers for cancer therapy (P4407)
Authors: Pratico Elizabeth , Urban Johannes , Johnson Laura , Sullenger Bruce , Nair Smita
Published: January 2023

Nucleic acid ligands (aptamers), both antagonists and agonists, can be used to target receptors on cells with high affinity and specificity. We describe the development of RNA aptamers for several cancer therapeutic applications: (1) targeting antigens to dendritic cells (DCs), (2) immune modulation, and (3) tumor-specific delivery of therapeutics. Our laboratory has successfully engineered a nuclease-resistant RNA aptamer that binds to the macrophage mannose receptor (MMR) expressed on DCs with excellent specificity and high affinity. MMR aptamer-mediated targeting of tumor antigen peptide to DCs resulted in a 4-fold increase of IFN-γ expression, which enhanced cross-presentation and T cell activation in vitro. For immune modulation of T cell function, we have now developed a human OX40 aptamer that is used as an agonist. A multimerized version of the human OX40 aptamer stimulates T cell proliferation and IFN-γ expression. Lastly, we have developed an aptamer to deliver toxic agents to a subset of leukemia and lymphoma cells. This aptamer is engineered into a chimeric molecule possessing two levels of specificity: the binding domain (aptamer) and the agent domain (saporin) that specifically targets and kills leukemia cells (K562) in vitro.
Evaluation of ebolavirus glycoprotein Fc fusion protein as a subunit vaccine (P4417)
Authors: Konduru Krishnamurthy , Shurtleff Amy , Bavari Sina , Kaplan Gerardo
Published: January 2023

Ebolavirus is a Filoviridae that induces severe hemorrhagic fever in humans. Several vaccines based on Filovirus glycoprotein (GP) are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, and subunit GP vaccines such as virus-like particles and soluble protein constructs. Here we describe the development of an ebolavirus subunit vaccine based on GP fused to the Fc fragment of human IgG. We expressed the ectodomain of the Zaire ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing as observed in the membrane-bound GP. In vaccinated mice, EBOVgp-Fc induced high levels of total and neutralizing anti-GP antibodies and elicited T-cell immunity characterized by the production of IFNγ (Konduru et al. 2011). Vaccinated guinea pigs developed a strong humoral response as measured by total and neutralizing antibodies to GP, and completely protected against lethal EBOV challenge. We are currently extending our studies to the monkey model. Our preliminary data indicates that EBOVgp-Fc induces neutralizing antibodies and recall CD8+ T-cell immunity, as assessed by the production of IFNγ and TNFα in PBMC. Further experiments, including protection against lethal challenge in monkeys, will be required to determine whether Filovirus GP-Fc fusion proteins could be developed as a candidate vaccine.
Evaluation of the genetically attenuated E. coli heat-labile enterotoxin (LTR192G) as adjuvant for mucosal and intradermal immunization (P4421)
Authors: Maciel Jr Milton , Dinesh Ravi , Poole Steven , Savarino Stephen
Published: January 2023

Among the challenges to developing a subunit vaccine against the diarrhea-causing pathogen enterotoxigenic E. coli (ETEC) is the selection of a proper administration route and adjuvant. We assessed the adjuvant LTR192G, an attenuated form of the heat-labile enterotoxin (LT), co-administered with dscCfaE protein, the prototype ETEC CFA/I fimbrial tip adhesin, by two different routes. BALB/c mice received dscCfaE+LTR192G by intranasal (IN) or intradermal (ID) immunization on days 0 and 21. Serum, spleen and bone marrow (BM) were collected on day 28. Both regimens elicited comparable serum IgG anti-dscCfaE and functional Ab levels, the latter measured by hemagglutination inhibition (HAI). IN immunization, however, resulted in higher serum and fecal IgA anti-dscCfaE Ab levels, and fecal HAI titers. IgG1 was the only adhesin-specific subclass detected in the ID group, while IN immunization also elicited low titers of IgG2a anti-dscCfaE Ab, suggesting a more balanced Th1/Th2 response. The number of IgG and IgA anti-dscCfaE antibody-secreting cells in spleens and BM were two-fold higher in the IN vs ID group. After in vitro stimulation with dscCfaE protein, similar numbers of IFN-γ spot-forming cells (SFC) were observed in both groups. The IN group showed lower numbers of IL-5 SFC compared to the ID group. The composite results suggest that ID immunization with dscCfaE+LTR192G elicits a mucosal immune response, but the magnitude and immune profile differ from the IN-immunized group.
Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8+CD28+ T lymphocyte responses (P4389)
Authors: Lu Xiaoling , Zhao Yongxiang , Zhou Nuo , Yu Xia , He Jian , Mongkhoune Sodaly , Peng Yi , Xie Yu an , Zhou Sufang , Xie Xiaoxun , Luo Guorong , Li Xi , Fang Yuan
Published: January 2023

Abstract Objective: To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte (CTL) responses. Methods: Cell-sized Dynabeads® M-450 Epoxy beads coated with H-2Kb:Ig-TRP2180-188 and anti-CD28 antibody were used as artificial antigen-presenting cells (aAPCs) to induce melanoma-specific CD8+CD28+ CTL responses with the help of IL-21 and IL-15. Dimer staining, proliferation, ELISPOT, and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs. Results: Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8+CD28+ CTLs. Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN-γ under the stimulation of H-2Kb:Ig-TRP2-aAPCs, IL-15, and IL-21. In addition, cytotoxic experiments showed that induced CTLs have specific killing activity of target cells. Conclusions: The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma. Our study provides evidence for a novel adoptive immunotherapy against tumors.
Ability of different isolates of Staphlococcal aureus to induce reactive metabolites in human polymorphovuclear leucocytes (P4429)
Authors: Javed Numan
Published: January 2023

Staphylococcus aureus is a gram positive bacterium that causes a number of diseases such as abscesses, infective endocarditis, septic arthritis, etc. It is acquiring resistance against many antibiotics like methicillin due to which its control is becoming difficult. Phagocytes interact with bacteria and phagocytose them. The phagocytosis leads to killing of bacteria by activation of respiratory burst during which different bactericidal metabolites like reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) are produced. For this one hundred different isolates of S. aureus were obtained from various hospitals of Lahore in which fifty isolates were methicillin resistant while rest of the fifty isolates were methicillin sensitive. Peripheral blood from healthy individuals was used to obtain polymorphonuclear leucocytes by dextran density gradient centrifugation. Superoxide concentration was determined by the reduction of ferricytochrome c and nitric oxide concentration was measured by Griess method.Methicillin sensitive S. aureus (MSSA) has showed more phagocytosis by human polymorphonuclear leucocytes as compare to methicillin resistant S. aures (MRSA). Similarly, a significant difference was observed between two isolates of S. aures to induce ROI and RNI by human PMNs. MRSA produced more mean value of superoxide and nitric oxide as compare to MSSA.
β-defensins that bind CCR6: Blunting the permissiveness of Th17 cells to HIV infection (P4432)
Authors: Christensen-Quick Aaron , Lafferty Mark , Goicochea Marco , Garzino-Demo Alfredo
Published: January 2023

HIV preferentially depletes IL-17-producing, CD4+ “T helper 17” (Th17) cells from the gut during the acute phase of infection. Th17 cells play a key role in mucosal barrier maintenance and protection against opportunistic infections commonly associated with HIV/AIDS. In response to IL-17, epithelial cells of mucosal barriers secrete β-defensins - a family of secreted, cationic peptides with potent broad-range antimicrobial activity against bacteria, fungi, and viruses including HIV. Our laboratory has shown that the expression of human β-defensin 2 (hBD2) is markedly decreased in the oral mucosa of HIV+ subjects, and that β-defensins, in addition to direct antimicrobial activity, upregulate the HIV restriction factor APOBEC3G via the chemokine receptor CCR6, which is expressed on Th17 cells. In order to characterize the effects of HIV infection on the Th17 subset and determine if β-defensins can protect Th17 cells from HIV infection, activated human primary CD4+ T cells are infected and/or treated with β-defensin, then characterized by flow cytometry. Our results show that IL-17A+ cells from peripheral blood are highly permissive to HIV, and are preferentially lost during infection. In addition, our model suggests that Th17-polarizing cytokines enhance HIV infection, and that β-defensins can protect these cells from infection. Our studies of the factors contributing to protection of Th17 cells may yield new therapeutic targets to protect the Th17 compartment.
In vitro characterization of GS-9620, a toll-like receptor 7 agonist (P4441)
Authors: Hesselgesser Joe , Frey Christian , Duatschek Paul , Halcomb Randall , Zheng Jim , Tumas Daniel , Wolfgang Grushenka
Published: January 2023

Efficient innate and adaptive immunity is necessary for initial containment and subsequent clearance of viral infections, including hepatitis B and C viruses. A feature of an effective anti-viral immune response is the interaction of single stranded viral RNA with toll-like receptor 7 and activation of plasmacytoid dendritic cells and B lymphocytes. In this study, the novel toll-like receptor 7 agonist GS 9620 was characterized in regards to its toll-like receptor selectivity and its ability to activate innate and adaptive immune responses in vitro. GS 9620 selectively activated human toll-like receptor 7 over other toll-like receptors and induced several cytokines and chemokines in a concentration dependent manner. In particular, GS-9620 was able to induce secretion of interferon-alpha at 55-fold lower concentrations compared to those that induce tumor necrosis factor in human PBMCs and isolated plasmacytoid dendritic cell cultures. We also detected GS-9620-dependent induction of interferon stimulated gene expression in PBMCs. Moreover, GS-9620 transiently induced the expression of the activation markers CD25 and CD69 in both CD3+CD4+ and CD3+CD8+ T lymphocyte subsets as well as up-regulated expression of CD69 and the co-stimulatory molecule CD86 on CD20+ B lymphocytes. In conclusion, GS-9620 is a potent and selective toll-like receptor 7 agonist that activates innate and adaptive antiviral immune responses which is currently being evaluated in human clinical trials.
The phosphatase and tensin homolog PTEN improves murine polymicrobial sepsis outcome by decreasing MyD88 expression. (P1344)
Authors: Sisti Flavia , Ferreira Ana , Wang Suojuan , Filgueiras Luciano , Wang Zhuo , Cunha Fernando , Alves-Filho Jose , Serezani C. Henrique
Published: January 2023

Sepsis is systemic inflammatory response caused by an infectious agent and may progress to multiple organ dysfunction and death. Therapeutic strategies to inhibit excessive inflammation in sepsis are needed. The lipid and protein phosphatase PTEN is a well-known tumor suppressor, but its role in host defense is poorly studied. Here, we investigated whether PTEN activation is involved in TLR activation in vivo and in vitro. PTEN expression is decreased in cells from the peritoneal cavity 24 h after cecum ligation and puncture (CLP). In vivo pretreatment with the PTEN inhibitor bpV(HOpic) 150 ug/Kg for 1 hour before cecum and ligation puncture decreases animals survival, associated with high bacterial number in the peritoneal cavity when compared to untreated mice. In vivo PTEN inhibition increases expression of the TLR adaptor MyD88 during sepsis. In vitro, LPS (TLR4) and PAM3Cys (TLR2) increase PTEN phosphorylation in thyoglicolate-elicited macrophages (MΦs). PTEN mRNA silencing further enhances MyD88 expression and TLR-2 and -4-induced IRAK and NFkB p65 phosphorylation. Transfection of MΦs with adenovirus expressing constitutively active PTEN decreases MyD88 expression. Together, our data show that PTEN is a key determinant of macrophage TLR activation by decreasing MyD88 expression, leading to reduced systemic inflammatory response and therefore improving sepsis outcome.
Cyclic GMP-AMP Synthase (cGAS) is a cytosolic DNA sensor that activates type I interferon pathway by generating a second messenger (P1346)
Authors: Sun Lijun , Wu Jiaxi , Chen Zhijian
Published: January 2023

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. Here we showed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production in vivo. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP represents the first cyclic di-nucleotide in metazoa and it functions as an endogenous second messenger that triggers interferon production in response to cytosolic DNA. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.
In dendritic cells Toll-like receptor-dependent induction of IL-12p70 is accompanied by a profound down regulation of sphingosine-1-phosphate lyase mRNA (P1348)
Authors: Radeke Heinfried , Arlt Olga , Ranglack Annika , Pfeilschifter Josef
Published: January 2023

Sphingosine-1-phosphate (S1P) is an immune modulator produced by sphingosine kinase (SphK)1 and SphK2 and dephosphorylated by two S1P phosphatases or irreversibly degraded by a lyase. We recently showed that Toll-like receptor (TLR)4-induced IL-12p70 is selectively counter regulated by SphK1 and extracellular S1P/S1PR1. Others demonstrated that specific SphK1-dependent binding of intracellular S1P to TRAF2 enhances TNF signaling. In an ongoing investigation to determine the influence of extra- and intracellular S1P on dendritic cell signaling our experiments revealed that activation of TLR4 by lipopolysaccharide dose and time dependently decreased S1P lyase mRNA expression of murine bone marrow-derived dendritic cells by up to 70%. This set of realtime PCR data was further confirmed by semi-quantitative RT-PCR using exon-specific primers for murine sgpl1. Systematic analysis with dose-optimized ligands of TLRs 1/2, 5, 2/6, 7/8 and TLR9 showed a differential pattern of S1P lyase down regulation with concomitant increase of intracellular S1P. Although S1P lyase defcient mice are immune compromised, we found that TLR-induced transient S1P lyase downregulation resulted in a TLR adaptor-dependent upregulation of IL-12p70, IL-23 and IL-6 expression on mRNA and protein level. Further experiments including cell compartment-specific quantification of S1P, hexadecenal and, possibly counter regulatory, sphingolipid enzymes are necessary to understand the role of sphingolipids in DC.
Sialylated ligands interact in cis to modulate PILRα inhibitory receptor in myeloid cells (P1358)
Authors: Sun Yonglian , Senger Kate , Sebrell Andrew , Sai Tao , Mazloom Anita , Chinn Yvonne , Luis Elizabeth , Zarrin Ali
Published: January 2023

Paired Ig-like type 2 receptor alpha (PILRα) is an inhibitory receptor expressed in myeloid cells. We show that an evolutionary conserved arginine-containing domain is critical for PILRα interaction with its sialylated ligands. In addition, PILRα is pre-engaged with its ligands in cis on the surface of primary cells and dissociation of PILRa-ligand interactions by sialidase-A treatment unmasks PILRα to bind soluble ligands. At the cellular level, the engagement of PILRα with monoclonal antibody suppresses the production of proinflammatory cytokines. Our data provides novel insights into nature of PILRα interactions and means to modulate the immune response by agonizing this inhibitory pathway.
Functionally distinct Gata3/Chd4 complexes coordinately establish Th2 cell identity. (P1340)
Authors: Hosokawa Hiroyuki , Tanaka Tomoaki , Kato Miki , Tamaki Yuuki , Nakayama Toshinori
Published: January 2023

Gata3 is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper (Th) 2 cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that Chd4 (chromodomain helicase DNA-binding protein 4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFNγ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4-NuRD repression complex at the Tbx21 locus in Th2 cells. We also demonstrated a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.
A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes (P1322)
Authors: Glasmacher Elke , Agrawal Smita , Chang Abraham , Murphy Theresa , Zeng Wenwen , Vander Lugt Bryan , Khan Aly , Ciofani Maria , Spooner Chauncey , Rutz Sascha , Hackney Jason , Nurieva Roza , Escalante Carlos , Ouyang Wenjun , Littman Dan , Murphy Ken , Singh Harinder
Published: January 2023

Interferon regulatory factor 4 (IRF4) and IRF8 regulate B, T, macrophage, and dendritic cell differentiation. They are recruited to cis-regulatory Ets-IRF composite elements by PU.1 or Spi-B. How these IRFs target genes in most T cells is enigmatic given the absence of specific Ets partners. Chromatin immunoprecipitation sequencing in T helper 17 (T(H)17) cells reveals that IRF4 targets sequences enriched for activating protein 1 (AP-1)-IRF composite elements (AICEs) that are co-bound by BATF, an AP-1 factor required for T(H)17, B, and dendritic cell differentiation. IRF4 and BATF bind cooperatively to structurally divergent AICEs to promote gene activation and T(H)17 differentiation. The AICE motif directs assembly of IRF4 or IRF8 with BATF heterodimers and is also used in T(H)2, B, and dendritic cells. This genomic regulatory element and cognate factors appear to have evolved to integrate diverse immunomodulatory signals.
Influence of site of antigen expression in polarised airway epithelial cells on induction of immune responses (P3339)
Authors: Herbert Rebecca , Norimatsu Mari , Kohl Wiebke , Herrler Georg , Iqbal Munir , Taylor Geraldine
Published: January 2023

Bovine respiratory syncytial virus (BSRV) is a major cause of respiratory disease in calves and is closely related to human RSV. Bovine viral diarrhoea virus (BVDV) is responsible for disease that affects the respiratory, reproductive, immune and enteric systems of cattle. These diseases are a huge problem globally for the livestock industry and a mucosally targeted, bivalent vaccine which prevents infection and spread of these viruses would be valuable. We investigated the influence of the site of expression of the BVDV E2 protein on induction of immune responses using recombinant (r)BRSV expressing wild-type and mutant forms of the E2 protein targeted to different sites within polarised epithelial cells. Calves vaccinated intranasally with rBRSV expressing the E2 protein at the basolateral surface induced higher levels of E2-specific serum antibodies than animals vaccinated with rBRSV expressing E2 at the apical membrane, intracellularly or when secreted from cells. Targeting E2 to the basolateral surface also induced E2-specific IgA antibodies in respiratory secretions without compromising RSV-specific responses. Calves vaccinated intranasally with rBRSV expressing E2 targeted to the basolateral surface were completely protected against challenge with BRSV, and were also protected against nasopharyngeal excretion of BVDV. These studies highlight the importance of the site of expression of foreign proteins in polarised epithelial cells on priming of immune responses.
CX3CR1+ intestinal mononuclear phagocytes regulate induction and maintenance of tolerogenic intestinal immune responses (P3343)
Authors: Diehl Gretchen , Galan Carolina , Longman Randy , Egawa Takeshi , Yang Yi , Littman Dan
Published: January 2023

The intestinal immune system must be tolerant towards commensal microorganisms and food while allowing for the induction of inflammatory responses against pathogens. The mechanisms governing this tolerance are not clearly understood, but loss of this balance can result in commensal- or food-mediated inflammation, leading to intestinal damage. The intestine contains many cell types that can initiate and regulate immune responses, and it is unclear what role each plays in maintaining homeostasis. Two main populations of mononuclear phagocytes reside within the intestine. One is defined by expression of the integrin CD103, and has been described as a migratory dendritic cell (DC). The other expresses the chemokine receptor CX3CR1 and has been compared to tissue resident macrophages. In contrast to previous reports, we recently demonstrated that CX3CR1+ cells migrate from the intestine to the mesenteric lymph node, suggesting that they can behave as DCs. We have generated novel mouse strains allowing depletion of each cell population so that their functions can be studied in vivo. We have found that CX3CR1 and not CD103+ cells are critical for the induction and maintenance of tolerogenic immune responses. We have further demonstrated that antigen presentation by CX3CR1+ cells is critical to prime tolerogenic T cells to intestinal antigens in vivo. This supports our hypothesis that CX3CR1+ cells are DCs that regulate induction of tolerogenic immune responses in vivo.
IFN-γ suppresses MUC5AC expression in human airway epithelium through inhibition of ERK1/2 and SP1 (P3349)
Authors: Takizawa Takumi , Oyanagi Takato , Aizawa Akira , Orosoo Solongo , Arakawa Hirokazu
Published: January 2023

Mucous secretion from airway epithelium plays a pivotal role in primary immune response against pathogens which infect the airway. However its excessive secretion can cause obstruction of airways and respiratory failure. Therefore it is vital to understand mechanisms underlying mucous secretion from airway epithelium and develop medical strategy to control the production of mucous. We have developed an in vitro viral-induced mucin production model using human airway epithelial cell line, NCI-H292, and found that TGF-α and double stranded RNA, polyI:C synergistically induce expression of MUC5AC (J Immunol 2009). Here we uncover IFN-γ suppresses transcription of MUC5AC induced by TGF-α and polyI:C in H292 cells and reveal its molecular mechanisms. IFN-γ inhibited activation of ERK1/2 provoked by TGF-a and polyI:C 9-12 hours after stimulation. IFN-γ decreased TGF-α and polyI:C-upregulated SP1 binging to its cognate site of the MUC5AC promoter. By using inhibitors against SP1 and ERK1/2 phosphorylation, we found that ERK1/2 activation and SP1 binding is independent from each other. These findings demonstrate IFN-g suppress dual pathways that are required for MCU5AC expression.
Improved click chemistry demonstrating EdU cell proliferation with GFP expressing cells and R-PE based immunophenotyping. (P3299)
Authors: Deveny Ruth , DeMarco Carolyn , Bradford Jolene , Clarke Scott , Grecian Scott , Singh Upinder , Gee Kyle
Published: January 2023

The current Click-iT® EdU kit, for measuring cell proliferation, cannot be use for the simultaneous detection of EdU, GFP and R-PE fluorescence due to the presence of copper and reactive oxygen species mediated damage to fluorescent proteins. We present modifications to the click reaction resulting in“copper safe” catalysis of the click reaction. Flow cytometry measurements of cell proliferation using Click-iT® EdU are demonstrated to be compatible with immunophenotyping panels using R-PE conjugates and GFP expressing proteins. The basis of the click chemistry improvement is the use of a copper (I) ligand combined with a modified dye azide detection reagent. We optimized components in the improved click reaction which best preserved R-PE fluorescence while obtaining a bright EdU signal. Jurkat cells were pulsed with EdU, labelled with PE conjugated antibodies and then fixed and permeabilized. EdU incorporation was then detected using click chemistry, demonstrating improved detection while maintaining PE signal. Additionally, BacMam transduced cells expressing GFP were treated with cell cycle inhibitors then pulsed with EdU and used to demonstrate GFP and click chemistry compatibility. The modified click reaction is an improvement over the original copper based click reactions and will further enhance the utility of EdU based cell proliferation applications in flow cytometry.
Brilliant UltraViolet™ Dyes: A new set of high sensitivity fluorescence reagents for multicolor flow cytometry with a UV laser (P3301)
Authors: Stall Alan , Gaylord Brent , Liang Yongchao , Uckert Frank , Leonard Barry , Li Haiqing , Tran Lan , Bartholomew Glenn , Chen Yu , Luan Fengjun , Widmann Stephanie
Published: January 2023

While UV lasers have been available for many years, their utility has been mainly restricted to DNA/RNA binding fluorochromes (e.g. DAPI, Hoescht 33342) for studying cell cycle or stem cell side populations. The low fluorescence intensity of standard fluorescent dyes, such as AlexaFluor™ 350 or AMCA, have made them impractical for routine immunological flow analyses. Here we report the development of a novel class of highly fluorescent polymeric reporters which are excitable by a UV (355nm) laser. The introduction of these dyes has the potential to dramatically extend the functionality of the UV laser adding up to 6 totally new colors for multi-color cytometric flow studies. The first Brilliant UltraViolet (BUV) polymer has an excitation maximum near 355nm with emission below 405nm. Cross laser excitation is minimal requiring very little compensation into the Brilliant Violet 421 channel. Furthermore these polymers can serve as the basis for developing a wide range of bright, ultraviolet excitable tandem reagents, which span the entire spectrum from UV to the near IR. Data will be presented to demonstrate how the superior staining performance and unique excitation / emission profiles of these new dyes offer a broader pallet of colors for those designing multi-parameter flow panels. Comparative flow performance will be presented as will multicolor staining panels which highlight their compatibility with conventional flow reagents.
Anti-CD3 antibody treatment induces hypoglycemia and super tolerance to glucose challenge in mice through T cell activation (P5218)
Authors: Xia Chang-Qing , Wan Suigui , Chernatynskaya Anna , Looney Benjamin , Clare-Salzler Michael
Published: January 2023

Anti-CD3 antibody has been used for various immune-mediated disorders through modulating immune responses. However, whether anti-CD3 administration leads to rapid metabolic alternation has not been well investigated. In the current study, we studied how anti-CD3 treatment affected blood glucose levels in mice. We found that anti-CD3 treatment induced immediate reduction of blood glucose as early as 3-4 hours post-administration. Furthermore, we observed that a single dose of anti-CD3 treatment was able to correct hyperglycemia in all nonobese diabetic mice with new-onset diabetes. This glucose-lowering effect was not attributable to major T cell cytokines secreted by anti-CD3 activated T cells. Furthermore, blood glucose levels of NOD-rag-/- mice lacking T cells remained unchanged after anti-CD3 treatment. Of interest, when tested in a normal strain of mice (C57BL/6), the serum levels of C-peptide in anti-CD3-treated animals were significantly lower than those of control animals. Paradoxically, the anti-CD3-treated animals were highly tolerant to the exogenous glucose challenge. In vitro studies demonstrated that anti-CD3 treatment lowered glucose levels in T cell culture media. Further studies showed that anti-CD3 antibody up-regulated GLUT-1 on activated T cells. This study indicates that anti-CD3 therapy-induced hypoglycemia is likely resulted from increased glucose consumption via enhancing glucose transportation into the activated cells induced by anti-CD3 treatment.
IL-21 neutralizing antibody inhibits human TFH proliferation and TFH-mediated maturation of naïve B cells (P5220)
Authors: Hussell Scott , Smith Patricia , Fox Brian , Serikawa Kyle , Levin Steven , Cox Jennifer , Lundsgaard Dorthe
Published: January 2023

The hallmark of T follicular helper (TFH) cells is the ability to help B cells generate humoral immune responses. Recent evidence suggests that aberrant function of TFH cells plays a key role in the generation of autoantibodies, which can inflict autoimmune pathologies. TFH cells are characterized by their unique master regulator (Bcl6), surface marker expression (CXCR5 and ICOS), and production of the pro-inflammatory cytokine interleukin-21 (IL-21). In mice, IL-21 is described as a regulator of TFH cell differentiation and B cell maturation, but its role in human TFH cells remains incompletely understood. In the present study, we sorted TFH subsets from human tonsils or TFH-like cells from human blood to characterize IL-21 production and the functional consequences of blocking IL-21 with a neutralizing antibody. IL-21 was highest in TFHHI cells (CXCR5++ICOS++), detectable in TFHINT cells (CXCR5+ICOS+), and minimal in non-TFH cells. Anti-IL-21 caused a moderate decrease in anti-CD3 stimulated TFH proliferation. Peripheral CXCR5+ TFH-like cells also had elevated IL-21 production, but anti-IL-21 had little effect on TFH-like proliferation. When TFHINT, TFHHI, or TFH-like cells were co-incubated with naïve B cells, anti-IL-21 decreased B cell maturation and Ig production in a dose-dependent manner. Together, these findings indicate that anti-IL-21 inhibits TFH generation and B cell help, and is a promising therapeutic for autoimmune disorders involving autoantibody production.
PD-1-mediated deletion induces tolerance of CD8 T cell response specific for H60 under helpless condition (P2225)
Authors: Choi Eun Young , Ryu Su Jeong , Kim Juhyun
Published: January 2023

H60 is a dominant minor H antigen, for which specific CD8 T cell response dominates over those for other minor H antigens in B6 anti-BALB.B response. The specific CD8 T cell response for H60 is CD4 help-dependent, as burst expansion of the specific cells during the primary and memory responses occurs only when CD4 T cell activation accompanies. When the CD4 help is not provided, primary expansion was limited and, then, recall expansion of the specific CD8 T cells is impaired even when the CD4 help is provided during the memory response. This tolerance phenomenon was ascribed to intrinsic property of H60-specific CD8 T cells activated under the helpless condition. The H60-specific CD8 T cells activated under helpless condition could develop into functionally active effector cells, migrating to inflamed sites. However, they were more prone to cell death with up-regulation of PD-1 on the H60-specific CD8 T cells activated under helpless condition. Recall expansion of H60-specific CD8 T was restored in the PD-1 deficient mice that had been primed under helpless condition, suggesting the significance of negative regulation by PD-1 in the tolerance induction of H60-specific CD8 T cell response after the helpless priming. These results will provide useful information for induction of tolerance after allogeneic transplantation.
Depletion of activated lymphocytes during the emergence of ovarian cancer (P2004)
Authors: Riggs James , Suarez Joyce
Published: January 2023

We are modeling the myeloid suppression found in tumors by characterizing how peritoneal cavity (PerC) macrophages (Mϕs) suppress lymphocyte activation. Cultured PerC Mϕs suppress the proliferation of C57BL/6J T cells by iNOS catabolism of arginine. To make our model more relevant to in vivo biology we initiated studies of ovarian cancer in the PerC. The ID8 cell line (mouse ovarian surface epithelial cell carcinoma) forms ovarian tumors and hemorrhagic ascites 5 weeks following i.p. injection into intact, syngeneic C57BL/6J mice. We found cancer development was accelerated in IL10-/- mice and delayed in iNOS-/- mice indicative that inflammation plays a role in cancer progression. Assessing mice before the ovarian cancer was fully developed we observed a depletion of CD44hi T and CD11b+IgMhi B cells in the PerC. With disease progression B and T cell depletion also became evident in the spleen. Lymphocyte depletion in the PerC correlated with ID8 expansion and the emergence of CD11b+F4/80+Gr-1+CD80+ myeloid suppressor cells (MSCs); splenic MSCs were CD80-. It was interesting to observe that GITR+ T cells, naturally enriched in the PerC of healthy, normal mice, persisted during MSC emergence in both the spleen and PerC. We are investigating what triggers the selective death of these important lymphocyte subsets. These results reveal an additional, challenging aspect of the immune suppression characteristic of myeloid cell emergence during cancer development.
Neutrophils inhibit tumor growth at early stages of tumor formation. (P2006)
Authors: Liu Yuhong , O’Leary Claire , Wang Liang-Chuan , Dai Ning , Kapoor Veena , Liu Peihui , Mei Junjie , Oliver Paula , Albelda Steven , Worthen George
Published: January 2023

Neutrophils are important innate immune cells involved in microbial clearance at sites of infection. However, the roles of neutrophils in cancer development are not clear. We hypothesized that neutrophils might mediate antitumor effects at the initial tumor formation stage. To test this hypothesis, we injected 2x106 mouse lung Lewis cancer cells into the flanks of C57BL/6 and Cxcr2-/- mice, the latter lack the receptor for ELR+ CXC neutrophil chemokines. We observed increased tumor growth in Cxcr2-/- mice as compared with controls within 3 weeks of injection. FACS analysis indicated fewer neutrophils and more IL-17-producing cells in the tumors of Cxcr2-/- mice than those of C57BL/6 mice. We further tested the roles of neutrophils using anti-Ly6G antibody depletion at the time of tumor cell injection. We demonstrated that tumors grew faster in the neutrophil depleted group, while the percentages of IL-17-producing cells in the tumors and the levels of G-CSF in the plasma were also increased in the absence of neutrophils. To test whether neutrophils have a direct cytotoxic effect on LLC cells, we performed in vitro cell killing assays. We demonstrated neutrophils isolated from both circulation and bone marrow exerted cytotoxic effects. In conclusion, by using Cxcr2-/- and neutrophil antibody-depleted C57BL/6 mice, we have demonstrated that initial tumor growth of LLC cells is enhanced in association with inaccessibility of neutrophils and activation of the IL-17/G-CSF axis.
Preclinical modelling of immune checkpoint blockade (P2012)
Authors: Stewart Ross , Mullins Stefanie , Watkins Amanda , Morrow Michelle , Sainson Richard , Robbins Paul , La Vallee Theresa , Wilkinson Robert
Published: January 2023

Harnessing the power of the immune system to identify and eliminate cancer is a promising area of development in cancer therapy. Increasing T-cell activation, in particular, is now a clinically validated approach to the treatment of cancer. Cytotoxic T lymphocyte antigen 4 (CTLA-4, CD152) and PD-L1 (B7-H1, CD274) are part of a complex system of receptors and ligands that are involved in limiting T-cell activation through the delivery of inhibitory signals. MedImmune is currently developing tremelimumab (anti-CTLA4) and MEDI4736 (anti-PD-L1), for the treatment of advanced or metastatic solid tumours. Both of these antibodies antagonise T-cell inhibition signals with the aim of enhancing anti-tumor T cell activity. We have established and characterised a panel of syngeneic mouse tumor models and employed these to explore the activity of anti-mouse CTLA-4 and PD-L1 antibodies using pharmacodynamic (PD) and tumor growth inhibition (TGI) endpoints. Published studies characterising the immunogenic nature of some existing cancer therapeutics provide reason to believe that a combination strategy, utilising checkpoint blockade together with current standard of care, or novel therapeutics, could result in increased activity. We have gone on to explore the value of such combinations in preclinical mouse models.
Hepatic T cell suppressors and their inhibition roles in hepatic viral clearance (P6353)
Authors: Tang Li , Liu Biao , Wang Min , Wang Xiaowen , He Fuchu
Published: January 2023

The unique microenvironment of the liver favors T cell inactivation, tolerance, and apoptosis. As such, it is a challenging environment in which to efficiently clear viruses, which may explain why HBV and HCV often establish a chronic viral presence. The principal mechanisms that account for hepatic T cell failure and thus influence the outcome of liver infections, including the identification of inhibitory molecules and their ligands, are beginning to be understood. We recently identified LSECtin from the liver and reported that LSECtin negatively regulates T cell receptor-mediated signaling and T cell Immunity. We then investigate whether LSECtin regulates anti-viral immunity and virus invasiveness in the liver. Using a mouse model infected with a hepatotropic adenovirus, we found that the absence of LSECtin led to a higher frequency of CTL, which produced higher levels of antiviral cytokines and cytotoxic factors and showed increased expression of T-bet and Runx3, thus mediated more efficient virus-specific cytotoxity than WT cells. As a consequence, LSECtin deficiency significantly accelerated liver adenovirus clearance. We further constructed an immunocompetent mouse model of acute hepatitis B viral infection to demonstrate that LSECtin significantly delayed clearance of HBV from blood and infected hepatocytes through limiting the frequency of HBV-specific IFN-γ-producing cells.
MMP-8 mediates vascular breakdown during viral infection of the nervous system (P6368)
Authors: McGavern Dorian , Gossa Selamawit
Published: January 2023

Viral infection of the central nervous system (CNS) routinely induces immune-mediated vascular damage that compromises the integrity of the blood brain barrier (BBB) and induces neurological complications. We have shown previously that cytotoxic lymphocytes massively recruit monocytes and neutrophils to the brain following infection with lymphocytic choriomeningitis virus (LCMV). These cells promote vascular leakage through synchronous extravasation. Here, we focus on identifying the mechanism by which innate myelomonocytic cells injure brain vasculature. Quantitative analyses revealed that 6 metalloproteinases (MMP-2, 3, 8, 12, 13, and 19) and 1 disintegrin (ADAM-8) were highly expressed in symptomatic mice with LCMV meningitis. Among these, the collagenase, MMP-8, was expressed greater than 2000-fold in neutrophils and 200-fold in monocytes. We therefore postulated that vascular collagenase activity during viral meningitis promotes myelomonocytic cell extravasation and vessel leakage. This theory was tested experimentally by administering a specific MMP-8 antagonist to LCMV-infected mice prior to the development of symptoms. Our results revealed that MMP-8 antagonism significantly reduced vascular damage, BBB breakdown, and the development of symptoms in infected mice. These data demonstrate that inhibition of collagenases might be a viable therapeutic approach to lessen the pathogenic effects of innate inflammatory cells following CNS viral infection.