bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR5 expression by CD11b+CD4- splenic dendritic cells enhances T cell responses to low dose flagellin immunization (158.15)
Authors: Atif Shaikh , Letran Shirdi , McSorley Stephen
Published: January 2023

Previous studies have demonstrated that TLR5 functions as an endocytic receptor, enhancing antigen presentation to flagellin-specific CD4 T cells. Here, we show that this enables flagellin-specific CD4 T cell activation to occur with low concentrations of antigen in vitro and in vivo. We used this sensitive functional read-out to examine TLR5 expression by splenic dendritic cells (DCs). While CD11b+CD4- DCs were able to activate flagellin specific T cells in a TLR5-dependent manner, CD11b+CD4+ or CD11b-CD8a+ DCs did not. We also examined the role of NLRC4 in flagellin-specific CD4 responses and have found that NLRC4 plays a modest additonal role in enhancing CD4 responses in vitro. Together, these data indicate that CD11b+CD4- DCs express TLR5 and are ideally poised to initiate adaptive immune responses to bacterial flagellins.
Genetic analysis of the CpG hyporesponsiveness in wild-derived MOLF/Ei mice (158.16)
Authors: Moseman Annie , Poltorak Alexander
Published: January 2023

CpG motif containing oligonucleotides are used to mimic the immunostimulatory properties of bacterial DNA in both innate and adaptive immune cells through the activation of toll-like receptor 9. Macrophages are one of the main cell types activated to elicit a pro-inflammatory response to CpG DNA. Downstream signaling of TLR9 is more well characterized and studied compared to upstreams events which remains largely unknown, such as what receptors and mechanisms are involved in CpG DNA receptor-mediated endocytosis and trafficking into endosomal compartments. While all classical laboratory mouse strains are responsive to CpG DNA activation a wild-derived inbred mouse strain, MOLF/Ei, is non-responsive. In this study we have used a forward genetics approach to analyze the CpG response in peritoneal macrophages from panels of N2 backcross mice [MOLF/Ei x (C57BL/6xMOLF/Ei)]. Our genome-wide linkage studies implicate, for the first time, the role of the mannose receptor, C type 1 (Mrc1) in the CpG hypo-responsiveness in MOLF/Ei mice. Furthermore, additional mapping studies have revealed a second receptor that is also involved in CpG DNA signaling for both classical and wild-derived mouse strains, implicating its role in CpG activation for the first time.
Epigenetic mechanisms underlying the reduced response of dendritic cells from aged humans to influenza infections (158.22)
Authors: Agrawal Anshu , Ulagaoozhian Sangeetha , Nguyen Michelle , Agrawal Sudhanshu , Gupta Sudhir
Published: January 2023

Increased susceptibility to respiratory infections is a major cause of age-related morbidity and mortality. Impaired age-associated T and B cell responses are prime suspect yet the mechanisms responsible for the increased risk are complex and are not well understood. Studies in recent years highlight the importance of innate immune cells such as dendritic cells (DC) in fighting infections and generating protective immunity. Our results indicate that monocyte derived DCs from aged subjects display a higher basal level of activation as evidenced by increased activation of NFkB at the basal level. Moreover, the response of aged DCs to influenza virus is impaired and there is reduced secretion of IFN-I and IFN-III compared to their young counterparts. During viral infections, IFNs are expressed and constitute perhaps the most important line of innate antiviral defense. Type III IFNs act specifically on mucosal epithelia and therefore, play an important role in the defense against infections of the respiratory tract. Our investigations using chromatin immunoprecipitation (CHIP-IP) with histone antibodies suggest that there may be increased association of IFN-I and IFN-III promoters with suppressor histone, H3K9 in aged DCs. Age-associated chromatin remodeling may therefore be responsible for the reduced secretion of IFNs by aged DCs and increased susceptibility to influenza infections.
Respiratory Syncytial Virus infection induces alternatively activated macrophages in the lung (158.3)
Authors: Raundhal Mahesh , Krishnamoorthy Nandini , Khare Anupriya , Yarlagadda Manohar , Ray Anuradha , Ray Prabir
Published: January 2023

Asthma is a chronic inflammatory disease of the airways characterized by a heightened response to normally harmless antigens. Respiratory Syncytial Virus (RSV) infections in children have been associated with predisposition to asthma in later life. However, the underlying molecular mechanisms are not well understood. RSV infection was recently shown to induce Th2 type cytokines such as IL-13 within 6 days after infection. Given this early induction of IL-13, we hypothesized that the source of RSV-induced IL-13 is a cell of the innate immune system. Our ongoing studies show rapid induction of alternatively activated macrophages (AAMs) in the lung following RSV infection. RSV induces expression of Arginase I (Arg I), a characteristic marker of AAMs and stimulates secretion of the Th2 cytokines IL-5 and IL-13 from the alveolar macrophages as early as 48 hours after infection. This was found to continue on day 5 after infection, being detectable in the lung tissue as well. These observations suggest that RSV, directly or indirectly, alters gene expression in alveolar macrophages very early after infection, which we hypothesize plays an important role in promoting an allergic immune response in the infected lungs.
Restoration of anti-Aspergillus defense by NETs in human CGD after gene therapy is calprotectin-dependent (158.5)
Authors: Bianchi Matteo , Niemiec Maria , Siler Ulrich , Urban Constantin , Reichenbach Janine
Published: January 2023

Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We have previously shown that Aspergillus hyphae which are too large for neutrophil phagocytosis are inhibited by reactive oxygen species dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) due to impaired phagocyte NADPH oxidase function. Here we determined the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD. We identified the host Zn2+ chelator calprotectin as neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A. nidulans growth at low concentration, and leading to fungal starvation at higher concentration. Specific antibody-blocking and Zn2+ addition abolished calprotectin-mediated inhibition of A. nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defence was confirmed in calprotectin knockout mice. Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate that calprotectin is a critical factor in the human innate immune defense to Aspergillus infection.
Human TLT2 is expressed on and potentiates chemotaxis of cells of both myeloid and lymphoid lineage (158.6)
Authors: Thomas Kimberly , King R. , Justement Louis
Published: January 2023

The TREM locus encodes a family of innate immune receptors, which play diverse roles in modulating the immune response. Trem-like transcript 2, or TLT2, is the only family member expressed on both myeloid and lymphoid cells, and is conserved among mouse and man, with 60% sequence homology between murine (m) and human (h) TLT2. Previous work by our lab defined the expression pattern for mTLT2 on cells of the immune system: granulocytes, macrophages, and B cells all express mTLT2, whereas monocytes and T cells do not. When mTLT2 is ligated with αTLT2 1C5 mAb, neutrophil chemotaxis is enhanced in a dose dependent manner in response to chemokines. To further understand the biological function of TLT2, our lab has characterized hTLT2 expression on peripheral leukocytes and studied its role in potentiating chemotaxis. Human granulocytes, monocytes, macrophages, NK cells, and B cells all express hTLT2 on their surface. Furthermore, interaction with antigen and differentiation downregulates surface expression of hTLT2 on B cells. In contrast, activation of granulocytes or monocytes with PMA upregulates hTLT2 expression. Lastly, naive, memory, and in vitro activated T cells do not express hTLT2, opposed to results from other groups. Functionally, ligation of hTLT2 augments B cell chemotaxis towards CXCL13, and neutrophil migration towards IL-8. In this regard, we observe hTLT2 and mTLT2 to have conserved phenotypic expression and function throughout the immune compartment.
Effects of novel TOR inhibitors on B cell differentiation (159.5)
Authors: Limon Jose , Lomon So , Ke Qiao , Corado Juana , Fruman David
Published: January 2023

A promising drug target frequently disregulated in many cancers is TOR (target of rapamycin). TOR kinase functions in two complexes, TORC1 and TORC2. TORC1 is sensitive to rapamycin that acts as an allosteric inhibitor. Rapamycin and its analogs (rapalogs) are potent immunosuppressants, but have shown limited efficacy in cancer therapy. We reported in Nature Medicine (PMID:20072130) that PP242, an active-site TOR inhibitor (asTORi), has improved anti-leukemic efficacy compared to rapamycin. Unexpectedly, PP242 at anti-leukemic doses had a lesser effect in vitro on lymphocyte proliferation and in vivo on lymphocyte function. Here we present new data comparing rapamycin with asTORi using in vitro assays of B cell differentiation. asTORi treatment increased the number of IgG switching B cells and decreased the differentiation of early antibody-secreting cells. IgM secretion was also decreased with asTORi treatment. We observed similar effects when B cells were treated with an inhibitor that targets Akt, a known TORC2 substrate. Rapamycin potently inhibited B cell differentiation at an early step before commitment to cell division. Gene expression analysis revealed an increased in FOXO target genes AID and Bcl6, and a decrease in BLIMP-1 expression in asTORi-treated B cells. These data indicate that asTORi alter the balance of B cell differentiation, likely through modulation of the TORC2/Akt/FOXO signaling axis.
Expression of a functional B cell receptor with the specificity of VRC01, a broadly neutralizing antibody against HIV (159.8)
Authors: Guo Xiaoti , Yang Zhiyong , Panet Amos , Liu Wanli , Pierce Susan , Nabel Gary
Published: January 2023

VRC01, a CD4-binding site antibody that neutralizes over 90% of HIV-1 isolates has recently been identified. One of its unusual features is that the variable domain goes through extensive somatic hypermutation (30%) from its germline precursors, which leads to greatly enhanced binding affinity and neutralization capacity. To understand the somatic hypermutation process of VRC01 during B cell development and to identify immunogens that drive the affinity maturation of VRC01, the B cell receptor (BCR) form of VRC01 and its germline precursor 02*02 was expressed and examined for antigen-binding, BCR microclustering and BCR signaling. Co-expression of surface IgM and kappa chain transgenes was detected on 293T and Ramos cells by either transfection or lentivirus-mediated transduction. The transgenic VRC01 BCR bound to different gp120 variants as determined by flow cytometry. The VRC01 BCR also exhibited higher binding affinity to the gp120 Resurfaced Stabilized Cores (RSC3) presented on lipid bilayers than to the mutant form ΔRSC3 as indicated by more BCR microclusters upon binding to RSC3. Stimulation of VRC01-BCR-expressing B cells with RSC3 or anti-IgM induced phosphorylation of the B cell signaling tyrosine kinases Fyn and Syk. In conclusion, a functional VRC01-BCR was successfully expressed on 293T and B cells. This transgenic system allows us to test the feasibility of guiding the maturation of VRC01 with appropriate antigens.
IL-17 expression in γδ T cells is negatively controlled by IL-27 (160.12)
Authors: Visperas Anabelle , Do Jeong-Su , O'Brien Rebecca , Min Booki
Published: January 2023

Interleukin 27 (IL-27) is secreted by activated antigen presenting cells in response to inflammatory signals and plays both pro- and anti-inflammatory roles. Particularly interesting is that IL-27 has been shown to antagonize Th17 differentiation by suppressing the expression of Th17-promoting transcription factor, RORγt. However, immunoregulatory roles of IL-27 on non-CD4 T cells have not been extensively examined. γδ T cells account for a small subset of peripheral T cells and have been recently considered ‘innate’ IL-17 producers because of their spontaneous IL-17 expression. We found that in the absence of IL-27ra (WSX-1) on γδ T cells, IL-17 production was dramatically enhanced compared to WT γδ T cells. These cells possessed a highly activated phenotype where a majority was CD44high, increased expression of CCR6 consistent with increased IL-17 production, and an altered Vγ repertoire. In vitro stimulation of γδ T cells in the presence of exogenous IL-27 failed to suppress IL-17 production, consistent with the notion that fully committed Th17 effector T cells are resistant to IL-27-mediated inhibition. We also found that enhanced IL-17 production by IL-27ra-/- γδ T cells was already pronounced in CD44high post-DN4 thymic γδ T cell stage. Our results propose a previously uncharacterized pathway in which IL-17 expression by γδ Y cells in the thymus is modified via IL-27.
The role of KLF7 in hematopoiesis and lymphopoiesis (160.2)
Authors: Burns Audrea , Park Chun-Shik , Yamada Takeshi , Puppi Monica , Lei Lei , Lacroazza Daniel
Published: January 2023

Krüppel-like factors (KLFs) are transcription factors that possess important roles in cell development and differentiation through the regulation of proliferation. Similar to KLF4, KLF7 regulates genes that control cell cycle arrest, specifically increasing p21 expression and downregulating cyclin D1. Based on our previous findings that KLF4 is important in hematopoiesis and negatively regulates CD8+ T cell proliferation, we hypothesize that KLF7 may also be important in hematopoiesis and T cell function. To test this hypothesis, qRT-PCR analysis of lineage negative, progenitor, and mature T cell subsets was performed. KLF7 mRNA expression increases throughout lineage commitment with the highest expression being in erythrocytes and mature T cells. Clonogenic assays revealed a decreased number of common myeloid progenitors (CMP) induced by ectopic expression of KLF7, which suggests that KLF7 is important for regulating commitment to a CMP fate. Further, to test whether KLF7 is important for effector T cell proliferation, KLF7-/- and KLF7+/+ fetal liver cells were transplanted into lethally irradiated congenic recipients (due to neonatal lethality) respectively. Although CFSE dilution assays demonstrated that CD4+ and CD8+ T cell proliferation was similar, thymic T cells from KLF7-/- recipients showed a developmental block from the DP to CD4SP and CD8SP stages in the thymus. Our findings suggest that KLF7 plays important roles in hematopoiesis and T cell development.
GATA-3 regulates the development, homeostasis, and activation of CD8 T cells (160.20)
Authors: Tai Tzong-Shyuan , Pai Sung-Yun , Ho I-Cheng
Published: January 2023

GATA3 is a master transcription factor for the differentiation of T helper 2 (TH2) cells and also essential for CD4 T cell development. GATA-3 is expressed in CD8+ T cells, but its role in this subset of T cells is largely unknown. We have studied the development and function of CD8+ T cells in mice that are deficient in GATA-3 only in T cells (G3KO mice). Although the total number of TCRhi CD8SP thymocytes was normal, we found that these mice contained significantly fewer TCRhiCD24lo CD8SP thymocytes, indicating that GATA-3 is required for the final maturation step of CD8SP thymocytes. Despite the dramatic reduction in thymic output, G3KO mice developed lymphoadenopathy due to abnormal expansion of CD8+T and B cells. This abnormal expansion of G3KO CD8+T cells is mediated by a CD8+T cell-extrinsic mechanism. Paradoxically, G3KO CD8+T cells displayed a cell-intrinsic defect in homeostatic proliferation and were hyporesponsive to TCR stimulation. There was no apparent impairment in signal transduction downstream of TCR in G3KO CD8+T cells, suggesting that GATA-3 may modulate the activation threshold of CD8+ T cells. Taken together, our data demonstrate that GATA3 also plays a critical role in regulating the development, homeostasis, and activation of CD8+ T cell.
TSE3 is an orphan gene essential for thymocytes development (160.3)
Authors: Lu Linrong , Wang Di , Zheng Mingzhu
Published: January 2023

The development of T cells in the thymus involves several differentiation and proliferation events, during which hematopoietic precursors give rise to single positive mature T cells. T cell antigen receptor (TCR) signaling during CD4+CD8+ double-positive stage determines cell survival (thymocytes selection) and lineage commitment (CD4-versus-CD8 lineage choice). However, this process is also regulated by other signaling pathways, which are not fully characterized. To explore this field, we analyzed several expressed sequence tag (EST) libraries from murine thymus and identified several ESTs specifically expressed in thymus including a previous uncharacterized transcript TSE3 (Thymic specific expression, number 3). TSE3 encodes a protein without any known domains or motifs but is conserved among vertebrates. We found that TSE3 is highly expression in different stages of T cell development especially in double positive thymocytes. In TSE3-deficient mice, thymocytes development was impaired reflected by decreased numbers and percentages of mature single positive CD4 and CD8 T cells in both thymus and peripheral lymphoid organs. We are further characterizing the TSE3 deficient mice to reveal the underlying mechanism used by TSE3 to participate in the thymic T cells development.
Developmental arrest of T cells in RpL22-deficient mice is dependent upon multiple p53 effectors (160.4)
Authors: Stadanlick Jason , Zhang Zhiqiang , Lee Sang-yun , Wiest David
Published: January 2023

αβ and γδ lineage T cells are thought to arise from a common CD4-CD8- progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We have demonstrated recently that a ubiquitously expressed ribosomal protein, RpL22, is selectively required for the development of αβ lineage T cells. Germline ablation of RpL22 impairs development of αβ lineage but not γδ lineage T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors employed by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22-/- thymocytes, including miR-34a, Puma, p21waf, Bax, and Noxa. Notably, the BH3-only member Bim, while not a direct p53 target, was also strongly induced in Rpl22-/- T cells. While gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in RpL22-deficient T cells, only a few p53 targets, when individually ablated by gene targeting or knockdown, alleviated developmental arrest. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21waf) and apoptosis (Puma) actually abrogated the partial rescue that ablation of Puma alone engendered, suggesting that the G1 checkpoint protein p21waf might actually facilitate thymocyte development in some contexts.
Regulation of Erk activation in T cells through the tail domain of RasGRP1 (160.5)
Authors: Fuller Deirdre , Zhang Weiguo
Published: January 2023

The Ras-guanyl nucleotide exchange factor RasGRP1 plays a critical role linking T cell receptor engagement to Erk activation. Previous studies have emphasized the importance of RasGRP1 for positive selection of thymocytes, activation of T cells, and control of autoimmunity. Unique from its other family members, RasGRP1 contains an approximately 600 nucleotide-long tail domain, the function of which is unknown. In order to determine the role of the tail domain in RasGRP1 function, we generated a knock-in mouse deleting this domain. Thymocytes containing the truncated form of RasGRP1 underwent aberrant selection processes and, following stimulation, were unable to activate Erk. Furthermore, the deletion of the tail domain negatively affected the ability of mature T cells to become activated and proliferate. Strikingly, these mice developed a severe autoimmune disease, marked by splenomegaly, high serum IgE levels, and the accumulation of T cells with an effector memory phenotype. Additionally, in an attempt to determine how the tail domain can regulate Erk activation, we observed that, in Jurkat T cells, the tail-deleted form of RasGRP1 demonstrated improper cellular localization following stimulation. While the DAG-binding C1 domain has long been viewed as the most important factor mediating RasGRP1 function, we have established an essential role for the tail domain, revealing a novel mechanism for the control of Erk activation through RasGRP1.
Dendritic cells patrol epithelium and the lumen of the crypts associated with the organized lymphoid tissue of the colon. (161.1)
Authors: Dzutsev Amiran , Koo Lily , Sui Yongjun , Gagnon Susan , Schwartz Owen , Belyakov Igor , Berzofsky Jay
Published: January 2023

Antigen capture and presentation by the gut dendritic cells (DCs) is a complicated process that often involves a direct sampling of the antigens from the lumen. Peyer’s patches (PP) and colonic patches (CP) contain two distinct population of DCs that are thought to participate in the antigen uptake and transport of antigens from the lumen. One group of the DCs sample the lumen by extending the transepithelial dendrites from the lamina propria. The other group of DCs is localized in the intraepithelial compartment itself. In our study we have shown, by intravital imaging of the colon, that the previously reported population of intraepithelial DCs belongs to the fraction of highly motile CD11c+ cells that circulate between crypts and the T-cell and B-cell zones of the CP. We observed that these CD11c+ cells come from the T-cell zone and make a brief contact with the lumen before retreating. Upon examination of the expression profiles of different chemokines we found that CCL20 is expressed on the apical portion of the colonic patch-associated epithelial cells. Administration of anti-CCL20 antibodies blocked homing of CD11c+ cells to the intraepithelial compartment. Motile CD11c+ cells were also able to uptake fluorescent particles and Salmonella bacteria from the lumen and rapidly transport them to the T- and B-cell zones. Thus intraepithelial DCs might play an important role in antigen uptake and trafficking in the gut lymphoid compartments.
Synergy between intraepithelial lymphocytes and lamina propria T cells drives inflammation during infection (161.12)
Authors: Egan Charlotte , Maurer Kirk , Cohen Sara , Mack Matthias , Simpson Kenneth , Denkers Eric
Published: January 2023

Oral infection of C57BL/6 mice with the protozoan parasite Toxoplasma gondii triggers severe necrotic lesions in the ileum within 7-10 days of cyst inoculation. Lesion development is mediated by Th-1 cytokines and CD4+ T cells. Pathology is associated with a shift in gut flora to predominantly gram-negative species and sub-epithelial bacterial translocation. As such, these features share striking similarity to inflammatory bowel diseases in humans, in particular Crohn’s disease. Recently, we uncovered an unexpected role for IFN-γ-dependent intraepithelial lymphocytes (IEL) in mediating ileal pathology after Toxoplasma infection. We show here that αβ and not γδ T cell receptor-positive IEL mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1-/- mice, we definitively establish that lesions are caused by pathogen-induced immunological factors rather than parasite-mediated tissue destruction. Furthermore, we demonstrate that IEL do not act alone to cause inflammatory lesions, but function to recruit CCR2-dependent CD4+ T lymphocytes from the lamina propria into the IEL compartment. Our data provide valuable new insight into mechanisms of intestinal inflammation triggered by microbial infection, findings that have important implications for understanding human inflammatory bowel disease.
Intranasal immunization of porcine reproductive and respiratory syndrome virus entrapped biodegradable PLGA- nanoparticles elicits virus specific cross-protective immune responses in pigs (161.21)
Authors: Dwivedi Varun , Manickam Cordelia , Binjawadagi Basavaraj , Patterson Ruthi , Dodson Katie , Gourapura Renukaradhya
Published: January 2023

Porcine reproductive and respiratory syndrome (PRRS) is an economically very important endemic chronic respiratory viral disease of pigs. Live PRRS virus (PRRSV) vaccines are not safe in pregnant sows and boars, and currently used killed vaccines are ineffective. In this study, killed PRRSV Ags were entrapped in poly-DL-lactide-co-glycolide (PLGA) nanoparticles (100-500nm by SEM) with an approximate 50% entrapment efficiency. Internalization and localization of nanoparticles-PRRSV Ags inside the endosomes of pig alveolar macrophages was demonstrated by confocal microscopy. Immune correlates of protection were analyzed in nanoparticles-PRRSV Ags immunized and genetically variant PRRSV challenged pigs, inoculated intranasally, at post-challenge day 15. In nanoparticles immunized pig lungs, reduced PRRSV load and reduced gross lung lesions were detected compared to pigs unimmunized or immunized with free killed PRRSV Ags and challenged. Immunologically, increased frequency of lymphocytes, CD4+, CD8+, γδ T cells, myeloid cells, and reduced frequency of Tregs were detected in lungs, blood, and tracheobronchial lymph nodes. In addition, increased secretion of cytokines, IFN-γ, IL-6, and reduced IL-10 and TGF-β by ex vivo restimulated mucosal immune cells was detected. Thus, our results suggested that mucosal delivery of nanoparticles- PRRSV killed Ags has the potential to control PRRS.
Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung (161.5)
Authors: Willinger Tim , Rongvaux Anthony , Takizawa Hitoshi , Yancopoulos George , Valenzuela David , Murphy Andrew , Auerbach Wojtek , Eynon Elizabeth , Stevens Sean , Manz Markus , Flavell Richard
Published: January 2023

Mice with a functional human immune system have the potential to allow in vivo studies of human infectious diseases and to enable vaccine testing. To this end, mice need to fully support the development of human immune cells, allow infection with human pathogens, and be capable of mounting effective human immune responses. A major limitation of humanized mice is the poor development and function of human myeloid cells and the absence of human immune responses at mucosal surfaces such as the lung. To overcome this, we generated human IL-3/GM-CSF knock-in (hIL-3/GM-CSF KI) mice. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis due to elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the PAP syndrome. Moreover, human alveolar macrophages mounted correlates of a human innate immune response against influenza virus. hIL-3/GM-CSF KI mice represent a unique mouse model which permits the study of human mucosal immune responses to lung pathogens.
IL-17-secreting neutrophils represent a novel lung cell subset playing a critical role in the resolution of inhalational anthrax (161.6)
Authors: Anne Quesnel-Hellmann , Garraud Kevin , Cleret Aurélie , Mathieu Jacques , Fiole Daniel , Gauthier Yves , Tournier Jean-Nicolas
Published: January 2023

Background: The role of IL-17 and neutrophils in the lung has been described as two intricate but independent players, IL-17 triggering downstream recruitment of neutrophils. Methods: A/J, C57BL/6, IL-17RA-/-, CX3CR1+/gfp mice were infected intranasally by B. anthracis Sterne 7702, its toxin-defective derivative mutant RP42 and the fully virulent 17JB strains. Cytokine productions were evaluated in the broncho-alveolar lavage and the population identified by intra-cellular staining. Results: Here, we identify neutrophils as the primary IL-17-secreting cell subset in an inhalation model of anthrax infection. Using IL-17RA-/- mice, we confirm that IL-17A/F signaling is instrumental in the self-recruitment of this population. Moreover, we show that IL-17A/F axis is critical for survival to pulmonary infection as IL-17RA-/- mice become susceptible to intranasal infection by anthrax Sterne spores. Eventually, by depleting neutrophils in wild type and IL-17RA-/- mice, we demonstrate the crucial role of IL-17-secreting neutrophils for mouse survival after anthrax infection in the Sterne strain model. In accordance, following intranasal infection with a fully virulent strain, neutrophils prove to be critical for survival. Conclusion: This work demonstrates the very intricate role of both IL-17 and neutrophils in the clearance of pathogen and survival to pulmonary anthrax.
Bone marrow mesenchymal stem cells calibrate the innate immune tone by inducing monocyte emigration in response to circulating TLR-ligands (162.1)
Authors: Shi Chao , Jia Ting , Mendez-Ferrer Simon , Frenette Paul , Pamer Eric
Published: January 2023

Inflammatory (Ly6Chi CCR2+) monocytes provide defense against infections but also contribute to autoimmune diseases and atherosclerosis. Monocytes originate from the bone marrow and their entry into the bloodstream requires stimulation of CCR2 chemokine receptors by MCP1. How monocyte emigration from bone marrow is triggered by infections in anatomically remote sites remains unclear. We demonstrate that low concentrations of TLR ligands in the bloodstream drive CCR2-dependent emigration of monocytes from bone marrow. Bone marrow mesenchymal stem cells (MSCs) rapidly express MCP1 in response to circulating TLR ligands or systemic bacterial infection and induce inflammatory monocyte trafficking into the bloodstream. Targeted deletion of MCP1 from MSCs impaired monocyte emigration from bone marrow upon LPS stimulation and increased susceptibility to Listeria monocytogenes infection. Our findings suggest that bone marrow MSCs respond to circulating microbial molecules and regulate bloodstream monocyte frequencies by secreting MCP1 in proximity to bone marrow vascular sinuses.
IL-13 regulates the in vivo generation of IL-17 producing T cells (163.15)
Authors: Kinyanjui Margaret , Shan Jichuan , Fixman Elizabeth
Published: January 2023

Several features associated with allergic airways disease are regulated by the Th2 cytokine IL-13. IL-17 is also known to regulate aspects of allergic disease since production of IL-17 is associated with severe asthma and correlates with an increase in airway neutrophilia. The mechanism by which these IL-17 producing cells develop during airways disease is still unclear. To better understand what factors regulate the development of IL-17 producing cells we used an acute model of IL-13-induced airway inflammation in which IL-13 was intranasally administered to anaesthetized mice. As expected, administration of IL-13 induced airway eosinophilia, airway hyperresponsiveness, and mucus production. In addition, IL-13 induced airway neutrophilia as well as increases in expression of neutrophil chemokines. IL-13 also induced increased mRNA expression of IL-17 and IL-6 in the lung. Intracellular cytokine staining of bronchoalveolar lavage cells showed that IL-13 increased the number of IL-17+ve cells when compared to control. These cells did not produce IL-22 or IFN-gamma and were identified as either CD4 or γδ T cells. Animals were then treated with IL-13 in the presence of BrdU to follow development of IL-17 expresing cells in vivo. The majority of these cells incorporated BrdU suggesting that these IL-17+ve cells proliferated in the lung. Further studies to characterize these IL17+ve T cells are under way.
Assessment of the role of basophils in promoting allergen-specific T-cell proliferation in allergic patients (163.3)
Authors: Eckl-Dorna Julia , Ellinger Adolf , Pavelka Margit , Blatt Katharina , Valent Peter , Womastek Irene , Valenta Rudolf , Niederberger Verena
Published: January 2023

The long standing view that basophils mainly play an effector role in allergy by releasing histamine and eicosanoids upon crosslinking of FcϵRI receptor bound immunoglobulin E with specific allergen has recently been challenged. Studies in mouse models indicated an additional role of basophils as antigen presenting cells (APCs) in the initiation of Th2 immunity. However, so far this question has not been addressed in allergic patients. Here we compared basophils and professional APCs with regards to their capacity in promoting in vitro T-cell proliferation upon allergen challenge. T-cells isolated from allergic patients were either cultured alone, together with APCs depleted of basophils or with purified basophils in the presence or absence of allergen. Proliferation was measured by 3H-thymidine incorporation. Stimulation with allergen-induced proliferation in both total PBMCs used as controls and T-cells co-cultured with APCs. In contrast, T-cells co-cultured with basophils did not proliferate in response to allergen stimulation. Addition of cytokines promoting basophil survival to the cultures did not have relevant effects on their capacity to promote T-cell proliferation. In summary, our data indicate that basophils do not play an important role in promoting T-cell proliferation in vitro upon allergen challenge in allergic patients.
Prevention of autoimmune myocarditis by angiotensin inhibition (164.13)
Authors: Schuessler Teresa , Engman David
Published: January 2023

Myocarditis, inflammation of the heart, is one of many cardiac pathologic states that can ultimately lead to heart failure. A variety of infectious and noninfectious agents can cause myocarditis, and certain individuals will develop autoimmune responses to various cardiac antigens. To study the autoimmune aspects of the disease, an animal model of experimental autoimmune myocarditis (EAM) has been developed in which immunization of susceptible mouse strains with cardiac myosin induces cardiac inflammation, hypertrophy, and fibrosis through mechanisms dependent on CD4+ T cells. We tested the ability of the antifibrotic, angiotensin-converting enzyme (ACE) inhibitor captopril to inhibit the massive fibrosis that develops during EAM. To our initial surprise, not only did captopril prevent fibrosis, but also it prevented inflammation primarily. Captopril treatment did not appear to directly affect T cell function, since T cells from captopril-treated mice proliferated and secreted proinflammatory cytokines. However, preliminary data indicate that immune cells from spleens of captopril-treated mice show reduced ability to transmigrate across endothelium ex vivo, suggesting that ACE inhibition somehow affects cell trafficking into the myocardium. Current studies are underway to further elucidate the mechanisms by which ACE inhibition mediates these effects with respect to how ACE and ACE products provide proinflammatory signals during EAM.
Docosahexaenoic acid modulates CD4+ T cell differentiation and is protective in experimental autoimmune encephalomyelitis (164.18)
Authors: Kong Weimin , Yen Jui-Hung , Adhikary Sabina , Ganea Doina
Published: January 2023

Protective anti-inflammatory effects of DHA were reported in clinical studies and animal models of colitis, sepsis, and stroke. In the present study, we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) in CD4+ T cell stimulation and differentiation. Pretreatment of DC with DHA prevented LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of costimulatory molecules and lack of proinflammatory cytokine production (IL-12p70, IL-6 and IL-23). DHA-treated DC were poor stimulators of antigen-specific T cells in terms of proliferation and Th1/Th17 differentiation. This was associated with an increase in p27(kip1), a cell cycle arresting agent. In contrast, T cells co-cultured with DC-DHA express higher levels of TGFβ and Foxp3, without exhibiting a functional Treg phenotype. In addition, DHA prevents up-regulation of VCAM-1 and ICAM-1 in bEND.3 cells (mouse brain microvascular endothelial cell line). Here we report for the first time a beneficial effect of dietary n-3 fatty acids in experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. The beneficial effect of DHA in EAE was associated with reduced numbers of IFNγ- and IL-17-producing CD4+ T cells in both spleen and CNS.
Interleukin-7 promotes autoimmune disease by enabling memory T cells to escape PD-1-mediated tolerance (164.21)
Authors: Dooms Hans , Penaranda Cristina , Kuswanto Wilson , Hofmann Jerry , Kenefeck Rupert , Narendran Parth , Walker Lucy , Bluestone Jeffrey , Abbas Abul
Published: January 2023

An important challenge in the therapy of immune-mediated inflammatory diseases is to control established disease, largely because autoreactive memory T lymphocytes are difficult to suppress or eliminate. Memory T cells depend on interleukin-7 (IL-7) for their generation and maintenance and genomic studies have associated the cytokine’s receptor, IL-7Rα, with increased risk for autoimmune diseases such as type 1 diabetes. Here we show that IL-7Rα blockade reverses hyperglycemia in the non-obese diabetic (NOD) mouse model of type 1 diabetes. Diabetogenic memory T cells deprived of IL-7 signals remained present in the lymphoid organs but expressed the inhibitory molecule Programmed Death 1 (PD-1), which is critical for preventing autoimmunity. Blocking the interaction of PD-1 with its ligand, PD-L1, in cured mice rapidly induced hyperglycemia, demonstrating that this mechanism inhibited memory responses to pancreatic islets after IL-7Rα blockade. Conversely, IL-7 suppressed PD-1 expression after T cell activation, indicating that IL-7 contributes to the pathogenesis of autoimmune diabetes by keeping memory T cells in a functionally competent, tolerance-resistant state. Our data uncover a novel link between IL-7 and the PD-1/PD-L1 tolerance pathway and identify IL-7Rα blockade as a potential therapeutic approach to induce PD-1-mediated inhibition of pathogenic memory T cells in established T cell-dependent inflammatory diseases.
Novel, orally active proteasome inhibitor, CEP-18770, protects and treats disease in preclinical mouse models of lupus nephritis (164.3)
Authors: Seavey Matthew , Lu Lily , Stump Kristine , Wallace Nate , Ruggeri Bruce
Published: January 2023

Since therapeutic blockade of the cellular proteasome has previously demonstrated preclinical efficacy against inflammatory diseases, the evaluation of a novel, orally active proteosome inhibitor, CEP-18770, was tested in models of systemic lupus erythematosus (SLE). Age matched, 8-week old (MRL/lpr) or 8 months old (NZM) mice with established SLE or lupus nephritis (LN), respectively, were treated with CEP-18770 either 3mg/kg 1-2x/wk, iv, or orally at 10mg/kg. Reference agent, bortezomib, was provided at 0.5mg/kg, 1x/wk, or 0.3mg/kg 1-2x/wk. Standard of care agents included dexamethasone or cyclophosphamide. Mice were monitored for clinical, pathological and immunological changes. Reductions in the frequencies of specific anti-chromatin, smith and dsDNA antibody secreting cells (ASC) and levels of the corresponding antinuclear antibodies (ANA), followed CEP-18770 treatment and were accompanied by decreases in spleen ASCs, plasma cells, and serum cytokines (IL-12, IL-17A, IL-1β, TNFα). CEP-18770-treatment also mitigated the incidence of renal histopathology, and subsequently, proteinuria, in both models. CEP-18770 treatment also extended survival for both models over bortezomib. Proteasome inhibition and IκBα accumulation in target tissues followed administration of CEP-18770 and bortezomib. CEP-18770 was equivalent to treatment with bortezomib; however, CEP-18770 resulted in greater tolerability and rate of response resulting in greater SLE disease stability overtime.
Egr-2 is a tumour repressive gene (165.1)
Authors: Miao Tizong , Wang Ping
Published: January 2023

EGR-2, a zinc finger domain containing transcription factor, plays an important role in the development of both centre never system and T lymphocytes. However, the mechanism of action is unknown. To investigate Egr-2 function in lymphocytes, we have developed lymphocyte specific Egr-2 KO (Knockout) and Tg (Transgenic) mice. In the absence of Egr-2, the homeostasis of T cells is disregulated with hyper-proliferation of effector phenotype cells. In addition to the increased effector phenotype lymphocytes, the development of spontanouse B and T cell lymphoma in Egr-2 KO mice has been found at later age. More than 30% Egr-2 KO mice aged more than 12-months developed either T or B cell lymphoma. Oncogene-database (Oncomine gene expression database) search showed that the expression of Egr-2 is either suppressed or deleted in haematopoietic tumours. To explore anti-tumour function, Egr-2 was transfected into HKE 293 and mouse melanoma B16 cell lines, respectively. The transfected cells exhibited small cell body and stopped cell division. In the undergoing mechanism study, we found that 1), The expression of p53 was increased in lymphocytes from Egr-2 Tg mice and p53 was phosphorylated in Egr-2 transfected HKE293 cells.2), Egr-2 binds to c-Jun, the key components of AP-1; 3), Egr-2 strongly inhibited STATs transcriptional activity. Our results suggest that tumour suppressive role of Egr-2 involved in multple cell signalling pathways.
Combining gp96-Ig vaccination and mTL1A-Ig fusion protein boosts anti-tumor immunity. (165.10)
Authors: Khan Samia , Schreiber Taylor , Deyev Vadim , Podack Eckhard
Published: January 2023

Tumor necrosis factor 25 (TNFR25) and its natural ligand, TL1A, inhibit the suppressive activity of regulatory T cells (Tregs) and block de novo biogenesis of antigen specific induced Treg (iTreg). TL1A/TNFR25 co-stimulates CD4+ and CD8+ T cells and promotes proliferation and cytokine production in the presence of antigen. Due to the role of TNFR25 as a co-stimulator of T cells, we hypothesize that combination therapy with g96-Ig vaccination will enhance the robust expansion of CD8 CTL observed with vaccine alone. As expected, gp96 vaccine with TNFR25 agonistic antibody, 4C12, enhanced CD8 CTL expansion and tumor rejection as compared to vaccine only. Here we genetically engineered murine TL1A-Ig fusion proteins that differ in retention of the cleavage site in the extracellular domain fused to the C-terminus of mIgG1 Fc. The in vitro potencies of the TNFR25 agonist fusion proteins were confirmed by caspase activation and binding assays. Apparent molecular weights were measured as 447 and 152 kDa by gel filtration, and biological half life was determined as 7 hours in mice. In vitro, mTL1A-Ig with anti-CD3 caused rapid proliferation of all CD4+ T lymphocytes, including CD4+FoxP3+Tregs. Enhanced antigen specific CTL expansion was observed in the spleen and peritoneal cavity when naïve mice were treated with vaccine and mTL1A-Ig. We will optimize dose and scheduling of mTL1A-Ig injection, which can be utilized in combination with the vaccine to enhance therapeutic benefit.
The pro-apoptotic effect of vitamin C on colon cancer cell lines, HCT116 via the regulation of SIRT1 expression (165.11)
Authors: Yu Yeon-sil , Bae Seyeon , Kim Hyemin , Kong Joo Myung , Kim Hang Rae , Kang Jae Seung , Hwang Young Il , Lee Wang Jae
Published: January 2023

Vitamin C is an essential nutrient for living organisms to protect from oxidative stress. In addition, It shows potent anti-tumor activity through the enhancement of immune system. However, there are few reports regarding the direct cytotoxic activity on tumor cells and its related mechanisms. Therefore, we investigated the direct cytotoxic activity of vitamin C on tumor cells especially through the regulation of SIRT1. Since It is generally known that the activity of SIRT1, a NAD-dependent deacetylase, is related with the survival and proliferation of tumor cells, we have first investigated whether vitamin C shows an anti-tumor effect through the regulation of SIRT1 activity in HCT116s. As a result, we found that extensive apoptosis is occurred in HCT116 in company with the suppression of SIRT1 mRNA expression by the treatment of vitamin C. Even though it is known that SIRT1 expression is regulated by p53, we could not find any differences between p53 wild type HCT116 and p53 null type HCT116. To clarify the direct regulatory effect of vitamin C on SIRT1 activity, we examined the change of phosphorylation on SIRT1 after treatment of vitamin C. We observed that vitamin C suppressed the phosphorylation of SIRT1 on the both of ser27 and ser47 residues. Taken together, our data suggest that vitamin C induces apoptosis in HCT116 via the down-regulation of SIRT1 expression and suppression of its phosphorylation.
CXC chemokine receptor 3 deficiency promotes cutaneous tumor formation (165.14)
Authors: Masui Yuri , Kai Hiromichi , Kadono Takafumi , Sato Shinichi
Published: January 2023

CXC chemokine receptor 3 (CXCR3), G protein-coupled, seven-transmembrane receptor, binds and is activated by the three IFN-γ-inducible C-X-C chemokines; MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11. CXCR3 is expressed on natural killer cells, plasmacytoid dendritic cells, myeloid dendritic cells, B cells, activated T cells, proliferating endothelial cells, and preferentially expressed by Th1-polarized activated/memory T cells. Although recent studies have correlated the expression of CXCR3 and its ligands with a variety of diseases or pathological states, little has been known about the engagements of CXCR3 in cutaneous immune responses, especially cutaneous anti-tumor immunity. In this study, we injected B16 murine melanoma cells into the skin superficial to the ear cartilage of CXCR3-deficient mice. The tumor formation in the ears from CXCR3-deficient mice was accelerated compared to that from C57BL/6 mice. Local cytokine mRNA expression levels of the ear tumors were measured by real-time PCR and immunohistochemical analysis was performed to evaluate leukocyte subsets within the tumors. Thus, CXCR3 may play an important role in tumor immunity.