bluuebunny/crossref_metadata_2025 huggingface dataset:
Thwarting HIV evasion of antibody avidity with novel antibody architectures (P4489)
Authors: Galimidi Rachel , West Anthony , Bjorkman Pamela
Published: January 2023

Despite decades of effort, no current vaccine elicits neutralizing antibodies at concentrations blocking HIV infection. In addition to structural features of HIV's envelope spike that facilitate antibody evasion, a hypothesis for the ineffective immune response lies in the low density and limited mobility of HIV envelope spikes, which impedes bivalent binding by antibodies, reducing avidity, minimizing the potential for high affinity binding and virus neutralization. We intend to engineer anti-HIV reagents that bind with high avidity to single spikes, overcoming potential problems with the low density of HIV spikes. Here, we demonstrate a strategy to use dsDNA as a rigid molecular ruler to map epitopes on the HIV envelope protein to gain insight into the relatively unknown spatial environment of the spike trimer. Upon determining the optimal separation distance between epitopes, the dsDNA linker will be replaced with a structured protein linker. This technique should allow for the development of a novel multivalent antibody reagent improving binding and increasing avidity. Optimal HIV binding proteins will be trimerized by attaching a trimerization motif, reducing the concentration required for sterilizing immunity. To date, we have several bispecific DNA reagents that have the ability to neutralize various strains of HIV with greater potency than its individual components. These results demonstrate the promise for discovery of optimal anti-HIV reagents using this technology.
HIV-1 gag specific exosome-targeted CD8+ T cell based vaccine stimulates specific CTL responses leading to long-term immunity (P4493)
Authors: Wang Rong , Xiang Jim
Published: January 2023

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4+T cell deficiency. We have recently demonstrated that Con A stimulated non-specific CD8+T cells could uptake OVA pulsed DC-derived exosome (EXO), act as CD8+Tc antigen presenting cells (Th-APCs), and capable of stimulating DC- and CD4+T cell-independent OVA-specific CTL responses leading to long-term immunity. This formed the new concept novel EXO-targeted CD8+T cell vaccine. The extreme conservation and intolerance of escape variants in HIV-1 Gag p24 make it a potential target for vaccine design. In this study, we constructed adenoviral vector AdV-gag and expression vector pEGFP-gag both expressing Gag. We generated Gag specific EXO-targeted T (Gag-Texo) cell vaccine by using C57BL/6 mouse CD8+ ConA T cells with uptake of AdV-gag transfected bone marrow DC (DCgag) released EXOgag. We then generated BL6-10gag cell lines by transfection of B16 melanoma cell line (BL6-10) with pEGFP-gag. We demonstrated that Gag-Texo vaccine is capable of stimulating efficient Gag-specific CD8+ CTL responses in vivo, leading to antitumor immunity against Gag expressing B16 melanoma (BL6-10 gag) in animal studies. Therefore, this novel HIV-1-specific EXO-targeted Gag-Texo vaccine may be useful in induction of efficient CTL responses in AIDS patients.
R-spondin3 prevents mesenteric ischemia/reperfusion-induced tissue damage by tightening endothelium and preventing vascular leakage (P5060)
Authors: Kannan Lakshmi , Kis-Toth Katalin , Thai To-Ha , Sehrawat Seema , Mayadas Tanya , Dalle Lucca Jurandir , Tsokos George
Published: January 2023

Inflammation and vascular injury triggered by ischemia/reperfusion (I/R) represent a leading cause of morbidity and mortality in a number of clinical settings. Wnt and its homologue partners R-spondins, in addition to regulating embryonic development have recently been demonstrated to serve as wound-healing agents in inflammation-associated conditions. Here we ask whether R-spondins could prevent inflammation-associated tissue damage in ischemic disorders and thus investigate the role of R-spondin3 (R-spo3) in a mouse model of mesenteric I/R. We demonstrate that R-spo3 ameliorates mesenteric I/R-induced local intestinal as well as remote lung damage by suppressing local and systemic cytokine responses and deposition of IgM and complement in intestinal tissues. We show that decreased inflammatory response is accompanied by the tightening of endothelial cell junctions and reduction in vascular leakage. We further provide evidence that R-spo3 preserves endothelial barrier function by maintaining VE-cadherin expression at adherens junction and decreasing the formation of stress fibers. We thus conclude that R-spo3 stabilizes endothelial junctions and inhibits vascular leakage during I/R and thereby mitigates the inflammatory events and associated tissue damage. Our findings, for the first time, demonstrate a protective effect of R-spo3 in I/R-related tissue injury and suggest a mechanism by which it may have these effects.
Inhibition of ADAM10 and 8 reduces transendothelial migration of the monocytic cell line THP1 in vitro (P5066)
Authors: Hess Franz , Alert Henriette , Prüssmeyer Jessica , Ludwig Andreas
Published: January 2023

Metalloproteases of the A Disintegrin And Metalproteases (ADAM) family are important molecular mediators of inflammation. ADAMs participate at all stages of inflammation via the proteolytic cleavage of cell surface molecules. It has been shown that leukocyte expressed ADAM8 and ADAM17 are important during the initial rolling of leukocytes on the endothelium. However, the role of leukocyte expressed ADAMs in transendothelial migration and chemotaxis remains to be investigated and in particular it is still unclear to what extent ADAM10 and ADAM9 contribute to leukocyte migration. In this study we show that treatment of leukocytes with a pharmaceutical ADAM10 inhibitor reduced both the in vitro transendothelial migration and the chemotaxis of the monocytic cell line THP1. The combined pharmaceutical inhibition of both ADAM10 and ADAM17 did not further suppress leukocyte migration in either experimental setting. The subsequent analysis of THP1 cells with a gene silencing of ADAM8, 9, 10 or 17 in transendothelial migration and chemotaxis experiments revealed that ADAM8 and ADAM10 are critically involved in THP1 cell migration. While ADAM9 did not seem to be required ADAM17 was only ofminor importance for the migration of THP1 cells. This study analyses the role of the four proteases ADAM8, 9, 10 and 17 in the migration of leukocytic cells. It shows that ADAM8 and ADAM10 are crucial for the in vitro transendothelial migration and chemotaxis of the monocytic cell line THP1.
Hydrogen peroxide as a signal induced cellular functions of macrophage in oxidative stress (P5038)
Authors: Wu Yu-Sheng , Chen Shiu-Nan
Published: January 2023

Experiments performed to show that one of the respiratory oxygen species, hydrogen peroxide was an attractant factor for macrophage that consequently affects the phagocytosis of macrophage and gene expression. Results obtained from the present study also show that LPS-induced inflammation would cause hepatocytes to produce hydrogen peroxide which subsequently attract macrophage migration to the inflamed region and promote scavenging function. Furthermore, this reaction is suggestive of macrophage interacts with oxidative stress which promotes the transcription factors activity to cause inflammation responses and suggested hydrogen peroxide act as cellular messenger to affect permeability of calcium ion channel to increase intracellular Ca2+ concentration as a migration messenger
Differential regulation of pro-inflammatory necrosis in males and females by Poly (ADP-Ribose) Polymerase-1 and 17β estradiol (P5074)
Authors: Jog Neelakshi , Caricchio Roberto
Published: January 2023

Cell death can be divided into the anti-inflammatory apoptosis and the pro-inflammatory necrosis. Necrosis as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)1 are two of the mediators. We previously showed that absence or inhibition of PARP-1 protects mice from immune mediated nephritis. Surprisingly, this protection showed a sex-bias, with only male mice being protected. Several studies have shown that the response of males and females to stress differs, although the basis for this sexual dimorphism remains to be determined. We hypothesized that these differences may be due to an inherent difference in the cell death program between the two sexes. We show here that in an immune mediated nephritis model, females show increased apoptosis compared to males. Treatment of males with estrogens inhibited necrosis but induced apoptosis to levels similar to those observed in females. Although PARP-1 was activated in both males and females, absence or inhibition of PARP-1 reduced necrosis only in males. Inhibition of RIP1 with Necrostatin-1 did not show sex bias. We demonstrate here that males and females are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of sex-bias in cell death. We therefore propose that clinical targeting of cell death should differ based on sex and could lead to better treatments for each gender.
Positive allosteric modulation of M1 muscarinic acetylcholine receptors suppresses lethal peripheral inflammation (P5082)
Authors: Pavlov Valentin , Ochani Mahendar , Dancho Meghan , Al-Abed Yousef , Nathanson Neil , Tracey Kevin
Published: January 2023

Peripheral inflammation can be regulated by activation of brain muscarinic acetylcholine receptor (mAChR)-dependent signaling functionally associated with a vagus-nerve mediated anti-inflammatory circuit (Proc Natl Acad Sci USA, 2006, 5219; Brain Behav Immun, 2009, 23:41). Here, we studied the specific role of the M1 mAChR subtype in this regulation by utilizing BQCA, a highly specific allosteric M1 mAChR activator that crosses the blood-brain barrier. Single drug (5,10 or 20 mg/kg, i.p) administration in mice 1h prior to endotoxin (8 mg/kg, i.p.) dose-dependently reduced serum and splenic TNF levels and significantly improved survival in mice as compared to vehicle-treated controls. Pharmacological blockade of brain mAChRs significantly abolished BQCA anti-inflammatory effects. Furthermore, BQCA (20 mg/kg, i.p.) significantly reduced serum and splenic TNF levels in wild type mice, but failed to alter TNF levels in M1 KO mice. Together these results indicate the anti-inflammatory role of increased functional activity of endogenous acetylcholine on the M1 mAChR by selective allosteric receptor activation. Our findings are of interest for further development of BQCA and other centrally-acting allosteric activators of the M1 mAChR as a novel class of experimental anti-inflammatory therapeutics.
Selenoprotein activity alters eicosanoid biosynthesis in macrophages (P5042)
Authors: Mattmiller Sarah , Sordillo Lorraine , Carlson Bradley
Published: January 2023

Selenium (Se) exerts anti-inflammatory properties largely through the activity of antioxidant selenoproteins. However, the effect of reduced selenoprotein activity on the eicosanoid signaling network is unknown. Therefore, the objective of this study was to characterize the biosynthesis of eicosanoids that enhance or resolve inflammation as a function of selenoprotein activity. Macrophages cultured in Se-sufficient or Se-deficient media or macrophages obtained from selenoprotein conditional knockout mice were used to assess inflammatory markers and eicosanoids. Reduced selenoprotein activity significantly increased free radical accumulation, pro-inflammatory cytokines, and cyclooxygenase and lipoxygenase enzyme expression. Reduced selenoprotein activity significantly diminished the production of anti-inflammatory eicosanoids, lipoxin A4 and protectin, hydroxyl eicosanoids from arachidonic acid, and hydroxyl and ketone eicosanoids from linoleic acid (LA). Since LA is a predominant lipid in the western diet and LA-derived metabolites were produced in substantial amounts by macrophages, studies are currently underway to determine the inflammatory effect of the LA ketone, 9oxoODE. Future studies aim at determining if LA eicosanoids are oxidized by lipoxygenases or by non-enzymatic means when selenoprotein activity is reduced. A better understanding of how Se regulates eicosanoid biosynthesis may uncover nutritional intervention strategies to counteract uncontrolled inflammation.
Ethanol extract of Alismatis Rhizoma reduces acute lung inflammation by suppressing NF-κB and activating Nrf2 (P5044)
Authors: Kwun Min Jung , Kim Kyun Ha , Choi Jun-Yong , Lee Jang Hoon , Joo Myungsoo , Han Chang Woo
Published: January 2023

The tuber of Alisma orientale has an anti-inflammatory effect. Here, we investigated a possible underlying mechanism and a protective effect on acute lung injury (ALI). A. orientale tuber was extracted in 80% ethanol and dried. The powder of the ethanol extract of A. orientale tuber (EEAO) was dissolved in PBS. The effect of EEAO on NF-κB and Nrf2 activities was analyzed with RAW 264.7 cells. The effect of EEAO on lung inflammation was determined by histologic and molecular biological analyses of the lung tissue of C57BL/6 mice that were gavaged once a day with 0.3 or 1.2g/kg of EEAO for 14 days, prior to an intranasal administration of LPS (0.01g/kg) for inducing ALI. EEAO pre-treatment of RAW 264.7 cells suppressed NF-κB activity and the expression of its dependent genes including COX-2, IL-1β and iNOS. Similar treatment enhanced Nrf2 activity and the expression of Nrf2-regulated genes including NQO-1, HO-1 and GCLC. LPS instillation induced acute neutrophilic lung inflammation, which was significantly suppressed by pre-treatment with EEAO. Analysis of the lungs revealed that EEAO pre-treatment induced the expression of Nrf2-regulated genes, with concomitant down-regulation of inflammatory gene expression. EEAO attenuated lung inflammation in LPS-induced ALI mice, which was associated with differential regulation of NF-κB and Nrf2 activities. We suggest that EEAO can be developed as a potential therapeutics for the treatment of ALI.
IL-33- and IL-25-responsive innate lymphoid cells are present in human peripheral blood. (P6237)
Authors: Bartemes Kathleen , Fox Stephanie , Kita Hirohito
Published: January 2023

Recent advances in the initiation and maintenance of type 2 immune responses have highlighted a new cell type, the type 2 innate lymphoid cell (ILC2). Studies using mouse models show that ILC2s mediate helminth clearance in the gut and development of airway hyperresponsiveness in the lung. Further, the epithelium-derived cytokines IL-25 and IL-33 are integral to this process. However, how ILC2s contribute to chronic human airway diseases such as asthma and how IL-25 and IL-33 mediate that effect are not well characterized. Here we show that human peripheral blood mononuclear cells (PBMCs) respond to IL-33 or IL-25 stimulation by making IL-5 and IL-13. CD3+ and CD16+ PBMCs are not required. Furthermore, an ILC2-like cell that is lineage-negative and CD44+CD127+ST2+ is present in the peripheral blood of a subject with asthma. IL-5 production was induced by IL-33 or IL-25 in PBMCs from normal individuals as well as from patients with allergic rhinitis or allergic asthma, however, PBMCs from the group with asthma released significantly higher amounts. Our data suggests that the number and function of ILC2s is greater in patients with allergic asthma than in healthy subjects or patients with allergic rhinitis. Thus, ILC2s may be involved in the development of type 2 airway diseases.
The size effects of amorphous silica nanoparticles on atopic dermatitis-like skin lesion (P6259)
Authors: Takahashi Hideki , Yoshioka Yasuo , Hirai Toshiro , Ichihashi Ko-ichi , Nishijima Nobuo , Yoshida Tokuyuki , Nabeshi Hiromi , Yoshikawa Tomoaki , Tsunoda Shin-ichi , Higashisaka Kazuma , Tsutsumi Yasuo
Published: January 2023

Exposure to environmental factors, such as microbes, diesel exhaust, and other chemicals are speculated to be associated with atopic dermatitis (AD), however, the detailed relationships between environmental factors and increase in AD are not well understood. Additionally, recent industrialization poses new threats to the development of AD. We recently revealed that amorphous silica nanoparticles (nSP) can penetrate the skin barrier and induce various immunological effects. Thus, nSP in various products may also be associated with development of the other allergic diseases such as AD. Here, we investigated the effect of nSPs on AD like skin lesions. We injected mite antigen (Dp) plus various-sized silica particles (SPs : diameter of 1,000, 300, 100, 70 and 30 nm) intradermally into the ear of NC/Nga mice which develops AD-like skin lesions induced by a mite allergen Dp. Ear thickness measurements and histopathological analysis revealed that a combined injection of SPs and Dp induced aggravation of AD in an SP size-dependent manner compared to that of Dp alone. Furthermore, these effects were correlated with the excessive induction of total IgE and Dp specific IgE. We suggested that these results are associated with the induction of IL-18 and thymic stromal lymphopoietin (TSLP), which leads to systemic Th2 response in the skin lesions. These results indicate that the size of SPs is critical determinant in terms of their safe use.
Chemerin receptor CCRL2 is required for maximal induction of peanut-induced anaphylaxis (P6253)
Authors: Lewen Susanna , Zhang Jian , Zabel Brian
Published: January 2023

Food allergy affects 6-8% of young children and ~2% of adults in the U.S., and is the leading cause of life-threatening anaphylactic reactions treated in hospital emergency departments. CCRL2 is an atypical attractant receptor expressed on vascular endothelial cells and mast cells that binds and concentrates chemerin on the cell surface. In vitro, this serves to significantly enhance adhesion of CMKLR1+ cells. We previously showed that CCRL2 is required for maximal tissue swelling and leukocyte infiltration in passive cutaneous anaphylaxis. We therefore hypothesized that CCRL2 plays a pathogenic role in experimental peanut-induced anaphylaxis (PIA). Wild-type (WT) or CCRL2 KO mice were sensitized by repeated oral inoculation with peanut protein and cholera toxin over four weeks, and challenged with systemic injection of peanut protein. CCRL2 was required for maximal peanut-specific immunoglobulin secretion, leukotriene B4 release, and vascular leakiness. Circulating chemerin levels were also significantly increased in the CCRL2 KO mice. Although circulating histamine levels were significantly elevated in CCRL2 KO mice compared with WT, clinical signs of anaphylaxis were significantly reduced. CCRL2 may therefore represent a novel drug target for preventing or treating food allergy in the clinic.
Antigen-specific T cell and B cell responses to the herpes zoster vaccine Zostavax® in the young and elderly. (P6330)
Authors: Sullivan Nicole , McCausland Megan , Chiu Christopher , Sidney John , Rouphael Nadine , Mehta Aneesh , Mulligan Mark , Peters Bjoern , Sette Alessandro , Pulendran Bali , Ahmed Rafi
Published: January 2023

Herpes zoster (HZ or shingles) is the clinical manifestation of varicella zoster virus reactivation. HZ typically develops as people age due to decreased cell mediated immunity. The goal of this study was to characterize adaptive immune responses to HZ vaccination in the young and elderly. Sixteen young (25-40yo) and 23 elderly (60-79yo) patients were enrolled and vaccinated. Longitudinal analysis of B cell responses demonstrated a transient increase in the HZ-specific plasmablast population 7 days post vaccination. Although both groups increased the number of HZ-specific plasmablasts, there was a significant reduction in the magnitude of response in the elderly group (p=0.0158). Next, modest T cell proliferation (Ki67+Bcl2-) was detected after vaccination (CD4 fold change: young=2.04±0.19; elderly=1.64±0.19)(CD8 fold change: young=1.31±0.17;elderly=1.47±0.17). No significant difference in CD4 and CD8 T cell responses was detected in the young vs. elderly (p=0.1511 and p=0.5172 respectively). Finally, we identified several new HZ-specific CD4 T cell epitopes using predictive epitope mapping followed by IFN-γ elispot. The majority of the CD4 epitopes identified were present in several glycoproteins and immediate early genes. Kinetic analysis showed increases in many of these epitopes up to 90 days post vaccination. Ongoing studies will further characterize the quality and quantity of the HZ-specific CD4 T cell response post vaccination.
SLC11A1 is expressed by innate lymphocytes and augments their activation (P4331)
Authors: Hedges Jodi , Kimmel Emily , Snyder Deann , Jerome Maria , Jutila Mark
Published: January 2023

SLC11A1 (formerly NRAMP1 and Bcg/Lsh/Ity locus) was thought to be exclusively expressed in monocyte/macrophages and to function in phagosome maturation and in cell activation through iron-dependent interference with protein tyrosine phosphatase (PTP) activity. We characterized the expression of SLC11A1 in the majority of γδ T cells and NK cells, and in CD3+CD45RO+ T cells. Consistent with a role for iron-dependent inhibition of PTPs, SLC11A1+ lymphocytes were more prone to activation, IFN-γ expression, and retained tyrosine phosphorylation. Transfection of SLC11A1 into a human γδ T cell-like line rendered the cells more prone to activation. Expression of functional SLC11A1 in vivo has been consistently associated with increased IFN-γ expression, during Salmonella infection in particular. We compared non-adherent splenocytes from wild type (Sv/129) to cells from Sv/129 (SLC11A1-/-) mice. Wild type cells expressed significantly greater IFN-γ in response to supernatant fluids from Salmonella-infected monocytes from either strain. Our data suggest that SLC11A1 has a heretofore unknown role in activation of a large subset of innate lymphocytes that are critical sources of IFN-γ. Commonly used inbred laboratory mice lack functional SLC11A1 protein. Expression of SLC11A1 in innate lymphocytes and its role in augmenting their activation may, in part, account for inconsistencies in studies of innate lymphocytes in different animal models.
Cytokine activation induces CD25 expression and a functional high-affinity IL-2 receptor on CD56dim human NK Cells (P4349)
Authors: Leong Jeff , Chase Julie , Romee Rizwan , Schneider Stephanie , Sullivan Ryan , Fehniger Todd
Published: January 2023

The heterotrimeric high affinity IL-2 receptor (IL-2Rαβγ) is expressed on CD56bright human NK cells and confers responsiveness to picomolar concentrations of IL-2. In contrast, resting CD56dim NK cells lack IL-2Rα (CD25), but constitutively express the intermediate affinity IL-2Rβγ chains. Here, we show that cytokine-activation induces rapid and prolonged expression of CD25 on CD56dim NK cells, especially the combination of IL-15 plus IL-18. This expression of CD25 correlated with STAT5 phosphorylation in response to picomolar concentrations of IL-2, indicating the presence of a signal-competent IL-2Rαβγ. Furthermore, picomolar concentrations of IL-2 acted synergistically with IL-12 to co-stimulate IFN-γ production by pre-activated CD56dim NK cells. Similarly, low dose IL-2 enhanced the cytotoxicity of pre-activated CD56dim NK cells against K562 tumor targets. Both effects were dependent on the expression of CD25. Preliminary experiments also suggest that the induction of CD25 on CD56dim NK cells enhances proliferative capacity and survival in cultures supplemented with low dose IL-2. Since CD56dim NK cells represent the major subset of human peripheral blood NK cells, these data support a rationale for immunotherapeutic strategies that include cytokine pre-activation prior to adoptive NK cell transfer, followed by low dose IL-2 therapy.
Malfunction of dendritic cells from aged mice leads to loss of activation of Natural Killer cells (P4353)
Authors: Guo Zhenhong , Strominger Jack
Published: January 2023

The interaction between dendritic cells (DC ) and Natural killer cells (NK) plays a key role in infectious disease and tumor immunology. However, the effect of aging on the crosstalk of DC and NK, a critical step in immune activation, has never been elucidated. In our study, we purified splenic DC and NK from young and old C57BL/6 mice and cocultured them in the presence of Poly I:C. Then the activation of NK cells including the expression of CD69 and IFN-gamma secretion was analyzed. The results showed that DC from old mice could not activate NK cells from either young or old mice; in contrast, DC from young mice could activate NK from both young and old mice. In vivo injection of Poly I:C efficiently activated NK cells in young mice, but not in old mice. Further investigation showed that NK cells from young and old mice secreted similar levels of IFN-gamma in response to IL-2. However, old DC secreted less IL-15, IL-18 and IFN-alpha upon stimulation by Poly I:C than young DC, but no significant difference in IL-12 and TNF-alpha production was seen. Moreover, CD48, CD86, MHC class II, GITRL and RAE expression were significantly down-regulated in old DC. All the data suggested that the lost activation of NK in NK/DC coculture using aged mice is caused by the malfunction of DC. Investigation by microarray analysis of DC differences from young and old mice and whether the defect in DC from aged mice extends to a defect in tumor eradiation will be carried out.
IL-25 and innate lymphoid cells induce pulmonary fibrosis (P4357)
Authors: Hams Emily , Armstrong Michelle , Barlow Jillian , Kubica Malgorzata , Saunders Sean , Cooke Gordon , Crotty Thomas , Hirani Nik , Flynn Robin , Donnelly Seamas , McKenzie Andrew , Fallon Padraic
Published: January 2023

Disease conditions associated with pulmonary fibrosis are progressive and have a poor long-term prognosis with irreversible changes in airway architecture leading to marked morbidity and mortalities. Currently there are limited effective therapeutics for pulmonary fibrosis due to an inadequate understanding of the pathogenic process and underlying mechanisms. We now show that in human idiopathic pulmonary fibrosis (IPF) the cytokine interleukin (IL)-25 positively correlates with periostin a marker human pulmonary fibrosis, and the presence of IL-13-producing type 2 innate lymphoid cells (ILC2) in the lung. Using murine models of pulmonary fibrosis we demonstrate an essential role for IL-25-elicited ILC2 in the generation of pulmonary collagen deposition, independent of an adaptive T cell response. Furthermore, we define a mechanism whereby IL-25 evokes ILC2 to induce pulmonary fibrosis via the alternative activation of macrophages in the lung. Collectively, we present a new innate mechanism for the generation of pulmonary fibrosis, via IL-25 and ILC2 that occurs independently of T cell-mediated antigen-specific immune responses. These results indicate that IL-25 and ILC2 are potential therapeutic targets for the treatment of human fibrotic diseases where antigen-independent damage responses may magnify the disease.
Effect of oenothein B, a cyclic dimeric ellagitannin isolated from Epilobium angustifolium, on the antiviral responses of innate lymphocytes. (P4365)
Authors: Ramstead Andrew , Schepetkin Igor , Robison Amanda , Quinn Mark , Jutila Mark
Published: January 2023

Oenothein B is an ellagitannin isolated from the Epilobium angustifolium plant, which has been shown to have immunomodulatory properties. This compound is known to activate myeloid cells, αβ T cells, and innate lymphocytes, such as NK cells, NKT cells, and γδ T cells. Previous work from our laboratory showed that oenothein B primes and enhances the production of interferon-γ by human and bovine NK cells in response to secondary stimuli, including tumor cells and IL-18. Here, we examined whether oenothein B enhances the response of innate lymphocytes to various pathogen associated molecular patterns. We found that oenothein B enhanced interferon-γ production by bovine peripheral blood mononuclear cells in response to several pathogen associated molecular patterns, including the Toll-like receptor 3 agonist poly I:C. We have also found that intranasal administration of oenothein B to mice before infection with influenza can reduce the morbidity of the infection. Our preliminary results suggest that oenothein B is capable of enhancing innate immunity against viral infections, possibly through the enhanced production of interferon-γ by NK cells. The effect of oenothein B on the antiviral activity, including interferon-γ production, of innate lymphocytes during viral infection is currently under investigation in both human cells and mouse models. This work is funded by NIH NCCAM P01 AT004986 and P20 GM103500.
Monkeypox virus infection of rhesus macaques induces massive expansion of NK cells, but suppresses NK cell function (P4369)
Authors: Song Haifeng , Josleyn Nicole , Janosko Krisztina , Skinner Jeff , Reeves R. Keith , Cohen Melanie , Blaney Joe , Jahrling Peter
Published: January 2023

In the current study of MPXV infection in rhesus macaques, we analyzed the NK cell responses in absolute cell numbers, cell proliferation, chemokine receptor expression, and function. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by similar increases in CD56+, CD16+ and CD16-CD56- double negative (DN) NK cells. Similarly, the frequency and NK cell number in the lymph nodes also largely increased with total NK cell number increasing nearly 50-fold. Ki67 staining showed that NK cells both in the blood and lymph nodes proliferated during acute infection. Chemokine analysis revealed an early reduction of CXCR3, CCR7 and CCR6 on NK cells, followed with an increased expression of CXCR3 and CCR5 at later time points (day 7). Furthermore, MPXV infection impaired NK cell degranulation and ablated secretion of IFN-g and TNF-a. Taken together, MPXV infection induced massive NK cell proliferation, but suppressed NK cell function.
Characterization of lacrimal gland NK cells (P4341)
Authors: Erick Timothy , Reilly Emma , Biron Bethany , Peterson Nicole , Brossay Laurent
Published: January 2023

The β-herpes virus murine cytomegalovirus (MCMV), a homologue of HCMV, is a well-characterized animal model of viral infection that results in a non-replicative, chronic infection of an immune-competent animal. MCMV is cleared efficiently by cytotoxic lymphocytes in most organs of the infected host but persists in salivary glands for several weeks after primary infection. Interestingly, it has also been shown that MCMV remains active in the lacrimal gland (LG) for several weeks after infection. Here, we investigate the phenotype of NK cells in the naïve LG and their response to MCMV infection. In naïve mice, we found that the LG contains a resident NK cell population with a mostly immature phenotype (CD27 high/CD11b low). These lymphocytes are absent in E4BP4 deficient mice and present in AhR deficient mice, indicating that these cells belong to the classical NK cell lineage. However, using a variety of cell surface markers we found that the phenotype of LG NK cells is distinct from splenic, liver, or salivary gland NK cells suggesting that the NK cell peripheral phenotype is shaped by the tissue microenvironment. LG NK cells become activated during MCMV infection and release low amount of cytokines. We are currently evaluating in more details the role of LG NK cells during MCMV infection.
High perforin levels in freshly isolated human CD56neg NK cells (P4345)
Authors: Barao Isabel , Redelman Doug , Hunter Kenneth , Hudig Dorothy
Published: January 2023

CD56neg NK cells are a subset that is elevated in the blood of patients with HIV and/or hepatitis C virus viremia, but drop when therapies reduce the viremias [Gonzalez VD, Clin Immunol 128: 46, 2008]. CD56neg NK cells of virally infected individuals are thought to be ‘dysfunctional’ with low cytolytic capabilities. Here we questioned whether the cytotoxic protein perforin is lower in CD56neg compared to CD56dim (perforin-high & highly cytolytic) NK cells and thus could affect cytotoxicity. We assessed the perforin levels of lymphocytes of 97 healthy donors by flow cytometry, staining peripheral blood cells with fluor-tagged anti- CD3, CD56 and perforin. The frequency of CD56neg NK cells of total CD3neg perforinpos NK cells ranged from 1.4 - 53.1% with a mean +/-1SD of 9+/-10%. Twelve donors had CD56neg NK frequencies above 1 SD of the mean, >20% of total NK cells, with counts 67-701 cells per ul of blood. Regardless of NK frequencies, for most donors their CD56neg NK cells were bimodal for perforin with the higher perforin mode equivalent to the perforin levels of CD56dim NK cells. Two donors with greatly elevated CD56neg NK cells had mostly perforin-low CD56neg NK cells. In conclusion, a substantial percentage of healthy donors (12%) have highly elevated CD56neg NK cells and the majority (83%) had CD56neg cells with high levels of perforin. Thus, it likely that for healthy donors, the cells of the expandable CD56neg NK subset have sufficient perforin to support killing.
Dendritic cell-expressed common gamma-chain recruits IL-15 for trans-presentation at the immunological synapse (P6286)
Authors: Choudhuri Kaushik , Beilin Chiara , Bouma Gerben , Llodra Jaime , Malinova Dessislava , Stokes David , Springer Timothy , Shimaoka Motomu , Dustin Michael , Thrasher Adrain , Burns Siobhan
Published: January 2023

Mutations of the common cytokine receptor gamma chain (gc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although these lineages are restored by stem cell therapy, residual immune defects are observed that may result from a selective persistence of gc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed gc to protective immune responses. We examined the importance of gc for myeloid lineage dendritic cell (DC) function, and show that DC-expressed gc is required for effective antigen-induced CD4+ T cell activation. Using a novel lipid bilayer system mimicking T cell interaction, we observed that gc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Ra colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on expression of gc in DC. These findings reveal a novel mechanism for recruitment of DC IL-15/IL-15Ra complexes to the immune synapse, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.
IL-7 suppresses CD127 gene transcription in human CD8 T cells by inducing expression of the transcription factor c-Myb (P6299)
Authors: Al-Ghazawi Feras , Faller Elliott , Parmar Parmvir , Sugden Scott , Kakal Juzer , Cherid Hafsa , MacPherson Paul
Published: January 2023

In view of the role interleukin (IL)-7 plays in T cell survival, homeostasis and function it is no surprise expression of the IL-7 receptor-α (CD127) is tightly regulated. Dysregulation of IL-7 signaling and CD127 expression has been implicated in a number of diseases such as multiple sclerosis, breast cancer, arthritis as well as in HIV infection. Thus it is important to understand how IL-7 regulates its own receptor expression. Here we elucidate the mechanism by which IL-7 suppresses CD127 transcripts in primary human CD8 T cells. We show by qPCR and nuclear run-on assays that IL-7 suppresses CD127 gene transcription in a time- and dose-dependent manner. The IL-7 mediated suppression of CD127 transcripts is dependent on JAK/STAT5 signaling. Notably, cycloheximide blocked IL-7’s ability to down-regulate CD127 transcripts suggesting IL-7 stimulates the de novo synthesis of a transcriptional repressor which in turn suppresses CD127 gene transcription. Through DNA microarrays and PCR arrays, the IL-7 inducible transcription factor c-Myb was identified. The region within the CD127 gene promoter required for IL-7 mediated transcriptional suppression was identified through progressive truncations using firefly luciferase as a reporter gene and is located from -1760 to -2406 bp upstream of the TATA box and contains c-Myb binding sites. Using siRNA-mediated knockdown and transient over-expression, we illustrate c-Myb suppresses CD127 gene transcription in primary human CD8 T cells.
Cytokine receptor signaling in CD4+ T cells (P6305)
Authors: dela Pena Myra Grace , Kartchner Laurel , Abel Kristina
Published: January 2023

Infants compared to adults exhibit reduced immune responsiveness and are biased towards a T helper 2 (Th2) response. We hypothesized that this is due to the presence of intrinsic differences in cytokine receptor signaling in CD4+ T cells. We utilized human adult and cord blood (CB) samples to determine whether age-related differences exist in (i) Th1 and Th2 cytokine receptor (R) expression, (ii) STAT phosphorylation, and/or (iii) JAK/STAT signaling. Our data show that a lower percentage of CB compared to adult CD4+ T cells expresses IFNγR1 (P=0.0053). However, absolute numbers of IFNγR1 are higher on CB compared to CD4+ T cells (P=0.0305). In contrast, we did not detect differences in the levels of IL4Rα expression. Consistent with this data, CB CD4+ T cells show lower phosphorylation levels of STAT1, but not STAT6 in response to IFNγ or IL4 stimulation. Furthermore, several genes of the JAK/STAT signaling pathway (e.g. jak1, ifng, stam, isg15 and oas1) were induced at higher magnitude and with faster kinetics in adult compared to CB PBMC after in vitro stimulation with IFNγ. Thus, our data demonstrate that unique age-related differences exist in CD4+ T cell cytokine receptor signaling that may underlie the reduced immune responsiveness and Th2-biased response of infants. Unraveling these molecular mechanisms may aid in pediatric vaccine and therapeutics design.
Rapid assessment of CD4 + T cell differentiation towards more specialized cell types using impedance based technology (P6325)
Authors: Santos Mark , Zhang Wenying , Whalley Jason , Hsu Matthew
Published: January 2023

Cellular differentiation is a fundamental process in developmental biology. Progenitor cells must have the ability to differentiate into more specialized cell types for the body to respond to infections during autoimmunity. In an immunological response to infections, CD4+ T cells can give rise to a variety of T helper cells depending on the nature of the immune response, and subsequently release a distinct subset of signature cytokines. Similarly, when monocytes are exposed to established lineage specific conditions in vitro, human monocytes differentiate toward mature dendritic cells. In all cases, a characteristic inherent in cell differentiation is that during this process cells undergo a dramatic change in cell size, metabolic activity, and responsiveness to signals. Current methods such as flow cytometry or phase contrast morphology for determining cell differentiation are informative; however, drawbacks to these methods are that either it is too subjective or labor intensive. Here we utilized the coulter principle of impedance based particle detection as an alternative method for rapidly assessing cell differentiation. This study outlines a method for implementing the coulter principle to rapidly analyze CD4+ T cell differentiation towards various Th cell lineages, as well as monocyte differentiaton towards DCs. We have employed impedance based technology for determining cell volume to investigate the relationship between cell differentiation and cell size changes.
An RNA binding protein, Y14 regulates TNF-α-induced NF-κB activation and IL-6 expression (P6329)
Authors: Togi Sumihito , Muromoto Ryuta , Kon Shigeyuki , Sekine Yuichi , Matsuda Tadashi
Published: January 2023

Y14 (also known as RNA-binding motif protein 8A, RBM8A) is an RNAbinding protein that binds preferentially to spliced mRNAs immediately upstream of the exon-exon junctions, and remains bound to the mRNA after nuclear export. Thereby, Y14 is thought to act mainly as a translational regulator. Recently, we demonstrated that Y14 specifically interacts to STAT3, and enhances IL-6/STAT3-mediated transcriptional activation. These results suggest that Y14 acts as not only translational regulator but also transcriptional regulator by interacting transcriptional factors. However, the roles of Y14 in cytokine signaling are still largely unknown. Here, we analyzed the effect of Y14 on another cytokine signaling mediated by TNF-α/NF-κB. Using small interfering RNA-mediated Y14 knockdown, we found that endogenous Y14 positively regulated TNF-α-induced IL-6 production in HeLa cells. To elucidate the mechanism of Y14-mediated regulation of NF-κB signaling, we screened the Y14-interacting proteins in NF-κB signaling and identified RIP1 and TRADD as a novel Y14-binding partner, both are known as novel regulators of TNF-α/NF-κB signaling. Furthermore, we found that Y14 facilitates the RIP1/TRADD complex formation and enhances RIP1/TRADD-mediated NF-κB transcriptional activity. These results demonstrate that Y14 is a novel regulator of TNF-α/NF-κB signaling by enhancement of the RIP1/TRADD complex formation.
Identification of signaling molecules responsible for IL-2-mediated cytokine expression in human T cells (P6343)
Authors: Kaminuma Osamu , Kitamura Noriko , Mori Akio , Hiroi Takachika
Published: January 2023

IL-2 is an important cytokine regulating growth, differentiation and function of T cells. IL-2 directly induces production of several Th2 cytokines by T cells, especially of human origin. The mechanisms of IL-2-mediated cytokine synthesis have not been clarified, though the contribution of IL-2 receptor (IL-2R)β has been suggested. Here, by employing a proteomic approach, the intracellular signaling molecules downstream of IL-2Rβ were investigated. IL-2Rα and IL-2Rγ were stably transfected in a T cell hybridoma established by fusing an antigen-specific human T cell clone and BUC cells. The resulting hybridoma was further transfected with full-length and intracellular component-truncated IL-2Rβ. Then, the differential protein tyrosine phosphorylation profiles in those cells following IL-2 stimulation were examined by two-dimensional gel electrophoresis and mass spectrometry. Several proteins specifically phosphorylated in full-length IL-2Rβ-expressing cells were identified. Among them, overexpression of protein phosphatase (PP)1β significantly augmented IL-2-induced IL-5 and IL-13 expression in human peripheral CD4+ T cells, whereas TCR-induced cytokine expression was not affected. In contrast, introduction of PP1 regulatory subunit 2 as well as treatment with tautomycin, a PP1 inhibitor, but not ocadaic acid, a PP2A inhibitor, inhibited the IL-2-mediated responses. Conclusively, PP1 is a key molecule responsible for IL-2-mediated cytokine synthesis in human T cells.
The evolution of TNF-induced apoptosis reveals 550 million years of functional conservation (P6345)
Authors: Quistad Steven , Stotland Aleksandr , Barott Katie , Grasis Juris , Wolkowicz Roland , Rohwer Forest
Published: January 2023

Coral reef health is in rapid decline worldwide yet the molecular mechanisms behind coral death remain poorly understood. The Tumor Necrosis Factor (TNF) receptor-ligand superfamily (TNFRSF/TNFSF) is a central mediator of apoptosis and it is hypothesized that the expansion of the TNFRSF/TNFSF occurred following the divergence of invertebrates and vertebrates. Here we challenge this hypothesis and identify more putative coral TNFRSF members than any organism described thus far, including humans. We then predicted Human TNFα (HuTNFα), a known inducer of apoptosis in humans, would also cause apoptosis in coral. Upon HuTNFα stimulation the coral proteome underwent an acidic shift, suggesting the induction signaling cascades. Stimulation of coral with HuTNFα also induced apoptotic blebbing, caspase activation and coral bleaching. This work identifies the first ligand/receptor system to be directly involved with apoptosis and bleaching in coral, and provides evidence for an ancient origin of the TNFRSF/TNFSF that has been functionally maintained for over 550 million years.
Jak3 is a key regulator of actin cytoskeleton dynamics in CCR7 and CXCR4 dependent signaling. (P6292)
Authors: Ambriz Peña Xochitl , Olguín Alor Roxana , Martínez Cuatepotzo Berenice , Macías-Silva Marina , García-Zepeda Eduardo , Soldevila Gloria
Published: January 2023

Jak3 plays an important role in the signaling pathways of chemokine receptors CCR7 and CXCR4. As cytoskeletal dynamics are essential to allow migration of T cell towards chemokines, and migration of T cells is severely impaired in absence of Jak3, here we analyzed the role of Jak3 in chemokine-dependent cytoskeleton reorganization. Our study demonstrated decreased actin polymerization in Jak3-pharmacologically inhibited murine and human T cells and in Jak3 deficient murine T cells in response to CCL19, CCL21 and CXCL12. This correlates with an alteration of the cofilin phosphorylation kinetic observed in the absence of Jak3, but not after Gαi inhibition by pertussis toxin (PTX). Furthermore, analysis of the activation and localization of small GTPases Rac1 and RhoA showed that Jak3 inhibited cells displayed increased basal levels of active Rac1, whose relocalization after chemokine stimulation was impaired. These effects did not occur in PTX treated cells, which showed only a minor activation of Rac1. In contrast, RhoA activation was severely affected both in Jak3-inhibited and PTX treated cells. In summary, our results provide strong evidence that T cells require Jak3 to achieve appropriate actin dynamics, cofilin kinetics and activation of the small GTPases Rac1 and RhoA in response to chemokines. These data supports the notion that Jak3 and the Gαi protein play complementary and independent roles in the chemokine receptor signaling pathways of CCR7 and CXCR4.
NF-κB controls IL-7 receptor expression in T cells (P6295)
Authors: Molinero Luciana , Miller Michelle , Mashayekhi Mona , Chen Luqiu , Zhou Ping , Tramontini Nicole , Michelotti Monica , Liu Xindong , Zhu Xiaoping , Alegre Maria-Luisa
Published: January 2023

IL-7 and tonic TCR signaling control naïve T cell homeostasis. The transcription factor NF-κB plays a key role during T cell activation, proliferation and survival, but little is known on the role of basal NF-κB in quiescent T cells. Using mice bearing NF-κB impaired T cells (IκBαΔN), we show that basal levels of NF-κB signaling in naïve T cells are required for cell survival both in vivo and in vitro. In vitro, IκBαΔN T cells display reduced survival to IL-7, IL-7-mediated STAT5 phosphorylation and Bcl2 upregulation. Indeed, protein levels of IL-7Rα, but not common γc subunit (CD132), are markedly reduced in IκBαΔN naïve T cells. Assessing mice deficient for NF-κB1 and NF-κB2 subunits, canonical (NF-κB1) but not alternate (NF-κB2) NF-κB pathway is required for IL7Rα expression in T cells. Conversely, constitutive activation of NF-κB in T cells (Lck-Cre x IKKβCA) enhances IL-7Rα expression and IL-7 induced T cell survival. RelA/p65 and NF-κB1/p50 might control transcription through a κB binding region located 3.5 kb upstream of il7r gene, as assessed by EMSA and chromatin immunoprecipitation. Notably, using a NF-κB pharmacological inhibitor we show that NF-κB also controls IL-7Rα expression in human T cells. Taken together, our data suggest that NF-κB regulates il7r transcription both in mice and human, therefore affecting homeostasis of naive T cells.