bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of α9 integrin on medullary sinus with its ligand regulates lymphocyte egress via regulating S1P transporter, Spns2 expression and subsequent S1P secretion under inflammatory condition (112.5)
Authors: Ito Koyu , Matsui Yutaka , Kurotaki Daisuke , Morimoto Junko , Kihara Akio , Uede Toshimitsu
Published: January 2023

Sphingosine 1-phosphate (S1P) is essential for lymphocyte egress from lymph node (LN). Despite its importance, it remains unclear how synthesis and secretion of S1P are regulated in LN. Here we report that α9 integrin is expressed on lymphatic endothelial cells (LEC) of medullary sinus in draining LN (dLN). In addition, Tenascin-C (TN-C), one of the α9 integrin ligands, was co-localized with LEC. The stimulation of LEC by TN-C in vitro led to the enhanced secretion of S1P. This α9 integrin-mediated enhancement of S1P secretion was not due to the augmentation of S1P synthesis, but rather induction of S1P transporter, Spns2. Abrogation of α9 integrin-mediated signaling by blocking anti-α9 integrin antibody (55A2C) in vivo resulted in the inhibition of lymphocyte egress from dLN under inflamed condition. Importantly, treatment of 55A2C did not affect lymphocyte egress under naive condition, indicating that blocking of α9 integrin-mediated signaling does not affect lymphocytes egress under non-inflammed condition. Consistent with those data, 55A2C attenuated severity of experimental autoimmune encephalomyelitis (EAE) with concomitantly accumulation of CD4+ T cell in dLN. Thus we propose that lymphocyte egress is regulated by α9 integrin-mediated signaling on LEC via inducing secretion of S1P from LEC under inflamed condition and that manipulation of α9 integrin-mediated signaling can be a potential therapeutic means for various inflammatory disorders.
Transcriptional profile of CD28 costimulation and Dec1 relevance in T-cell activation (113.14)
Authors: Martinez-Llordella Marc , Esensten Jonathan , Bluestone Jeffrey
Published: January 2023

Antigen recognition by the T cell receptor and costimulatory signal through CD28 receptor are required for a complete T-cell activation. CD28 ligation regulates a wide range of biochemical processes that drive T-cell proliferation, prevent apoptosis and enhance IL-2 production. However, the direct effect of CD28 signaling on the global gene transcription after T-cell activation remains controversial. We described the transcriptional profile of CD28 costimulation employing microarray analysis of human naïve CD4+ T cells. Although we demonstrated that CD28-dependent signaling had quantitative effects on gene expression during the first stages of activation, strong qualitative changes on transcription were observed by 24 hours after activation. Among the genes directly induced by CD28 costimulation we identified the transcription factor Dec1. We described the critical role of Dec1 for the transcriptional response to CD28 costimulation in both Jurkat T cells and primary mouse CD4+ T cells. Importantly, we showed that the effects of Dec1 deficiency impact specifically on CD28 costimulation-dependent gene expression. Further, we proved that Dec1 is necessary to induce experimental autoimmune encephalitis in mice after MOG peptide immunization. This observation validates the in vivo importance of Dec1 for immune system function in a CD28-dependent disease.
Regulation of naïve T cell tolerance and regulatory T cell function by GRAIL (113.21)
Authors: Nurieva Roza , Lozano Guillermina , Dong Chen
Published: January 2023

CD4+ T cells are the master regulators of adaptive immune responses, and many autoimmune diseases arise due to a breakdown of self tolerance in CD4+ cells. Gene related to anergy in lymphocytes (GRAIL), the E3 ubiqutine ligase, has acknowledged as one of key molecules implicated in T cell activation and tolerance. In order to understand the physiological function of GRAIL in immune regulation, we have generated and analyzed GRAIL deficient mice. Naive T cells lacking GRAIL showed greatly enhanced proliferation and cytokine production after T cell receptor (TcR) activation. In addition, lack of GRAIL abrogated suppressive function of regulatory T (Treg) cells. We found that GRAIL deficient naive and Treg cells after TcR activation expressed substantially higher amounts of NFATc1 compared to wild-type cells, whereas the activation of other factors in AP-1 and NFκB pathways were normal. Our data also suggested that sustained TcR cell-surface expression in the absence of GRAIL led to selective NFATc1 expression in both naive T cells and Treg cells. In contrast to naïve T cells, GRAIL, through controlling NFATc1 expression, inhibits IL-21 production and upregulation of Th17-specific genes in Treg cells. Thus, the immune regulation by GRAIL in both naive and Treg cells is absolutely critical as evidenced by the failure of T cell tolerance induction and greatly increased susceptibility to autoimmune diseases of GRAIL deficient mice.
TCR-actin association mediated via unique motifs within the ζ-chain plays a central role in T cell activation (113.24)
Authors: Klieger Yair , Wang Lynn Wang , Almogi-Hazan Osnat , Baniyash Michal
Published: January 2023

Cytoskeleton rearrangement initiated upon specific T cell-antigen presenting cell (APC) interactions, is required for immunological synapse (IS) formation/maintenance, T cell activation and polar cytokine secretion. Following activation, most T cell antigen receptors (TCRs) are internalized and degraded though some long-lived T cell-APC conjugates are maintained, by a yet unknown mechanism. T cells express at least two TCR forms, non-cytoskeleton (non-cska) and cytoskeleton (cska) associated; the latter are linked to actin via the ζ-chain. However, the mode of association and the functional significance of the cska-TCRs are currently enigmatic. We discovered two positively charged motifs within the ζ-chain intracytoplasmic region mediating its binding to F-actin and inducing actin bundling. T cells expressing ζ-chain mutated in these motifs, although displaying normal levels of cell surface TCRs and some immediate signaling events, they are devoid of cska-TCRs and are unable to present activation-dependent TCR cap formation, IS establishment and cytokine production/secretion. Accordingly, we demonstrate that APCs also display abnormal activation patterns. Based on the cumulative data showing that T cells expressing mutated ζ-chain are unable to display cellular events critical for the execution of an optimal immune response, we propose a model for the key roles of the cska-TCRs in TCR-mediated activation.
Anti-CD8 antibodies can trigger T-cell effector function in the absence of TCR engagement and improve pMHCI tetramer staining (113.28)
Authors: Wooldridge Linda , Ladell Kristin , Price David , Sewell Andrew , Clement Mathew
Published: January 2023

Cytotoxic T lymphocytes (CTLs) recognize foreign peptides presented at the cell surface bound to major histocompatibility complex class I (MHCI) molecules. Antigen recognition involves the binding of both T-cell receptor (TCR) and CD8 co-receptor to the same peptide-MHCI (pMHCI) ligand. Specificity is determined by the TCR, whereas CD8 mediates an effect on antigen sensitivity. Anti-CD8 antibodies have been used extensively in previous studies to examine the role of CD8 in CTL activation. However, it is unclear from the literature whether anti-CD8 antibodies per se are capable of inducing effector function. Here, we report on the ability of seven monoclonal anti-human CD8 antibodies to activate six human CTL clones with a total of five different specificities. Six out of seven anti-human CD8 antibodies tested did not activate CTLs. In contrast, one anti-human CD8 antibody, OKT8, induced effector function in all CTLs examined. Moreover, OKT8 was found to enhance TCR/pMHCI on-rates and, as a consequence, could be used to improve pMHCI tetramer staining. The observed heterogeneity in the ability of anti-CD8 antibodies to trigger T-cell effector function provides an explanation for the apparent incongruity observed in previous studies and should be taken into consideration when interpreting results generated with these reagents. Furthermore, the ability of antibody-mediated CD8 engagement to deliver an activation signal underscores the importance of CD8 in CTL signalling.
Differential expression of IL-17A and Il-17F in inflammatory bowel disease/colon cancer using a new IL-17A/F-dual-color reporter mouse (114.1)
Authors: Shen Wei , Li Wenqing , Feigenbaum Lionel , Hixon Julie , Durum Scott
Published: January 2023

Interleukin (IL) 17A and IL-17F are products of Th17 cells which are associated with autoimmunity and many inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Some studies have suggested that production and activity of these two cytokines may be different and we sought to visualize them simultaneously in the inflamed bowel. A bacterial artificial chromosome was modified to introduce two fluorescent reporter genes, tdTomato and GFP, into the linked IL-17A and IL-17F genes respectively. Mice were generated that harbor the BAC construct. In vitro, splenic T cells cultured under Th17 conditions showed single expressing cells and dual expressing cells. In vivo colonic intraepithelial cells in DSS-induced acute mouse colitis showed slightly more IL-17A reporter than IL-17F, while in AOM/DSS induced chronic mouse colitis model, much more IL-17A reporter than IL-17F was expressed. In the T cell (CD4+CD45RBhigh) transfer colitis model, only expression of the IL-17A reporter could be detected in intestinal intraepithelial cells of Rag1-/- recipients. These IL-17A/F-dual-color reporter mice should prove useful in determining the sites of microenvironmental triggers of activation of Th17 cells and other cells that produce IL-17A and F.
Phenotypic and functional differences in Foxp3+ cells expressing different levels of the IL-2 receptor α-chain CD25 (115.20)
Authors: Brand David , Luo Jiwen
Published: January 2023

Regulatory T cells (Tregs) are key players in tolerance and immune homeostasis. The identification of this unique cell subset was originally established through identification of CD4+ T cells expressing high levels of the IL-2 receptor α-chain CD25. CD4+CD25+ cells from naïve mice are predominantly Tregs. CD4+CD25- cells are predominantly conventional T cells. With the identification of the nuclear transcription factor Foxp3 as a protein that plays a key role in the biology of Tregs and with GFP as a means of selection, pure populations of Foxp3 expressing cells can be obtained. Among murine CD4+ T cells that express Foxp3, there is a continuum of CD25 expression from negative (or very dim) to very bright. Our analyses of peripheral Foxp3+CD4+CD25dim/- cells suggest that while their transcription of many cytokine, chemokine and receptor genes are similar to Foxp3+CD4+CD25bright T cells, they do not have identical phenotypic and functional properties, with the latter capable of a greater degree of suppression and an enhanced transcription of Interleukin-7. Parenteral administration of a heterologous type II collagen to induce tolerance in a TcR tg mouse model of Collagen-Induced Arthritis increases the percentage of Foxp3+CD4+CD25bright T cells and protects the mice from disease. Adoptive transfer of a Foxp3+cells from CII-tolerized mice confers a greater degree of protection when compared with transfer of equivalent numbers of Tregs from naïve mice.
Role of helper T cells in the loss of B cell anergy mediated by the absence of Tregs (115.6)
Authors: Gauld Stephen , De Santis Jessica , Hartog Nicholas , Josephson Jennifer , Leonardo Steven
Published: January 2023

Regulatory T cells (Tregs) are crucial for the maintenance of immune tolerance and the absence of Tregs results in significant autoantibody production. This suggests that Tregs play a key role in B cell tolerance. The mechanism by which Tregs regulate B cell tolerance has yet to be resolved but has important implications for our understanding of autoimmunity. Using both constitutive and inducible models, we show that the absence of Tregs results in autoantibody production, due to the loss of B cell anergy. Further studies show that this is dependent on the presence of helper T cells. We propose that the absence of Tregs results in the dysregulation of helper T cells that drive the loss of B cell anergy. Interestingly, our studies show the expansion of a CD4+GL7+ population in the absence of Tregs. Subsequent analysis shows that many of these cells express CXCR5 and thus resemble germinal-center TFH cells, an observation confirmed by their expression of Bcl6 and IL-21. We hypothesize that the absence of Tregs drives the expansion of TFH cells that play a role in the loss of B cell anergy. Characterization of this proposed mechanism may unveil new strategies for the treatment or prevention of autoantibody-mediated autoimmune diseases.
Aryl hydrocarbon receptor-mediated epigenetic modulation of Foxp3 and IL-17 genes results in differential regulation of regulatory T cells and Th17 cells and amelioration of experimental autoimmune encephalomyelitis (115.8)
Authors: Singh Narendra , Singh Udai , Rouse Michael , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

AhR was earlier identified for its role in xenobiotic metabolism, recent studies have suggested that AhR may regulate T cell differentiation. To understand this further, we examined the effect of AhR activation on micro RNAs (miRNAs) profile in T cells, epigenetic modulation of Foxp3 and IL-17, and differentiation of Tregs and Th17 cells, using TCDD, an AhR ligand. Upon AhR activation, there was a significant difference in the miRNAs profiles in T cells. Analysis of some of these miRNAs showed their role in regulation of various genes, including Foxp3 and IL-17. Further examination of epigenetic effects of AhR activation on Foxp3 and IL-17 gene promoters, we observed significant demethylation of Foxp3 promoter but methylation of IL-17 promoter in the presence of TCDD. Also, culture of T cells in the presence of TCDD led to different degrees of T cell differentiation. There was a significant increase in Tregs but decrease in Th17 cells in the presence of TCDD. Using an EAE murine model of multiple sclerosis, we examined the effects of AhR activation on EAE symptoms. There was significant amelioration of EAE symptoms in mice treated with TCDD, compared to vehicle-treated mice. Also, there was significantly higher number of Tregs but lower number of Th17 cells in mice treated with TCDD. Together, these data demonstrated that AhR activation regulates epigenetic modulation and can have differential effects on T cell differentiation into Tregs/Th17 leading to immune suppression.
The interaction of PKC, S100A4, and the cytoplasmic domain of FcγRIIIa (CD16A) α-chain regulates receptor mediated functions (116.1)
Authors: Li Xiaoli , Baskin Julie , Mangan Erin , Yang Xiaoli , Su Kaihong , Gibson Andrew , Ji Chuanyi , Edberg Jeffrey , Kimberly Robert
Published: January 2023

FcγRIIIa(CD16A) pairs with FcR-common γ-chain for surface expression and function. Since many functionally distinct immune receptors utilize γ-chains as signaling subunit, we hypothesized that elements for specificity in receptor signaling may be determined by the distinct FcγR cytoplasmic domains (CY). In silico analysis identified a PKC phosphorylation consensus motif within CD16A-CY; and in vitro kinase assays demonstrated that it is specifically phosphorylated by PKC. Non-phosphorylated CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation, while phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk and pro-inflammatory cytokine production. S100A4, a specific protein binding partner for CD16A-CY, inhibits phosphorylation of CD16A-CY by PKC in vitro. Reduction of S100A4 levels in vivo enhanced receptor phosphorylation upon cross-linking. Taken together, we show that among IgG and IgA receptors, PKC-mediated phosphorylation of CD16A is specific and modulates distinct signaling pathways engaged by the receptor. Calcium activated binding of S100A4 to CD16A, promoted by the initial calcium flux and attenuating the phosphorylation of CY, may serve as a negative feedback on cytokine production pathways during sustained receptor engagement and favor a shift to degranulation, consistent with the importance of degranulation following conjugate formation between CD16A+ effector cells and target cells.
Adenosine promotes alternative macrophage activation by triggering an A2B receptor-initiated p38-C/EBPβ signaling cascade (116.2)
Authors: Csoka Balazs , Koscso Balazs , Nemeth Zoltan , Selmeczy Zsolt , Deitch Edwin , Hasko Gyorgy
Published: January 2023

There are two types of macrophage (MΦ) activation. Classical MΦ activation (M1) is driven by TLR ligands and IFNγ, while alternative MΦ activation (M2) is triggered by Th2 cytokines. Adenosine exerts its actions by activating the G protein-coupled receptors, A1, A2A, A2B and A3. The role of adenosine in regulating M1 MΦ activation is well established; however, our understanding about its impact on M2 MΦ activation is not clear. Therefore, we investigated the effect of the adenosine on IL-4-driven M2 MΦ activation. We found, that adenosine enhanced the IL-4-induced expression of M2 MΦ activation markers, Arginase-1 and TIMP-1. We demonstrated that the A2B receptor is required for this stimulatory effect. The stimulatory effect of adenosine on M2 MΦ activation was unlikely to be mediated by STAT-6, because we found that IL-4-induced STAT6 phosphorylation was decreased by adenosine. Conversely, the stimulatory effect of adenosine on IL-4-induced arginase activity, and TIMP-1 production was blocked by p38 inhibition, indicating a role for p38. In addition, because adenosine increased IL-4-induced C/EBP luciferase activity, and deficiency of C/EBPβ reversed the stimulatory effect of adenosine on arginase activity, we conclude that C/EBPβ has an essential role in the mediation of adenosine signaling. In summary, our results show that adenosine up-regulates M2 MΦ activation via an A2B receptor-p38-C/EBPβ axis.
Myeloperoxidase acts as a double-edged sword in rotenone-exposed brain-resident immune cells (116.32)
Authors: Chang Chi Young , Song Mi Jeon , Yoon Hee Jung , Jeon Sae-Bom , Suk Kyungho , Kim In-Hoo , Choi Dong-Kug , Park Eun Jung
Published: January 2023

Rotenone exposure has emerged as an environmental risk factor for inflammation-associated neurodegenerative diseases. However, the underlying mechanisms responsible for the harmful effects of rotenone in the brain remain poorly understood. Here, we report that myeloperoxidase (MPO) may have a potential regulatory role in rotenone-exposed brain-resident immune cells. We show that microglia, unlike neurons, do not undergo death, but instead exhibit distinctive activated properties under rotenone-exposed conditions. Once activated by rotenone, microglia show increased production of reactive oxygen species (ROS), particularly hypochlorous acid. Notably, MPO, a hypochlorous acid-producing enzyme that is undetectable under normal conditions, is significantly increased after exposure to rotenone. Interestingly, our studies with MPO inhibitors and MPO-knockout mice reveal that MPO deficiency potentiates, rather than inhibits, the rotenone-induced activated state of glia, and promotes glial cell death. Collectively, our data provide evidence that MPO acts a double-edged sword under rotenone-exposed conditions, playing a critical regulatory role in modulating pathological and protective events in the brain.
IL-17 promotes expression of vascular adhesion molecule in vascular smooth muscle cells (117.1)
Authors: Yu Shiguang , Zhang Haitao , Chen Jing , Liu Xiaoming , Awar Lena , Nagarajan Shanmugam
Published: January 2023

Several recent reports showed that IL-17 plays a pro-atherogenic role in atherosclerosis. However the underlying mechanism by which IL-17 promotes the pathogenesis of atherosclerosis remains unclear. The expression of vascular adhesion molecule (VCAM-1) in aortic vascular smooth muscle cells (SMCs) is important for the development of atherosclerosis. We hypothesized that IL-17 may regulate the expression of VCAM-1 in SMCs, as one of potential mechanisms for contributing to atherosclerosis. In this study, we report that IL-17 induces expression of VCAM-1 in SMCs is dependent on NF-κB, but independent of Akt and AP-1 activation. Notably, resveratrol inhibits IL-17-mediated upregulation of VCAM-1 expression. Therefore, blocking IL-17-induced VCAM-1 expression in SMCs may be highly beneficial in treatment of atherosclerosis as VCAM-1 plays a key role for recruitment of inflammatory cells.
An orally active natural product based anti-inflammatory small molecule (RNV-2005) (117.19)
Authors: Nag Bishwajit , Nijjar Sundeep , Sen Ananda , Surve Bhushan , Vutukuru Sriteja , Gulati Shweta , Kr Partha , Srinivasan Narasimhan
Published: January 2023

RNV-2005 is a natural product based molecule originally designed based on its interaction with glucocorticoid receptor (GR). The effective dose of RNV-2005 was found to be in the range of 0.3-10 mg/kg in rodent models. Unlike most analgesic and NSAID compounds, RNV-2005 was non-ulcerogenic up to a tested dose of 200 mg/kg. In an acute toxicity study, RNV-2005 did not show any adverse effect at a highest tested dose of 2000 mg/kg. The LD50 dose of RNV-2005 is therefore not established. A 70-80% reduction in paw edema was observed in animals treated with RNV-2005 at a dose of 10 mg/kg in carrageenan induced paw edema rat model. The analgesic properties of RNV-2005 was measured using tail flick latency and acid induced writhing reflex in Albino mice at doses 0.3-3 mg/kg. In a LPS induced ex-vivo study in SW mice, reduction of cytokines such as TNFα, IL-1β, IL-6 and IL-12 was measured in plasma. Further studies of RNV-2005 in collagen-induced arthritis in DBA/1J mice, adjuvant induced arthritis in Lewis rats, PK studies and in vitro inhibition assays for COX-1 and COX-2 in cell based assays will be presented. Similarly, the molecular mechanisms underlying GR mediated inflammatory responses, with an emphasis on NF-κB and AP-1 and their respective signaling pathways will be discussed. These results with preliminary safety data encourage the clinical potential of RNV-2005 towards development of a novel anti-inflammatory molecule with high therapeutic index.
Individual heterogeneity determines the interaction between human dendritic cells and natural killer cells (147.23)
Authors: Guo Zhenhong , Zhang Hong , Kawamoto Norio , Strominger Jack
Published: January 2023

Previous research has shown that human dendritic cells (DC) can promote activation of natural killer cells (NK), including IFN-γ secretion and enhanced expression of activation markers. However, our recent research suggests that the heterogeneity of individual human donors determines the effect of DC on NK. In our experiments, DC, generated from peripheral blood monocytes and matured by LPS, were co-cultured with autologous NK cells purified from peripheral blood for 24 hours. In the 31 samples, only two donors’ mature DC significantly promoted the secretion of IFN-γ by NK. DC from an additional ten donors promoted the secretion of IFN-γ in the presence of a very low dosage IL-2 or IL-15 which by themselves had no effect. However, all DC could increase expression of CD69 on NK to varying degrees. Further, the DC, not the NK cells, were the key factor for increased IFN-γ secretion because those DC with higher ability to activate autologous NK could also activate allogeneic NK which had otherwise failed to be stimulated with their own autologous DC. Moreover, our current samples did not suggest that the age of the donor was significant for the interaction of NK and DC. Since both NK and DC are very important in infectious disease and cancer immunology, the heterogeneity of NK and DC crosstalk might be related to the susceptibility of some individuals to infection and cancer. However, the mechanisms that determine the effect of DC on NK activation remain to be investigated.
Myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice are only immunosuppressive under serum-free conditions (147.25)
Authors: Hamilton-Valensky Melisa , Lam Vivian , Banáth Judit , Krystal Gerald , Bennewith Kevin
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are defined by their ability to suppress T cell responses. However, different in vitro culture conditions have been used to assay MDSC function, which may explain why some labs have been unable to demonstrate the suppressive nature of these cells. To address this, we isolated MDSCs from BALB/c mice bearing syngeneic 4T1 metastatic murine mammary tumors and compared the ability of these 4T1-MDSCs to suppress T cell proliferation under serum-containing (10% FCS) and serum-free conditions. Interestingly, we found that these MDSCs only suppressed T cell proliferation under serum-free conditions. Conversely, peritoneal macrophages and tumor-associated macrophages were able to effectively suppress T cell proliferation under both serum-free and serum-containing conditions. These results indicate that serum profoundly alters the immunosuppressive properties of 4T1-MDSCs. We investigated the components of serum responsible for inhibiting the suppressive phenotype of MDSCs and found that 4T1-MDSCs failed to suppress T cell proliferation in serum-free medium supplemented with bovine serum albumin (BSA), yet MDSC suppression in 10% FCS medium was restored when BSA was depleted from the serum. Taken together, these data highlight the importance of testing different in vitro culture conditions on MDSC phenotype to ensure that the presence of serum is not masking the immunosuppressive properties of MDSCs.
Paradoxical role for the lipid phosphatase SHIP1 in control of helminth infection (147.29)
Authors: Hadidi Sima , Hughes Michael , Antignano Frann , McNagny Kelly , Zaph Colby
Published: January 2023

Immunity to helminth infections is mediated by polarized Th2 responses that lead to clearance of infection. One of these responses is the development of AAMs that are associated with intestinal repair and parasite killing. SHIP1 is a lipid phosphatase that negatively regulates immune receptor signaling. Ship1-deficiency in macrophages results in a skewing towards a Th2-type response and the spontaneous development of AAMs. Thus, we hypothesized that macrophage-specific Ship1 knockout mice (Ship1ΔMac) mice would display increased resistance to Trichuris infection. Following infection with Trichuris, cells isolated from Ship1ΔMac mice produced equivalent levels of IL-4, IL-13 and IFN-γ compared to control Ship1fl/fl mice, suggesting that lack of Ship1 in macrophages had little effect on the development of Th2 responses in vivo. Strikingly however-and in contrast to our expectations-Ship1ΔMac mice were more susceptible to infection with Trichuris than control Ship1fl/fl mice. These results demonstrate that macrophage-specific expression of Ship1 is critical for resistance to Trichuris independently of a normally protective Th2 cell response and suggest an alternative role for AAMacs in helminth infection. Thus, these studies identify a novel and paradoxical role for Ship1 in the development of immunity to Trichuris and suggest that manipulation of macrophage function could be critical for inducing or limiting immune responses in the intestine.
Human CD8 T cells drive Th1 responses through differentiation of TNF/iNOS-producing dendritic cells (147.4)
Authors: Chong Shu Zhen , Wong Kok Loon , Lin Gen , Yang Christopher , Wong Siew-Cheng , Angeli Veronique , MacAry Paul , Kemeny David
Published: January 2023

TNF/iNOS-producing (Tip) dendritic cells have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8 T-cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs which express high levels of TNF-α and iNOS. These cells exhibited T-cell priming ability, expressed high levels of MHC class II, up-regulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40, CD83 expression and TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4 T-cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC’s production of TNF-α and NO. Our study hence identifies a role for CD8 T-cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.
Human multipotent stromal cells inhibit the activation of antigen specific murine CD4+ and CD8+ T cells (147.7)
Authors: Nazarov Cristina , LoSurdo Jessica , Bauer Steven , Wei Cheng-Hong
Published: January 2023

Multipotent stromal cells (MSCs) are known to have immunosuppressive activity. They do not induce allospecific T cell responses, making them promising tools for reducing the severity of graft vs host disease. Although the immunosuppressive effect has been extensively studied, the molecular mechanisms remain elusive. In our in vitro model, we culture CD4+ or CD8+ T cells from two T cell receptor transgenic mice strains together with human MSCs, followed by flow cytometry analysis of different activation markers present on T cells, real time RT-PCR for important transcription factors and ELISA for Th1 and Th2 cytokines. First, proliferation of both CD4+ and CD8+ T cells is clearly inhibited by human MSCs. These T cell subsets are, with some exceptions, similarly affected by MSCs. Both CD25 and CD69 activation markers are significantly inhibited on the surface of CD4+ and CD8+ T cells. Second, factors associated with a T regulatory phenotype (CTLA-4, CD62L) have an increased expression, while IFN-gamma and other cytokines are inhibited by MSCs in both T cell subsets. Third, real time RT-PCR data analysis showed that transcription factors Tbet and GATA3 are significantly inhibited by MSCs. Also, MSCs induce a slight increase in annexin V positive CD4+ T cells, but have no effect on the viability of CD8+ T cells. Moreover, the effects seem to be specific to MSCs; incubating the T cells with two different controls has no effect on T cell proliferation and activation.
Identification and characterization of myeloid-derived suppressor cells in neonates (147.8)
Authors: Jacobson Amanda , Mitchell Caroline , Xu Min , Frenkel Lisa , Horton Helen
Published: January 2023

The neonatal immune system is regarded as immature and consists of impaired T cell and antigen presenting cell function. During pregnancy, suppression of both the maternal and fetal immune responses is necessary to allow successful viviparity. The neonatal immune response may have to overcome this modulated immune environment in the first months of life in order to respond at the level required for protection. It is unclear whether infant immune responses are simply immature or are being actively suppressed. The primary goal of this study was to elucidate mechanisms of infant immune deficiencies, in particular those that are present at birth. We have identified a prevalent population of arginase 1-positive myeloid-derived suppressor cells (MDSC) within neonates (7.7%+/-6.9% of cord blood vs 0.7%+/-0.5% of adult blood mononuclear cells) that may negatively influence infant immune responses. This cell population present at birth has not previously been described in infants, but similar cells have been described to suppress T cell responses in adult cancer patients. In addition, we demonstrate that neonatal MDSC are functional and suppress allogeneic T cell proliferative responses in an arginase 1-dependent manner by an average of 38%. These data suggest that suppressor cell populations, like MDSC, can suppress infant T cell responses. This may be one mechanism behind reduced neonatal immunity resulting in inefficient responses to both infection and vaccination early in life.
CD38 involvement in autoimmune disease using a murine model for systemic lupus erythematosus (148.13)
Authors: Romero-Ramirez Hector , Gaxiola-Centeno Ricardo , Santos-Argumedo Leopoldo , Rodriguez-Alba Juan Carlos
Published: January 2023

Mouse models contribute in our understanding of human diseases. By this way, several strains of systemic lupus erythematosus (SLE)-prone mice including New Zealand Black (NZB), F1 hybrids of NZB x New Zealand White (NZW) (B/W F1), MRL/Mp-lpr/lpr (MRL/lpr), and BXSB mice are important in the research of the autoimmune diseases. Several genes and signaling pathways molecules have been related with SLE pathologies such as: Lyn, CD22, SHP-1 or the sle1, sle2 and sle3 congenic models. CD38 is a transmembrane receptor able to induce activation, proliferation, and survival of human and mouse lymphocytes; this molecule is expressed along the B cell ontogeny, in addition, CD38 is an ectoenzyme involved cell adhesion and is used as a disease marker for leukemia and myeloma, also is a dependable negative prognostic marker for chronic lymphocytic leukemia (CLL). The function of CD38 in autoimmunity has been proposed in human but there are not reports that clear the authentic role of this molecule in SLE. To study the implication of CD38 in the SLE pathogenesis we compare the proteinuria, serum ANA and anti-dsDNA antibody production, body-weight, nephritis and mortality in the CD38-deficient compared to the wild type (WT) mice. Subsequently, we expect that the pathologic manifestations of nephritis appear significantly earlier in the WT mouse as well survival rate changes.
AID dysregulation in lupus-prone MRL/Faslpr/lpr mice promotes interchromosomal c-Myc/IgH loci translocations, increased class switch DNA recombination and production of anti-dsDNA IgG autoantibodies: modulation by HoxC4 (148.25)
Authors: White Clayton , Hawkins J. , Pone Egest , Al-Qahtani Ahmed , Mai Thach , Zan Hong , Casali Paolo
Published: January 2023

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR), which are critical in the generation of pathogenic autoantibodies. In these studies, we addressed the regulation of AID by HoxC4 in lupus. We found that SLE patients and lupus-prone MRL/Faslpr/lpr mice displayed increased expression of HoxC4, which directly activates the promoter of the AID gene, and increased expression of AID. Dysregulation of these proteins was associated with increased CSR in B cells of MRL/Faslpr/lpr mice. In the HoxC4-/- MRL/Faslpr/lpr mice we generated, HoxC4-deficiency resulted in reduced AID expression, impaired CSR and decreased serum anti-dsDNA IgG, particularly IgG2a production, in association with a reduction in IgG deposition in kidney glomeruli. Consistent with our previous findings that upregulated AID expression is associated with extensive DNA lesions, we found a high frequency of c-Myc to IgH translocations in MRL/Faslpr/lpr mice. The frequency of c-Myc to IgH translocations was significantly reduced in autoimmune MRL/Faslpr/lpr mice deficient in HoxC4. Our findings showed that HoxC4 is upregulated in lupus B cells, and this plays a major role in dysregulating AID expression, increasing CSR and autoantibody production, and promoting c-Myc to IgH translocations.
Lovastatin exhibits synergy with Vitamin D hormone to confer protection from experimental autoimmune encephalomylitis (148.5)
Authors: Paintlia Ajaib , Paintlia Manjeet , Singh Avtar , Singh Inderjit
Published: January 2023

Recent studies provide sufficient evidence that disease modifying agents (DMAs) i.e., vitamin D hormone (VDH) and statin (lovastatin) were immunomodulatory to attenuate experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Interestingly, irrespective of their different mechanism of action these DMAs produce similar protective activities to attenuate EAE. Therefore, we asked whether statin regulate the vitamin D precursor (VDP; vitamin D3) homeostasis in treated EAE model. Interestingly, we observed a significant increase in the serum level of both VDP and VDH in lovastatin treated EAE animals as compared to those treated with vehicle. It was associated with diminished level of CYP24A1 (VDH inactivating enzyme) and elevated level of CYP27B1 (VDH synthesizing enzyme) involved in VDH metabolism in the blood cells and spinal cord of EAE treated with lovastatin than vehicle. Next, we tested the potential of lovastatin and VDH in combination to confer protection from EAE. Interestingly, suboptimal doses of these DMAs significantly attenuated acute clinical exacerbations, cellular infiltration, inflammatory response, axonal loss and demyelination in the spinal cord of EAE animals compared with their individual treatment analyzed by using quantitative and qualitative analyses. Altogether, these findings provide initial evidence that lovastatin maintains VDH homeostasis and its synergy with VDH enhances neuroprotection in EAE/MS brain.
CXCL10 is an important therapeutic target to treat bone metastasis (149.18)
Authors: Lee Zang Hee , Lee Jong-Ho , Ha Hyunil
Published: January 2023

CXCL10 is a chemokine, which takes a significant role in migration and proliferation of T cells. It binds to the receptor CXCR3 and is induced in a wide variety of cell types. We have reported in our previous study that RANKL promotes the expression of CXCL10 in osteoclast precursors and, in turn, CXCL10 mediates RANKL expression in T cells in the synovium. Auto-amplification loop of CXCL10 has been acknowledged to be involved in bone destruction of rheumatoid arthritis. However, the role of CXCL10 in bone metastatic destruction remains largely unknown. We are currently conducting further research on the potential role of CXCL10 in bone resorption and bone metastasis. In this study, the synergistic production of CXCL10 was found in co-culture of bone marrow cells and cancer cells in the presence of RANKL. In addition, the serum level of CXCL10 was increased in bone metastatic mice. The CXCL10 deficient mice showed less metastatic lesions in long bone compared to wild mice. Furthermore, treatment with neutralizing antibody to CXCL10 and antagonist for CXCL10 and CXCR3 interaction significantly inhibited the infiltration of tumor cells into long bone and increased the mice survival rate. Therefore, CXCL10 might be an important therapeutic target in the prevention of bone destruction in bone metastasis.
Formyl peptide receptor 1 is functionally expressed on human lens epithelial cells and promotes lens homeostasis (149.19)
Authors: Schneider Erich , Gaur Sonia , Murphy Philip , Gao Ji-Liang
Published: January 2023

Formyl peptide receptor 1 (FPR1) is a chemoattractant receptor specific for phagocytes, without clearly defined function for non-hematopoietic cells. We found that the lens of Fpr1-/- mice degenerates by 6 months of age without infection or inflammation. Thus, we hypothesized functional expression of FPR1 on lens epithelial cells, the only cell type present in lens. Flow cytometry, qPCR and PTX sensitive fMLF-induced calcium flux assays showed functional FPR1 expression in fetal human lens (FHL-124) cells and in the human lens epithelial cell line SRA 01/04. Despite strong anti-FPR1 binding to lens cells by FACS, similar to that observed for human neutrophils or hFPR1-transfected HEK 293 cells, fluorescent FPR1 ligand fNLFNYK-Fl only poorly bound to FHL-124 cells, with > 80% non-specific binding; by contrast, human neutrophils and hFPR1-transfected HEK-293 cells exhibited >70% specific binding. The KD of fNLFNYK-Fl at neutrophil FPR1 and hFPR1-transfected HEK-293 cells was 2-3 nM, but ~ 0.2 nM in FHL-124 cells. fMLF internalized ~80% of neutrophil FPR1, but only ~25% of FHL-124 FPR1. FPR1 protein in FHL-124 cells resisted siRNA-mediated knockdown, despite strongly reduced mRNA levels, and could only be detected by Western blot using alkali-stripped membranes, suggesting high-molecular complexes of FPR1 and peripheral membrane proteins. The data suggest different conformations of FPR1 in neutrophils versus lens epithelial cells and a key function in lens maintenance.
Differential up-regulation of CXCR3 on effector CD4+Foxp3- T cells and regulatory CD4+Foxp3+ T cells during blood-stage Plasmodium chabaudi AS infection (149.5)
Authors: Berretta Floriana , Piccirillo Ciriaco , Stevenson Mary
Published: January 2023

The chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are critical for trafficking of pathogenic CD8+ T cells to the brain during experimental cerebral malaria in Plasmodium berghei ANKA infected mice. We investigated the role of chemokines and their receptors during blood-stage P. chabaudi AS infection in C57BL/6 (B6) mice which control and resolve the infection with a peak of parasitemia at days 8-9 post infection (p.i.). Real-time PCR analysis revealed significantly increased CXCL9 mRNA expression in spleen cells at days 6, 8 and 11 p.i. while FACS analysis showed significantly increased CXCR3 expression on splenic CD4+ T cells at days 8 and 11 p.i. compared to naïve mice. To determine if CD4+ T cell populations differentially expressed CXCR3 during P. chabaudi AS infection, we examined CXCR3 expression on splenic CD4+Foxp3- T cells (Teffs) compared to CD4+Foxp3+ T cells (Tregs). In naïve mice, 15% of Tregs and 15% of Teffs were CXCR3+, at day 8 p.i., 30% of Tregs vs 45% of Teffs were CXCR3+, and at day 11 p.i., CXCR3+ Tregs were still 30% while CXCR3+ Teffs were 55%. Increased CXCR3 expression on Teffs correlated with increased protective CD4+Tbet+IFNγ+ Th1 cells in the spleen. These findings indicate that CXCR3 expression is up-regulated on both Teffs and Tregs during blood-stage malaria. Higher expression of CXCR3 on Teffs might allow these cells to migrate more efficiently to the spleen, the critical tissue for immune control and resolution of the infection.
CXCL17: a novel mucosal chemokine with antimicrobial activity (149.6)
Authors: Burkhardt Amanda , Kamdar Karishma , Tai Kenneth , Valle-Rios Ricardo , Hevezi Peter , Ouellette André , Zlotnik Albert
Published: January 2023

The mucosal immune network is a crucial barrier preventing pathogens from entering the body. The network of immune cells that mediates the defensive mechanisms in the mucosa is likely shaped by chemokines. Chemokines attract a wide range of immune cells to various sites of the body. Chemokines have been divided into homeostatic or inflammatory depending upon their expression patterns. Additionally, several chemokines have also been shown to mediate direct killing of invading pathogens. An example of the latter is CCL28, a mucosa-associated chemokine that exhibits antimicrobial activity against a range of pathogens. CXCL17 is the last chemokine ligand to be described, and the 17th member of the CXC chemokine family. We have identified CXCL17 as a novel, homeostatic, mucosa-associated chemokine. Its strategic expression in mucosal tissues suggested that it is a barrier protein involved in the maintenance of the homeostasis and/or sterility of the mucosa. To test this hypothesis, we tested CXCL17 in various antimicrobial assays against a panel of pathogenic and opportunistic bacteria, and compared it with CCL28. Our results indicate that CXCL17 has strong antimicrobial activity and that its mechanism of antimicrobial action involves direct killing of the bacteria. We conclude that CXCL17 is a novel antimicrobial chemokine that likely plays significant roles in protecting the mucosa from bacterial invasion, and in shaping the microbiome in the intestine.
Critical roles of chemokine receptor CCR10 in regulating memory IgA responses in intestines (149.7)
Authors: Hu Shaomin , Yang Kangkang , Xiong Na
Published: January 2023

CCR10 is expressed by nearly all the intestinal IgA-producing plasma cells and was suggested to position these cells into the lamina propria. However, the in vivo function of CCR10 in the process is unclear and interfering with CCR10 or its ligand did not affect the intestinal IgA production under homeostatic conditions or during infection. Using CCR10-knockout/EGFP-knockin (CCR10EGFP/EGFP) mice we generated recently, we investigated expression and effect of CCR10 on IgA+ cells of different differentiation stages. We found that in the CCR10EGFP/EGFP mice IgA+ plasma cells were defective in migration to the intestine; but there was a compensational increase in generation of IgA+ plasma cells within intestinal isolated lymphoid follicles, resulting in normal levels of intestinal IgA production and increased localization of IgA+ plasma cells in bone marrow and other internal lymphoid organs under homeostatic conditions and in primary responses to the C. rodentium infection. In addition, the CCR10-deficiency also impaired maintenance of IgA+ memory B cells specifically in the intestine. As a result, IgA memory responses to the bacterial re-infection were severely compromised in the CCR10EGFP/EGFP mice. These findings demonstrate that CCR10 is critical in IgA memory responses by regulating migration and maintenance of IgA+ plasma cells and memory B cells and provide a mechanistic basis to manipulate the CCR10/ligand axis for the vaccine development against mucosal pathogens.
Recognition of a dendritic cell ligand by an MCMV-encoded MHC-I-like protein (154.17)
Authors: Revilleza Maria Jamela , Mans Janet , Iizuka Koho , Mage Michael , Carey Rosalene , Boyd Lisa , Natarajan Kannan , Margulies David
Published: January 2023

Mouse cytomegalovirus (MCMV), a valuable model for studying virus-host interactions, encodes several glycoproteins with predicted MHC-I structure, which may function as immunoevasins. We recently determined the crystallographic structure of a member of this family, m153, and showed that this is a cell surface homodimeric protein with structural similarity to MHC-I, which is expressed independently of beta-2m or peptide. To determine the role of m153 in the viral infection of the host, we wished to identify host proteins with which m153 interacts. We constructed both a birA-tagged version of m153 for staining experiments as well as a reporter cell containing the m153 extracellular domain fused to the human T cell receptor zeta chain. Using both approaches we show that CD11c+ splenic dendritic cells (DCs) and bone marrow-derived DCs express a cell surface ligand for m153. To our knowledge this is the first example of a putative viral immunoevasin that targets a DC expressed molecule. We describe our efforts to identify this molecule using a variety of approaches, including antibody blocking, cDNA library screening, and pull-down experiments on DC lysates. Identification of the m153 ligand on DCs offers to elucidate mechanisms the host employs to defend against viral infection, and also to clarify the evolution of viral immunoevasion of the immune response. This research was supported by the Intramural Research Program of the NIH, NIAID.