bluuebunny/crossref_metadata_2025 huggingface dataset:
Homeodomain Interacting Protein Kinase 1 (HIPK1) Binds O‐GlcNAc Transferase and is O‐GlcNAcylated
Authors: Copeland Ronald , Hart Gerald
Published: June 2021

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification consisting of the addition of a single N‐acetylglucosamine (GlcNAc) moiety to serine and/or threonine residues of numerous nuclear and cytoplasmic proteins. O‐GlcNAc is involved in various cellular processes by regulating aspects of a protein's function including affecting a protein's stability/activity, localization, and protein‐protein interactions. Using a Y2H screen with O‐GlcNAc transferase (OGT), the enzyme responsible for the O‐GlcNAc modification as bait, we have identified homeodomain‐interacting protein kinase 1 (HIPK1) among others as interacting with OGT. HIPK1, a nuclear protein kinase shown to be elevated in breast cancer cells, serves to phosphorylate various transcription factors including p53. Others have shown using HIPK1 −/− and +/+ MEFs that HIPK1 may play roles in cancer and tumorgenesis by regulating p53 and/or Mdm2 function. Herein, we begin to investigate what roles O‐GlcNAc may play regulating HIPK1 function. Y2H results were confirmed as HIPK1 co‐immunoprecipitates in both directions with OGT in vivo. We also demonstrate using an immunoblotting technique with an O‐GlcNAc specific antibody (110.6) and by an enzymatic labeling method that HIPK1 is O‐GlcNAc modified. Currently, we are working to map the O‐GlcNAc site(s) on HIPK1 and also map its OGT interacting domain. Results obtained will allow us to generate O‐GlcNAc site‐specific antibodies and begin to uncover the roles that O‐GlcNAc may play in regulating HIPK1 function, specifically as it relates to cancer and tumorgenesis. Supported by NIH NCI 42486 and DK 61671 to G.W.H. (Dr. Hart receives a share of the royalty received by the university on the sales of the 110.6 antibody. Terms of the arrangement are managed by JHU).
Regulation of catalytic activity of S6 kinase 2 during cell cycle
Authors: Joseph Alton Jude , Boyer Derek , Quintanilla Rene Harold , Lee‐Fruman Kay K
Published: June 2021

Ribosomal S6 kinase 2 (S6K2) is a homolog of S6 kinase 1 (S6K1), and both S6K1 and S6K2 are regulated by mammalian target of rapamycin (mTOR). Even though there are many similarities between the two kinases, evidence suggests that S6K1 and S6K2 may have non‐overlapping functions, but the biological function of S6K2 besides phosphorylation of S6 is yet to be determined. In order to shed light on potential cellular function of S6K2, we set out to determine cell cycle phase(s) during which S6K2 is most active. To date most research on mTOR and S6K2 had focused on G1 phase of the cell cycle since the mTOR inhibitor rapamycin blocks cell cycle progression at G1 phase. Our data shows that S6K2 is active throughout the cell cycle with higher activity in G2 and M phases, which suggests that S6K2 may have yet‐unidentified role(s) in these cell cycle phases. We are currently analyzing the signaling mechanisms that control S6K2 activity throughout the cell cycle, and we are also assessing potential regulation of S6K2 activity via sub‐cellular localization throughout the cell cycle. This research was supported by NIH SCORE Grant 2 S06 GM063119.
A requirement of growth factor receptor PTK and metalloproteinase activation in triggering endothelin‐1 (ET‐1)‐induced ERK1/2 and PKB phosphorylation In VSMC.
Authors: Bouallegue Ali , Srivastava Ashok K
Published: June 2021

A role of growth factor receptor transactivation and metalloproteinase in angiotensin II‐induced signaling events has been well established in vascular smooth muscle cells (VSMC). However, an involvement of receptor transactivation and metalloproteinase activation as upstream mediators of ET‐1‐induced signaling in VSMC has not been explored. In this study, we examined if similar to angiotensin II, ET‐1‐induced ERK1/2, PKB and GSK‐3 phosphorylation also requires transactivation of IGF‐1‐R, EGF‐R and PDGF‐R in VSMC. This possibility was tested by using a series of pharmacological inhibitors. We found that AG1024 and AG1478, inhibitors of IGF‐1‐R, and EGF‐R protein tyrosine kinases (PTKs) respectively, decreased ET‐1‐induced ERK1/2, PKB and GSK‐3 phosphorylation in a dose‐dependent fashion whereas, AG1295 an inhibitor of PDGF‐R‐PTK had no effect on ET‐1‐induced signaling events. Furthermore, PP‐2 a specific inhibitor of Src‐family PTK also blocked ET‐1‐induced phosphorylation of these protein kinases. Doxycycline and GM6001 two specific inhibitors of metalloproteinases, markedly attenuated ET‐1‐induced ERK1/2, PKB and GSK‐3 phosphorylation. These data suggest that, in VSMC, the phosphorylation of ERK1/2, PKB and GSK‐3 induced by ET‐1 is mediated through Src family PTKs, IGF‐1‐R and EGF‐R transactivation as well as metalloproteinase activation and not through PDGF‐R transactivation. (Supported by a grant from CIHR).
Demonstration of the interaction between AKAP18 and PKC
Authors: Sinnamon John , Mayer Nicole , Dodge‐Kafka Kimberly
Published: June 2021

The A kinase‐anchoring protein AKAP18 coordinates the beta‐adrenergic stimulation of the L type calcium channel in the heart by localizing PKA in close proximity to the channel, enhancing phosphorylation. L‐type calcium channels are regulated by additional serine/theronine kinases including protein kinase C. These observations led to the hypothesis that AKAP18 has the ability to not only localize PKA to the L type calcium channel, but PKC as well. Here we show that AKAP18 associates with both the alpha and epsilon isoforms of PKC in HEK293 cells. Futhermore, PKC binds to several isoforms of the anchoring protein, indicating the presence of a conserved PKC binding domain. PKC activity co‐precipitates with AKAP18 complexes isolated from both rat and human heart extract, as well as from HEK293 cells expressing both proteins, showing no loss of function upon binding. The co‐expression of AKAP18 increases the ability of PKA to pulldown PKC, indicates AKAP18 functions as a bridge to localize the two kinases into the same signaling complex. Together, these data suggest that AKAP18 functions as a scaffolding protein in the heart to coordinate dual phosphorylation of the L type calcium channel by anchoring both PKA and PKC.
Investigation of GST fusion proteins for screening protein interactions
Authors: Thomsen Kristen , Barnett Jason , Elder Darcie , Caldwell Benjamin D , Ducey Michael W
Published: June 2021

Proteins of interest are commonly expressed as fusion protein conjugates with Glutathione S‐Transferase (GST) due to the relative ease with which GST can be purified. Expression vectors may have proteolytic cleavage sites engineered into the proteins sequence so that the protein of interest may be separated from GST and isolated for further study; however, these enzymes can be expensive and cleavage may be incomplete, reducing the usable yield. We investigated fluorescence anisotropy as a method for examining the interaction of GST fusion proteins. Fluorescence studies often use ligands specific for the protein of interest; however some proteins may not have natural probes suitable for fluorescence studies. We investigated the possibility of measuring the interaction of GST fusion proteins via glutathione (Glu‐Cys‐Gly, or GSH) labeled with a fluorecein‐maleimide. Titration with GST demonstrated an increase in fluorescence anisotropy, indicating GSH‐Fl ligand binding. Results suggest that fluorescence anisotropy of GST‐fusion protein interaction could be a preliminary or complementary screening method for examining protein interactions, especially those whose natural ligands are not spectroscopically observable.
Microwave‐assisted synthesis of N‐vanillylformamide
Authors: Keller Brent , Bobylev Mikhail
Published: June 2021

The Leuckart reaction is a fast and convenient method of synthesis of various formamides and amines and has been successfully used for the synthesis of many biologically active compounds, including agrochemicals and pharmaceuticals. Surprisingly, it has never been used for the synthesis of vanillylamine (I) from vanillin (II) via the intermediate N‐vanillylformamide (III). In the literature, the intermediate III is described only once, when it was obtained via the formylation of I, not by the reductive amination of II. In this work, we report a successful synthesis of III from II via a microwave assisted Leuckart reaction. This method provides a new synthetic pathway to I, which is a valuable intermediate in the synthesis of a number of biologically‐active compounds. This method can also be used for the synthesis of other hydroxy‐substituted benzylamines.

The project is supported by NIH grant P20 RR016741 from the NCRR.
The receptor deformation model of TCR triggering
Authors: Ma Zhengyu , Janmey Paul A , Finkel Terri H
Published: June 2021

Signal initiation from the T cell antigen receptor (TCR) by its ligand, peptide antigen presented by major histocompatibility complex (pMHC), is a critical step in immune responses. How TCR is “triggered” by ligand binding is still unclear. Using lipid bilayer and plastic based artificial antigen presenting systems, we demonstrate that TCR can be triggered by a very low number (<10) of monomeric agonist pMHCs anchored on a surface. TCR triggering was not dependent on the presence of endogenous pMHCs. It was, however, critically dependent on active T cell adhesion to the surface and intact actin cytoskeleton function. Based on these observations, and the dynamic T cell/APC interaction observed in vivo by other groups, we propose the receptor deformation model of TCR triggering. In this model, TCR signaling is initiated by conformational changes of the TCR/CD3 complex, induced by a pulling force originating from the cytoskeleton, and transmitted through pMHC‐TCR binding interactions with enough strength to resist rupture. The increased dissociation rate of pMHC‐TCR interaction under force should facilitate fast and efficient TCR serial triggering by a single agonist pMHC, thus explaining the sensitivity of TCR triggering. This model also offers “pMHC‐TCR binding strength under force” as the parameter that defines the specificity of TCR triggering, i.e., the ability and potency of certain pMHCs to trigger TCR.
Palmitoylation‐dependent plasma membrane transport, but lipid raft‐independent signaling function, of linker for activation of T cells (LAT)
Authors: Hundt Matthias , Harada Yoshuke , Hayashi Keitaro , De Giorgio Lauren , Tanimura Natsuko , Zhang Weiguo , Altman Amnon
Published: June 2021

LAT is a dually palmitoylated transmembrane (TM) adaptor protein that is essential for T cell development and peripheral T cell activation. LAT‐deficient Jurkat T cells (J.CaM2) have been used to explore the role of LAT palmitoylation in its function. Consistent with previous reports, a non‐palmitoylated LAT mutant (LAT C26/29A) did not reconstitute TCR signaling in J.CaM2 cells and did not localize in lipid rafts. Surprisingly, this mutant was not found in the plasma membrane (PM) but, rather, was restricted to the Golgi apparatus. To further examine the importance of LAT palmitoylation, we analyzed a chimeric LAT construct (Src‐LAT) consisting of the membrane‐targeting N‐terminal region of Src kinase and the cytoplasmic tail of LAT. Src‐LAT localized in the PM (but not in lipid raft). Unexpectedly, and similar to another, TM LAT chimera (LAX‐LAT), Src‐LAT was capable of restoring TCR signaling in J.CaM2 cells, and T cell development in Src‐LAT‐reconstituted LAT−/− bone marrow chimeras, despite being expressed as a peripheral membrane protein. Thus, functionality of LAT correlates with its PM localization (even as a peripheral protein), but is independent of its lipid raft localization. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its transport to the PM.

Supported by the Leukemia and Lymphoma Society.
Neutrophils Sequestered in the Liver Moderate Proinflammatory Cytokine/Chemokine Production by Kupffer Cells
Authors: Holub Martin , Wintermeyer Philip , Gregory Stephen H.
Published: June 2021

The liver plays a major role in clearing bacteria from the bloodstream. Clearance is often attributed to Kupffer cells, which line the hepatic sinusoids and comprise ≈35% of the nonparenchymal liver cell population in mice. Rather, we reported previously that most bacteria taken up by the liver are killed by immigrating neutrophils. Ingestion by Kupffer cells and timely elimination of these neutrophils suppresses inflammation and tissue injury that might otherwise ensue. To determine the effect of ingested neutrophils on cytokine production by Kupffer cells, mice were treated with anti‐neutrophil monoclonal antibody. Kupffer cells isolated from antibody‐treated control (non‐infected) mice, as well as from ‐treated mice at 2 hours postinfection with Listeria, exhibited a marked increase in intracellular myeloperoxidase activity demonstrating the role of Kupffer cells in eliminating dead neutrophils. Neutrophil ingestion exerted a significant effect on cytokine/chemokine mRNA expression and protein production by Kupffer cells subsequent to Listeria infection. IL‐6, IL‐12, IL‐10, KC and MIP‐1α production by neutrophil‐containing Kupffer cells was sharply diminished. These findings demonstrate the role of neutrophils in regulating cytokine/chemokine production by Kupffer cells and, thus, moderating the proinflammatory response to bacteria taken up by the liver. Supported by NIH Grant DK068097.
The analysis of interrelationship of parameters of immune homeostasis with the system of LPO‐AOS
Authors: Dushanova Gavhar A
Published: June 2021

Taking into account very important role of mebraine patology in processus of immunogenesis, investigation of existence of parallels between immune system status and indexes of intensity of AOS and enzymes activity of antiradical defence systems are subject of our research.

Investigation of correlation analysis of indexes of immune system connected with parameters of LPO‐AOS of totally healthy pacients in general group, showed existence of negative correlation between serum Lg classes M with indexes of MDA.

Further investigations on biological significance of system as a central genetic apparatus of immune system functioning, showed great interest of existence of some relationship of antigen HLA with the level of activity of metabolic processus, related to LPO‐AOS.

Thus, the level of MDA differ in groups of HLA‐DR opposit pacients, representing more lower indexes among antigen positive individuals. Pacients with had HLA antigen B in their fenotypes had higher activity of SOD enzyme, as compared to pacients without this antigen in fenotype.
Definition of the DRB1*0901 peptide binding motif within novel DRB1*0901 restricted T cell epitopes by peptide binding and structural modeling
Authors: James Eddie A , Moustakas Antonis K , Papadopoulos George K , Nouv Randi S , Kwok William W
Published: June 2021

HLA‐DRB1*0901 is a common allele among Asian populations that has been associated with Type 1 Diabetes. However, its peptide binding motif is only partially characterized. This study further defined the binding motif for DR0901. The motif was deduced from the binding affinities of peptides containing amino acid substitutions at anchor positions and confirmed by structural modeling. In agreement with previous studies, pocket 1 preferred aromatic anchor residues while pocket 4 preferred small aliphatic or polar amino acids. In contrast to previous studies, pocket 9 was not completely permissive, as Pro, Asn, and Arg substitutions were unable to bind. The previously uncharacterized pockets 6 and 7 accommodated a wide range of residues with only subtle influences on overall binding. Modeling studies revealed features within the peptide binding pockets consistent with these findings, including a constrained pocket 4 and a uniquely spacious pocket 9. The resulting motif defined T cell epitopes within 27 of 29 novel antigenic peptides identified by tetramer guided epitope mapping. The observed motif also predicted an epitope within the published GAD65 200–217 peptide and other portions of the protein. This work was supported by NIH contract HHSN266200400028C
Quantitative Analysis of Cellular Antigen and Determination of the Half‐Life of Hsp90‐peptide complexes in vivo
Authors: Kelly John , Srivastava Pramod
Published: June 2021

Heat shock proteins (HSPs) are ubiquitous, highly‐abundant components of cells, functioning as molecular chaperones for nascent or denatured proteins, as well as short protein fragments. HSPs can be biochemically purified in complexes with short polypeptides (8–20 AA) that include the precursors to MHC Class I‐binding octa/nonamers. Chaperones protect these peptides on their journey to MHC‐I for direct presentation, and HSP‐peptide complexes specifically activate the immune system through delivery of the antigenic peptides to antigen‐presenting cells (APC) for cross‐presentation. To study the cell biology of HSP‐peptide formation we developed a novel cell line, PIK23, which employs a modified version of ovalbumin (KOVAK) under the control of an inducible promoter system. Using this cell line we have identified MHC‐I binding peptide precursors associated with gp96 and Hsp90. In the present study we have quantified the peptides bound to biochemically‐purified Hsp90 and found: (1) Hsp90‐chaperoned KOVAK peptides are abundant even in cells synthesizing minute quantities of antigen, (2) these peptides increase in proportion to antigen synthesis, (3) these peptides represent a mixture of proteasomal products, and (4) the half‐life of Hsp90‐peptide association is ~12 hours. These findings shed new light into the mechanics of Hsp90's chaperone role in direct and cross presentation of antigen.
Computational modeling of the effect of receptor‐ligand complex diffusion on immunological synapse formation in B cells
Authors: Tsourkas Philippos K , Longo Marjorie , Raychaudhuri Subhadip
Published: June 2021

B cell receptors have been shown to cluster at the intercellular junction between a B cell and an antigen‐presenting cell (APC) in the form of a segregated pattern of B cell receptor/antigen (BCR/Ag) complexes known as the immunological synapse. We use a Monte Carlo model to study the effect of diffusion of surface‐bound molecules on B cell synapse formation. Our results show that B cell synapse formation is optimal for a limited range of receptor‐ligand complex diffusion coefficient values, typically one‐to‐two orders of magnitude lower than the diffusion coefficient of free receptors. By tracking individual antigen molecules in our simulation, we have found a nonlinear relationship between the diffusion coefficient and mean‐square displacement of receptor‐ligand complexes. This can be used to deduce the order of magnitude of the diffusion coefficient of receptor‐ligand complexes, something which is difficult to determine experimentally due to the finite lifetime of receptor‐ligand bonds. Our analysis also shows how tracking of individual antigen molecules can provide insight into the mechanism of B cell synapse formation. Thus, our results can guide single molecule tracking experiments to elucidate the synapse formation mechanism in B cells, and potentially in other immune cells.
IL‐17 Upregulates Regulator of G‐protein Signaling (Rgs)13 and Rgs16 for the Formation of Autoreactive Germinal Centers in BXD2 Mice
Authors: Mountz John D , Yang PingAr , Wu Qi , Guentert Tanja , Wang John , Hsu Hui‐Chen
Published: June 2021

The lupus and arthritis prone BXD2 mice develop a large number of spontaneous germinal centers (GCs) in the spleens and dramatically elevated sera levels of autoantibodies. Sera from BXD2 mice exhibited significantly elevated IL‐17, but not IFN‐γ or IL‐4, compared to B6 mice. Under the optimal TH‐17 polarization condition, 9% and 20% of purified CD4 T cells from B6 and BXD2 mice were polarized into TH‐17 cells, respectively, in vitro. Supplementation of IL‐6 and IL‐23 was not required for the development of TH‐17 CD4+ T cells from the unfractionated spleen cells from the BXD2 mice but was required for their development from the spleens of B6 mice. Quantitative real‐time PCR analysis indicated that purified CD4 T cells and purified B cells from BXD2 mice express high levels of Il17 and Il17r, respectively. In situ confocal fluorescent microscopy showed that TH‐17 CD4 T cells and IL‐17R+ IgM+ B cells were localized preferentially in the PNA+ GC region in the spleens of the BXD2 mice. Treatment of BXD2 mice with anti‐IL‐17 neutralization antibody (250 μg/mouse q.3 days × 4) resulted in dissipation of B cells from CD4 T cells, decreased percentage of PNA+Fas+ GC B cells, and decreased number of B cells that produce IgG antibodies against histone, BiP, and DNA. IL‐17 upregulated the expression of Rgs13 and Rgs16 and suppressed the CXCL12‐induced migration response to both CD4 and B cells. Our results demonstrate a novel pathogenic mechanism of IL‐17 independently of its chronic inflammatory effects in autoimmune disease. IL‐17 induced spontaneous formation of GCs provided the optimal microenvironment for the production of pathogenic autoantibodies. This work is supported by Arthritis Foundation, VAMC Merit Review, ACR, and Daiichi‐Sankyo Co. Ltd.
NKT cells constitutively express IL‐23 receptor and RORγt, and rapidly produce IL‐17 upon receptor ligation in an IL‐6‐independent fashion
Authors: Caspi Rachel R , Rachitskaya Aleksandra V , Horai Reiko , Li Zhuqing , Luger Dror , Villasmil Rafael , Nussenblatt Robert B , Hansen Anna M
Published: June 2021

Th17 cells require IL‐6 and TGFβ for lineage commitment, and IL‐23 for maintenance. Unexpectedly, naive IL‐6−/−splenocytes stimulated with anti‐CD3 and IL‐23 produced normal amounts of IL‐17 during the first 24 h of culture. These rapid IL‐6‐independent IL‐17 producers were identified as NKT cells by phenotype (TCRβ+/DX5+) and function (response to α–GalCer). Human NKT cells also produced IL‐17 upon stimulation. NKT cells constitutively expressed IL‐23R and RORγt. Ligation of either TCR or IL‐23R triggered IL‐17 production, and both together had an additive effect suggesting independent pathways. IL‐17 was produced exclusively by the NK1.1− fraction, but was not restricted to a particular subset of NKT cells, as the NKT populations of Jα18−/− and of CD1d−/− mice, which lack certain NKT subsets, produced IL‐17. Importantly, in vivo administration of α–GalCer triggered a rapid IL‐17 response in the spleen. These data suggest an important biological role for innate IL‐17 production by NKT cells that is rapid and precedes the adaptive IL‐17 response.
Type I IFN inhibits the expansion of Th17 lymphocytes from both healthy subjects and Multiple Sclerosis patients
Authors: Novelli Francesco , Conti Laura , Clerico Marinella , Contessa Giulia , Durelli Luca
Published: June 2021

Murine Th17 cells are responsible for EAE, the model of multiple sclerosis (MS).IFNβ is used in the therapy of MS but the mechanism of its action have not been clarified yet. The kinetics of IFNs receptors during the differentiation of IL‐23‐driven differentiation of Th17 from healthy PBMC was evaluated. On developing Th17 lymphocytes a correlation exists between the early up‐regulation of IFNγR2 surface expression and the transient ability of IFNγ to activate STAT1 and suppress Th17 differentiation. Later on, developing Th17 cells display an increased IFNα receptor (IFNαR1) surface expression compared to developing Th1 cells, and a strong ability of type I IFNs to induced‐STAT1 activation and apoptosis in differentiated Th17. Analysis of PBMC from never‐treated patients with relapsing‐remitting (RR)‐MS showed that Th17 cells are expanded during the active phases of MS, and decreased during stability, while Th1 cells are slightly expanded in both active and stable patients compared to healthy donors. IFN‐αR1 is more expressed on Th17 than on Th1 cells from MS patients, and IFNβ treatment strongly reduces the number of Th17 cells, while expands Th1 cells. These data indicate that Th17 cells play a role in the pathogenesis of MS. The enhanced IFNαR1 expression and IFNβ responsiveness on Th17 cells may explain the mechanism of the effectiveness of its administration in MS patients.
TLR activated APC enhance the response of aged naïve CD4 T cells via an IL‐6 dependent mechanism
Authors: Jones Stephen Charles , Roberts Deborah , Swain Susan L
Published: June 2021

Toll like receptor (TLR) ligands hold great potential as adjuvants to enhance the aged antigen specific CD4 T cell response. We established an in vitro model to determine whether TLR ligand stimulated bone marrow derived dendritic cells were capable of enhancing the response of aged naïve CD4 T cells. Antigen presentation by non‐stimulated bone marrow derived dendritic cells (BMDCs) results in an aged naïve influenza‐specific Tg CD4 T cell response characterized by reduced expansion, reduced IL‐2 production, and increased cell death of activated cells. Upon stimulation with TLR ligands BMDCs express higher levels of CD80, CD40 and MHC class II, and secrete increased amounts of TNFα, RANTES, IL‐12p70, IL‐12p40, IL‐6 and KC. Aged Tg naïve CD4 T cells activated by TLR ligand stimulated BMDCs exhibited enhanced expansion and reduced cell death that correlated with the increased expression of the anti‐apoptotic molecule, Bcl‐2. Analysis of culture supernatants revealed an early increase in the levels of IL‐2, IL‐6 and KC when aged cells were activated by TLR ligand treated BMDCs. The addition of anti‐IL‐6 blocking antibodies at the initiation of the culture completely prevented the rescue of the aged response, indicating that inflammatory cytokines are a central component to enhancing the aged CD4 T cell response. We feel that these are exciting data that clearly establish a mechanism for how TLR ligands may serve as adjuvants for enhancing the function of aged CD4 T cells.
PCB77‐induced adhesion molecule expression in primary endothelial cells is mediated through caveolae signaling
Authors: Choi Yean Jung , Lim Eun Jin , Zheng Yuanyuan , Majkova Zuzana , Oesterling Beth , Eum Sung Yong , Toborek Michal , Hennig Bernhard
Published: June 2021

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that can activate endothelial cells thus leading to atherogenic events. Induction of vascular adhesion molecules is a common feature in inflammatory environments, leading to the early development of atherosclerosis. We have investigated PCB77‐induced up‐regulation of adhesion molecules through caveolae‐regulated signaling in primary mouse aortic endothelial cells (MAECs) and porcine pulmonary artery endothelial cells (PAECs). PCB77 significantly increased the expression of vascular cell adhesion molecule‐1 (VCAM‐1) and intracellular adhesion molecule‐1 (ICAM‐1) measured by both Western blot and RT‐PCR in a dose dependent manner in both MAECs and PAECs. In addition, the adhesion of monocytes to PCB77‐activated endothelial cells was increased in a dose‐dependent pattern. Phosphorylation studies of caveolin‐1 and related kinases were conducted in endothelial cells containing either functional or siRNA silenced caveolin‐1 protein. Activation of these caveolae‐associated signaling pathways was decreased in absence of functional caveolae. These data provide the evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs. (Supported by grants from NIH/NIEHS (P42ES07380), the University of Kentucky AES and KRF‐2007‐357‐F00045)
Type I Natural Killer T (iNKT) Cells Regulate Inflammatory Immune Response in Mice Liver Injury Induced by CCl4
Authors: PARK OGYI , JEONG WON‐IL , GAO BIN
Published: June 2021

Natural killer T (NKT) cells are the predominant lymphocyte population (up to 30 %) in the mouse liver and play an important role in Concanavalin A (Con A) and α‐Galactosylceramide (α‐GalCer) induced liver injury by producing inflammatory cytokines, such as IFN‐γ and TNF‐α and activating other cell populations including NK and T cells. In this study, we examined the role of NKT cells in carbon tetrachloride (CCl4) liver injury using type I, or invariant NKT (iNKT) KO mice (Ja18−/−) and WT C57BL/6 mice. Liver injury in mice was induced by intraperitoneal single injection of 1 ml/kg body weight of 10% CCl4 and samples were collected at different time points. The administration of CCl4 to mice induced liver injury with depletion of NKT cells and elevation of NK cells in the liver in both mice groups. Compared to WT mice, Jα 18−/− mice showed severe liver damage and increased neutrophil infiltration at early time point. Serum levels of inflammatory cytokines, such as TNF‐α, MCP‐1, IL‐4 and IL‐6, as well as mRNA expression were remarkably increased in Jα18−/− mice after CCl4 administration compared to WT mice. These findings suggest that type I NKT cells may protect against inflammatory immune response via suppressing the production of proinflammatory cytokines in early stage liver injury.
Mitogenic CD28 induces granzyme B and a cytokine storm in human TH1 cells
Authors: Medina Miguel A. , Martin Ashley , Couturier Jacob , Camell Christina , Rodgers John , Lewis Dorothy
Published: June 2021

Phase I clinical trials with a humanized super‐agonistic CD28 monoclonal antibody resulted in multi‐organ failure with loss of lymphocytes in the periphery.

We investigated the mechanism of mitogenic CD28 induced activation and cell death in T cells. Mitogenic but not conventional CD28 induced activation and apoptosis of the Jurkat but not H9 T cell line. In vitro polarized human T‐helper (Th1) cells were activated by mitogenic CD28, but unlike their TH2 counterparts, produced significant amounts of granzyme B (10 ug/ml) from 5 x 105 cells. Negatively selected human Th1 cells exhibit a Th1 like cytokine profile following PMA and ionomycin stimulation and express extracellular markers, CXCR3 and CCR5, a profile consistent with Th1 cells. Upon mitogenic CD28 stimulation more than half of the cells are activated and about 30% apoptotic similar to activation induced cell death seen after anti‐CD3 stimulation. Following mitogenic CD28 stimulation negatively selected TH1 cells produced a cytokine profile consistent with a cytokine storm. Therefore, activated inflammatory Th‐1 cells are potential producers of large quantities of granzyme B and inflammatory cytokines, which may be associated with multi‐organ failure in the phase I clinical trial.

This study was supported by NIH grants R21‐AI054251, R37‐AI36682 and Center for AIDS Research grant P30 AI36211.
Critical contribution of T cell‐derived MMPs to influenza virus pathology
Authors: Baaten Bas J. G. , Lin Melissa M. , Hughes Chris C. W. , Bradley Linda M.
Published: June 2021

Influenza viruses cause significant annual illness and death with recurrent seasonal epidemics and sporadic devastating pandemics. The efficiency of highly pathogenic influenza viruses has been attributed to potent and aberrant inflammatory responses, which can result in severe immunopathological damage and death in humans. T cells can contribute to pulmonary damage by direct lysis of infected cells and the production of pro‐inflammatory cytokines. Migration of T cells and their effector responses in situ are crucial steps towards immunity, but could also contribute to the high morbidity and mortality associated with pathogenic strains. We hypothesized that the ability of T cells to efficiently cross the lung endothelial barrier and move through the basement membrane, which is essential for their function, is regulated by matrix metalloproteases (MMPs). MMPs have the proteolytic capability to mediate cleavage of the collagen network that makes up the basement membrane. We demonstrate that T cells express MMP2 and MMP9 activity, which is crucial during influenza virus‐induced pathogenesis. Furthermore, we discuss the factors that affect the induction of MMPs in T cells. The discovery of the roles of MMPs in T cell function during influenza virus pathogenesis could elucidate the balance between immunity and pathology and may therefore have considerable therapeutic relevance.
In vitro and in vivo activation of CD4+CD25−Foxp3+ Tregs in NZB/W mice
Authors: Scalapino Kenneth John , Daikh David I
Published: June 2021

PURPOSE: CD4+Foxp3+ Tregs increase with onset of lupus in (NZBxNZW)F1 (B/W) mice, but many Tregs lack CD25 expression. Absence of CD25 is correlated with impaired Treg function. The ability to restore CD25 expression and enhance Treg function was assessed.

METHODS: CD4+CD25−CD62LLO T cells were stimulated with anti‐CD3/anti‐CD28 and IL‐2. Cells up‐regulating CD25 were sorted and tested for Foxp3 expression, suppression capacity, and survival. In vivo up‐regulation of CD25, Foxp3, and suppression capacity following a brief course of IL‐2 to mice was then assessed.

RESULTS: 44% of Tregs in sick B/W mice were CD4+CD25−CD62LLO. Stimulation of these cells in vitro resulted in up‐regulation of CD25 on 85% of Tregs. Activated Tregs expressed high levels of CD25, Foxp3, and had enhanced in vitro suppression and in vivo survival following adoptive transfer. Activation of CD4+CD25− Tregs in vivo by IL‐2 increased per cell Foxp3 expression and the fraction of Tregs that were CD25+ (75%). Purified CD4+CD25+ T cells from treated mice were highly suppressive when tested in vitro.

CONCLUSION: Inadequate CD25 expression on B/W Tregs may correlate with impaired Treg function and disease development. This problem is reversible, producing Tregs with prolonged survival and suppression capacity. These results suggest the potential for lupus therapies directed towards Treg activation.

Supported by Department of Veterans Affairs.
Targeted deletion of fgl2 leads to impaired regulatory T cell activity and development of autoimmune glomerulonephritis
Authors: Shalev Itay , Liu Hao , Koscik Cheryl , Bartczak Agata , Javadi Mojib , Wong Kit Man , Maknojia Asif , He Wei , Liu Ming Feng , Diao Jun , Winter Erin , Manuel Justin , McCarthy Doug , Gommerman Jennifer , Cattral Mark , Clark David A , Phillips James , Gorczynski Reginald R , Zhang Li , Downey Greg , Grant David , Cybulsky Myron I , Levy Gary
Published: June 2021

Mice with targeted deletion of fibrinogen‐like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild‐type recipients reconstituted with fgl2−/− bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, T helper 1 (Th1) polarization, and increased numbers of antibody‐producing B cells following immunization with T‐independent antigens. Dendritic cells (DC) were more abundant in fgl2−/− mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2−/− mice, but their suppressive activity was significantly impaired. Antibody to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited DC maturation and induced apoptosis of B cells through binding to the low affinity FcγRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.

This work was supported in part by grants from the Heart and Stroke Foundation of Canada and the Canadian Institutes for Health Research.
IL‐17 augment type I IFN‐mediated antibody production in BXD2 autoimmune mouse
Authors: Wang John , Mountz John
Published: June 2021

Type I IFN and IL‐17 are thought to induce distinct autoimmune diseases. However, patients with Th17‐mediated diseases could also present with a type I IFN signature, suggestive that IL‐17 and type I IFN may not be mutually exclusive for the induction of disease. As a novel model of autoimmune disease, BXD2 mice exhibit an increased presence of both IL‐17 and type I IFN. BXD2 mice displayed significantly increased serum IL‐17 (5–6 fold), and Th17 population in the spleen of BXD2 mice relative to that of non‐autoimmune mice (14% vs. 0.1%). Blood mononuclear cells showed increased type I IFN message levels (6‐fold), and treatment with CpG increased serum IFN‐á (5–50‐fold) in BXD2 mice. Confocal microscopy showed the presence of an increased population (2–4 fold) of plasmacytoid dendritic cells (pDCs) that primarily produce IFN‐á in BXD2 spleen (~80% of IFN‐á+ cells are pDCs in BXD2 mouse). When treated with CpG, serum IFN‐á (2–3 fold) was decreased in mice in which IL‐17 signaling was disrupted compared to normal BXD2 and B6 mice. In vivo blocking of IFN‐á in NP‐CGG immunized BXD2 mouse also decreased antibody serum titers to pre‐immunization levels, demonstrating that IFN‐á is essential to antibody formation. Our study indicates that IL‐17 signaling promotes IFN‐á expression and thereby may provide a novel pathway by which IL‐17 promote antibody production through type I IFN.
Phenotype switching by inflammation‐inducing polarized Th17 cells, but not by Th1 cells
Authors: Shi Guangpu , Cox Catherine A. , Vistica Barbara P. , Wawrousek Eric F. , Gery Igal
Published: June 2021

Analyses of inflammation inducing Th cells revealed that a portion of these cells express both IFN‐γ and IL‐17, suggesting that Th1 and/or Th17 may switch their phenotype. Here, we examined phenotype retention in a system in which polarized Th1 or Th17 cells specific against hen egg lysozyme (HEL) induce ocular inflammation in recipient mice expressing HEL in their eyes. Whereas no switching to Th17 was detected in Th1 recipient eyes, substantial proportions of transferred Th17 expressed IFN‐γ, or both IFN‐γ and IL‐17 in Th17 recipient eyes. The phenotype switch of Th17 cells increased with time and was attributed to exposure to IL‐12 that is expressed in the inflamed eyes. Incubation in vitro with Th1 polarizing cocktail (IL‐2+IL‐12) converted most Th17 into IFN‐γ or IFN‐γ/IL‐17 expressing cells, but no reciprocal conversion was noted with Th1 cells. Furthermore, whereas Th1 cells did not express ROR‐γt following incubation with Th17 polarizing cocktail, Th17 expressed T‐bet following incubation with Th1 cocktail. Thus, polarized Th1 cells fully retain their phenotype, while Th17 switch and express IFN‐γ or both IFN‐γ and IL‐17, as well as T‐bet, following exposure to IL‐12.

This study is supported by NEI/NIH intramural program.
Exploring the transfer of experimental autoimmune encephalomyelitis using MOG TCR transgenic T cells
Authors: Williams Jessica Lynn , Kithcart Aaron , Song Fei , Gienapp Ingrid , Shawler Todd , Smith Kristen , Whitacre Caroline
Published: June 2021

Experimental autoimmune encephalomyelitis (EAE), a widely studied animal model for multiple sclerosis, is mediated by CD4+ T cells directed against myelin antigens such as myelin oligodendrocyte glycoprotein (MOG). A MOG T cell receptor transgenic (MOG Tg) mouse on a C57BL/6 background was recently reported. Difficulties have been encountered in transferring EAE using MOG Tg T cells. We explored a number of avenues for effecting transfer of disease such as varying culture time, density of cells in culture, antigen concentration and number of cells transferred with negative results in spite of robust proliferation and production of IFNγ. We hypothesized that encephalitogenic cells were being over‐stimulated and dying. Dilution of encephalitogenic cells with irradiated wildtype cells prior to transfer did not result in EAE transfer. Additionally, the cytokine cocktail was perhaps not adequately stimulating the encephalitogenic T cells. MOG Tg T cells were activated using IL‐2, IL‐7, IL‐12, IL‐18, and anti‐CD3/CD28 and pertussis toxin given to recipients. This protocol did not result in disease transfer. We are currently exploring additional cell activation parameters including generation of Th‐17 cells and suppressive mechanisms operative in the recipient. (Supported by NIH grant AI 064320, National MS Society grant RG3272 and NIH grant T32 AI055411)
FoxP3 Mediated Induction of Pim 2 Allows T Regulatory Cells to Preferentially Expand in Rapamycin
Authors: Riley James , Basu Samik , Golovina Tatiana , Mikheeva Tatiana , June Carl
Published: June 2021

Addition of rapamycin to cultures of expanding T regulatory cells helps maintain their suppressive activity, but the mechanism behind this observation is unclear. Human pim 2, similar to murine pim 2, is a serine/threonine kinase that can confer rapamycin resistance. Unexpectedly, pim 2 was found to be constitutively expressed in freshly isolated, resting T regulatory cells but not in CD4 T effector cells, explaining why addition of rapamycin prior to T cell activation favors T regulatory cell expansion. Introduction of FoxP3, but not FoxP3Ä2, into effector CD4 T cells induced pim 2 expression and conferred preferential expansion in the presence of rapamycin, indicating that FoxP3 can regulate pim 2 expression and that functional differences do exist between the two prominent FoxP3 isoforms. Finally, we correlated the ability of natural T regulatory cells to expand with FoxP3 expression and observed positive correlation in the presence of rapamycin and a negative correlation in the absence of rapamycin. Together, these results indicate that T regulatory cells, because of their constitutive expression of FoxP3, are programmed to be resistant to rapamycin and this provides further rationale why this immunosuppressant drug should be used to augment T regulatory activity.
Influence of CD4 T cell help and Tregs in diabetes induction in RIP‐GP mice
Authors: Oldham Janine , Martinic Marianne M. , Bresson Damien , Fousteri Georgia , von Herrath Matthias G.
Published: June 2021

Recently, we have developed a virus‐free protocol, which induces Type 1 Diabetes (T1D) in two different RIP‐GP strains with different disease kinetics and incidence. RIP‐GP Arm and RIP‐GP Berlin mice express the glycoprotein (GP) from the lymphocytic choriomeningitis virus (LCMV) strains Armstrong and WE, respectively, under control of the rat insulin promoter resulting in pancreatic LCMV‐GP expression. Our diabetes induction protocol induced T1D in all RIP‐GP Berlin mice seven to twelve days after treatment. In contrast, only maximally 35% of RIP‐GP Arm mice developed transient T1D from day nine onwards. We then analyzed whether LCMV‐specific Smarta CD4 T helper cells would increase diabetes incidence in RIP‐GP Arm mice and whether Smarta CD4 regulatory T cells (Tregs) would decrease T1D incidence in RIP‐GP Berlin mice. Transfer of Smarta T helper cells indeed increased T1D kinetics and incidence in RIP‐GP Arm mice whereas transfer of Smarta Tregs reduced T1D incidence in RIP‐GP Berlin mice to below 30%. In the future, we will analyze the mechanisms of how LCMV‐specific CD4 T helper and Treg cells influence T1D development in the two RIP‐GP strains.
The role of glycolysis in lymphocyte survival and autoimmunity
Authors: Saltis Mark , Tchatchouang Claudia , Levina Irina , Quoc‐Pham Michael , Davidson Wendy
Published: June 2021

It is well established that constitutively enhanced glycolytic activity in tumor cells confers a selective advantage and inhibitors of glycolysis, such as 2‐deoxyglucose (2‐DOG), can inhibit tumor growth. The growth and survival of normal B and T lymphocytes following activation also is dependent on upregulation of glycolytic activity and is inhibited by 2‐DOG. Here, we ask whether diseases characterized by chronic lymphocyte stimulation such as systemic autoimmunity are associated with dysregulated glycolysis. Studies were performed with mice deficient in FasL (gld/gld) that develop lymphoproliferative disease and autoimmunity. We found that B cells from gld mice had significantly elevated levels of mRNA transcripts for glycolytic enzymes and increased hexokinase II, a key regulatory enzyme in glycolysis. To determine the effects of glycolysis inhibition on activated lymphocytes in vivo, we treated mice for 10 days with 2‐DOG. This resulted in a significant reduction in both T and B cells with selective depletion of activated CD4+ and CD8+ (CD44hi) and B cells (CD23loCD21lo), suggesting these cells are particularly dependent on enhanced glycolysis for survival. Studies are in progress to determine whether prolonged therapy with glycolysis inhibitors reduces autoantibody titers in autoimmune models. This work was supported by an Arthritis Foundation Chapter Research Grant awarded to W.F.D.
Single dose tolerance protects against Experimental Autoimmune Encephalomyelitis (EAE) via immune deviation and regulatory T (Treg) cells
Authors: Rynda Agnieszka , Maddaloni Massimo , Callis Gayle , Riccardi Carol , Pascual David W.
Published: June 2021

Recently it was shown that when reovirus protein sigma 1 (pσ1) is engineered to deliver ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether pσ1‐based tolerogen can prevent and/or treat autoimmune disease, a genetic fusion between proteolipid protein (PLP) peptide, PLP130–159 and OVA‐pσ1, termed AR1, was produced and used to nasally dose EAE‐susceptible SJL/J mice in a prophylactic or therapeutic paradigm. In either case, clinical EAE and histology of spinal cords was greatly reduced in AR1‐dosed mice. FACS analysis of regional lymph nodes showed >40 % increase in FoxP3+CD4+CD25+and CD25− T cells in mice given nasal AR1. Cytokine analysis showed >8‐fold reductions in IFN‐γ, IL‐17, and IL‐21, but greatly elevated IL‐4 and IL‐10 in AR1‐dosed mice compared to controls. AR1‐derived Treg cells produced IL‐10, and IL‐4 was produced by Th2 cells. Adoptive transfer of AR1‐specific Treg cells fully protected recipient SJL/J mice from the EAE development, but partial protection was also conferred by IL‐4‐producing CD25−CD4+ T cells. These data show that a single nasal dose of pσ1‐based tolerogen induces specific Treg cells and Th2 cells that contribute to efficacious prevention and treatment of EAE.
Downregulation of Cellular Energy Sensor during Disease condition results in Alteration of Lipid Homeostasis in Immune Cells
Authors: Giri Shailendra , Nath Narender , Khan Mushfiquidin , Singh Avtar K , Singh Inderjit
Published: June 2021

AMP‐activated protein kinase (AMPK), an energy sensing metabolic switch of mammalian cells, was observed to be down regulated during T cells mediated autoimmune disease. A catalytic subunit of AMPK, alpha1, was predominantly expressed in T‐ and antigen presenting cells. During EAE disease, activity of AMPK was significantly downregulated in thymocytes, spleen cells and brain. Decreased AMPK activity results increased lipid biosynthesis and their content in total T cells, adherent and non‐adherent spleen cells at disease state. Increased lipids biosynthesis leads to the higher proliferative state of these cells was evident from active form of Akt, p42/44 and reduced levels of p21 and p27. AMPK activators restored lipid alteration in total spleen cells by reversing the AMPK activity and inhibited production of pro‐inflammatory cytokines under in vitro stimulation. All together, our results demonstrated that downregulation of AMPK‐mediated altered lipid metabolism in immune cells, resulted an inflammatory environment may be one of the critical aspects of pathogenesis during EAE or Multiple Sclerosis and identified AMPK as a novel therapeutic target for autoimmune diseases.

Grants support: Mainly by RG 3810‐A‐1 and PP1283 from the MS Society (SG) and in part by (NS‐22576, NS‐34741, NS‐37766 and NS‐40810) from NIH (IS).