bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of a Th1 Response from Th2 Polarized T Cells by Activated Dendritic Cells: Dependence on TCR:pMHC Interaction, ICAM-1, IL-12 (92.7)
Authors: Radhakrishnan Suresh , Wiehagen Karla R , Pulko Vesna , Knutson Keith , WIlliam Faubion , Pease Larry R 
Published: January 2023

Abstract
Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of co-stimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this report we demonstrate that addition of activated DC to spleen cultures containing established Th2 polarized CD4+ T cells was sufficient to suppress Th2 and to induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite antigen specificity. The DC activator B7-DC XAb was more than 10,000-fold more efficient at inducing phenotype reversal than the TLR agonists, CpG-ODN and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR: pMHC complex, the expression of the co-stimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFNƒ×ƒnby the activated DC. Requirement for the co-stimulation/adhesion molecule SLAM was found to be quantitative. Thus, activation of DC, particularly by cross linking B7-DC, can modulate well established Th2 T cell responses in an antigen specific manner.
Similarities and differences in tapasin interaction with disparate murine MHC class I allotypes (93.11)
Authors: Groening Laura Christina , Wang Xiaojian , Tuli Amit , Ormsbee Briana D. , McIlhaney Mary M. , Solheim Joyce C. 
Published: January 2023

Abstract
Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide-loading complex. In this study, we first assessed the influence of the tapasin C-terminus on H2-Kb, -Kd, and -Ld in mouse cells. Truncated mouse tapasin (lacking the transmembrane and C-terminal region) was unable to associate with TAP or any of three murine MHC class I allotypes, and did not assist murine MHC class I folding and surface expression. A tapasin mutant with a single substitution in the C-terminus (K408W) increased the amount of Kd (but not Ld) in the open, peptide-free form, relative to wild type tapasin. We investigated whether Kd was also particularly responsive to other tapasin mutations, and found that a C95S substitution in tapasin also caused an elevation in open Kd (but not open Ld or Kb). Folded Kd was found to be amply present in association with wild type tapasin, and even more so with tapasin K408W and C95S. In summary, our results suggest that the C-terminus of mouse tapasin plays a vital role in the surface expression of murine MHC class I molecules, and that mouse MHC allotypes vary in their sensitivity to specific alterations in tapasin sequence and in their release from tapasin after folding. Overall, our results reveal new complexity in tapasin’s interactions with polymorphic MHC class I molecules. (NIH Grant GM57428)
Tapasin functions to assemble MHC class I subunits but does not affect their lifetime on the cell surface (93.12)
Authors: Everett Maya W , Edidin Michael 
Published: January 2023

Abstract
Proper assembly of MHC class I (MHCI) molecules is dependent on the dedicated ER-resident chaperone tapasin (tpn). Tpn binds both MHCI and TAP through different domains. We created three fluorescently tagged constructs: wild type tpn, soluble tpn, which no longer interacts with TAP, and N300 tpn, which no longer interacts with MHCI. Despite previous biochemical data, using fluorescence microscopy we find that soluble tpn is mostly located within the ER, similarly to wild type tpn. FRAP analysis shows that soluble tpn is more mobile than both wild type and N300, possibly because the latter constructs interact with TAP. Transfecting tpn negative cells with soluble tpn does not increase MHCI surface expression to the same level as wild type. When wild type is transfected in combination with N300, MHCI surface levels are reduced to those found with soluble, suggesting that N300 blocks wild type tpn access to TAP. Although resulting MHCI surface levels differ between wild type, soluble, and N300 tpn constructs, MHCI surface lifetimes are the same. Our data suggests that tpn functions to bring together the MHCI subunits, a process which is aided by proximity to TAP, but does not act to increase stability of the resulting MHCI complex.
The double mutation of DRBW61A/N82A changed the MHC II-peptide complex stability and DM catalytic activity (93.24)
Authors: Zhou Zemin , He Xiao , Chen Xinjian , Jensen Peter E. 
Published: January 2023

Abstract
The MHC class II protein HLA-DM (DM) is required for efficient antigen presentation; however the molecular basis of how DM facilitates this process remains incompletely understood. We have generated the HLA-DR1 β chain double mutant, replacing W61 and N82 with alanine (DRβ61/82), and a single mutant, DRβH81A (DRβ81), to investigate the role of conserved hydrogen bonds in the DM mechanism. Previous in vitro experiment showed that DRβ61/82 is relatively DM independent in peptide binding or dissociation. Expression of the DRβ mutants with DRα in MHC class II deficient T2 cells showed that there is no detectable CLIP on the surface of DRβ61/82 T2 cells, while CLIP is present on the surface of DR-WT or DRβ81 T2 cells. When DM was transduced into the DR-WT, DRβ81 or DRβ61/82 T2 cell line, the amount of CLIP on the surface of DM-DR-WT or DM-DRβ81 T2 cell line was decreased substantially, indicating functional interaction with DM. Our results suggest that the two sites, W61 and N82, but not H81, play an important role in DR/CLIP complex stability and DM-catalyzed peptide exchange
(Supported by NIH grant AI030554).
The TCP-1 Ring Complex (TRiC) binds antigenic peptides and facilitates their cross-presentation by APCs (93.5)
Authors: Vatner Ralph Eric , Srivastava Pramod K 
Published: January 2023

Abstract
The multimeric chaperonin TCP-1 Ring Complex (TRiC), an hsp60 homologue, was recently found to associate with the precursors of the ovalbumin-derived SIINFEKL epitope during processing and loading onto MHC I. The TRiC-peptide association is essential for loading of MHC I. Here, we have sought to characterize the peptide-binding properties of TRiC in vitro, and study the behavior of TRiC-peptide complexes in cross-presentation assays. TRiC is shown to bind a variety of peptides in vitro in much the same manner as previously described peptide chaperones of the hsp90 family. TRiC-peptide complexes are efficiently taken up by APCs and the peptides are processed and represented on MHC class I for recognition by specific CD8+ T cells. These results as well as the immunological properties of TRiC-peptide complexes formed in vivo shall be reported.
Developing a novel approach to follow the processing of viral antigens for MHC class I presentation. (93.7)
Authors: CARDINAUD Sylvain J , SHASTRI Nilabh 
Published: January 2023

Abstract
Cytotoxic T cells recognize their targets by the presence of unique peptides displayed by MHC class I molecules on the surface of antigen presenting cells. The peptides are generated by the antigen processing pathway and some proteins are known to inhibit this processing to escape the immune response. This is the case of EBNA1 (EBV nuclear antigen), a viral antigen detected in EBV-associated tumors. It is now established that the glycine-alanine repeat sequence present in EBNA1 is responsible for the absence of cell surface antigen presentation. Nevertheless how the antigen processing is modified remains unknown. Indeed, using conventional T cell assays, only the final peptide can be efficiently detected on the cell surface. We developed a novel assay, based on HPLC fractionation, allowing the detection of antigenic precursors in vivo. So far, we have successfully used this system to isolate, quantify and characterize antigenic intermediates derived from EBNA1 proteolysis. Using inhibitors as well as RNA interference, we can determine for the first time how EBNA1 interacts intracellularly with key steps of its antigen processing: degradation of newly synthezed proteins (DRiPs), proteasome proteolysis, TAP transport, and the role of chaperonin molecules.
Role of IL-18 on phosphorylation of STAT-6 in monocyte/macrophages (94.11)
Authors: Tamma Seetha M , Bhavsar Alap , Patel Tejal , Costa Kevin 
Published: January 2023

Abstract
Cytokine interactions in health & disease are very complex. There are conflicting reports on the cytokine IL-18 and its role in Th1 versus Th2 shift. The hypothesis that STAT-6 is expressed in monocyte/macrophages and that cytokine IL-18 has an effect on the phosphorylation of STAT-6 is addressed. Monocyte/macrophages were isolated by negative selection. The data indicates that the optimum concentration of IL-18 required for stimulation is 10μg/1x106 cells and 72 hour culture period is the optimum time for the induction of maximum levels of STAT-6 phosphorylation. We also observed total tyrosine phosphorylation of several intracellular proteins. Based on these studies, we propose that macrophages respond to IL-18 via STAT-6 phosphorylation and that IL-18 induces mitotic activity in monocyte/macrophages. Our data suggest that IL-18 actsin a more subtle manner as a costimulatory factor in the activation of STAT-6 in monocytes and provides evidence for a new immunoregulatoryeffect of the proinflammatory cytokine IL-18 on monocytes.
Grant from C.W.Post Campus Research Committee of Long Island University, New York supported this work.
Role of IL-18 on Th-2 shift and on phosphorylation of STAT-6 (94.12)
Authors: Tamma Seetha M , Bhavsar Alap 
Published: January 2023

Abstract
STAT-6 is a central mediator of IL-4-induced gene responses. It has been shown that STAT-6 is expressed in B cells and T cells upon stimulation with IL-4. Interleukin-12 (IL-12) and IL-18 are the key factors for the induction of Th1 Cells, early signals being involved in Th2 differentiation if any by IL-18 are less well characterized. We have investigatedthe mechanisms employed by IL-18 and IL-4 to control expression of STATs. Since we have shown in earlier studies that Th2 cytokines protect cells from apoptosis, we explored the possible role of the controversial cytokine IL-18 on Th2 shift and analyzed the effects of IL-18 on CD4+ T cells. Cells were cultured either with IL-18, IL-4, IL-18 + CD28 for different time points. The optimum time required for the phosphorylation of STAT-6 when stimulated with IL-18 was 72 hours. This effect of STAT-6 phosphorylation was blocked by addition of anti-IL-18 antibody in the culture. However, phosphorylation of STAT-1 and STAT-4 was seen by 24 hours. Furthermore, when cells were stimulated with anti-CD28 antibody plus IL-18, there is accelerated and enhanced phosphorylation of STAT-6. These studies may suggest that IL-18 serves as a negative feed back mechanism on inflammatory responses.
Grant from C.W.Post Campus Research Committee of Long Island University, New York supported this work.
Exploring the cross-talk between p38 MAPK and canonical Wnt signaling pathways (94.15)
Authors: Flick Lisa M , Florczyk Stephen J 
Published: January 2023

Abstract
The Wnt signaling pathway is active in many stages of embryonic development and tissue maintenance in adult organisms including bone homeostasis. Further understanding of the Wnt pathway could allow for improvements in therapeutics for metabolic bone disease and biomaterials used for bone replacement. To explore the potential crosstalk between the canonical Wnt and p38 mitogen-activated protein kinase (MAPK) pathways, MC3T3-E1 pre-osteoblasts were cultured and stimulated with LiCl in the presence or absence of the p38 MAPK inhibitor SB239063. Activation of the canonical Wnt signaling pathway was assessed by quantifying the lymphoid enhancer binding factor 1 (LEF1) binding activity in a non-radioactive colorimetric assay. The p38 MAPK pathway is typically considered independent of the Wnt signaling pathway and therefore, the SB239063 was hypothesized to have no effect on the LEF1 binding activity. Interestingly, cell extracts from cultures stimulated with LiCl and SB239063 showed a 37% increase in LEF1 binding activity compared to LiCl treated controls. The data suggests that p38 or a molecule downstream from p38 acts as an inhibitor on the canonical Wnt pathway. One candidate repressor protein is the transcriptional repressor high-mobility group box protein 1 (HBP-1) which has been shown to be a target of p38 MAPK and can also suppress LEF1 activity resulting from canonical Wnt signaling.
Cyclosporin A and FK506 Inhibit IL-12p40 Production through the Calmodulin/Calmodulin-dependent Protein Kinase-activated PI3K in LPS-Stimulated Human Monocytic Cells (94.19)
Authors: Ma Wei , Mishra Sasmita , Gee Katrina , Mishra Jyoti Prasad , Angel Jonathan , Kumar Ashok 
Published: January 2023

Abstract
Cyclosporine-A (CyA) and FK506 are immunosuppressive agents that are widely used to treat inflammatory and autoimmune disorders because of their ability to suppress the production of Th1 cytokines including IL-12. Both CyA and FK506 are potent inhibitors of calcineurin in the calcium signalling pathway. However, the role of calcium signalling in the regulation of human IL-12p40 production remains unknown. Interestingly, calcium and phosphoinositide 3-kinase (PI3K) signalling pathways have been shown to negatively regulate LPS-induced murine IL-12p40 production. Contrary to these observations, we show here that LPS-induced IL-12p40 production in human monocytic cells is positively regulated by calcium activated calmodulin- (CaM) and CaM-dependent protein kinase-II (CaMK-II)-activated PI3K. Furthermore, LPS induced the production of IL-12p40 by NFκB and AP-1 transcription factors through the activation of upstream CaM/CaMK-II. LPS-induced IL-12p40 production is known to be regulated by the c-Jun-N-terminal kinase (JNK) pathway. Importantly, both CyA and FK506 downregulated LPS-induced IL-12p40 transcription by inhibiting CaM/CaMK-II-activated PI3K and their downstream transcription factors NFκB and AP-1 independent of the JNK pathway.
C3a desArg/Acylation stimulating protein (ASP) and C5L2 correlation with expression of inflammatory factors and metabolic factors in human adipose tissue (94.25)
Authors: Cianflone Katherine , Lu HuiLing , MacLaren Robin , Smith Jessica 
Published: January 2023

Abstract
C5L2 is a recently identified receptor for C5a and C3a/C3adesArg (ASP). C5a/C5adesArg binds C5L2 with high affinity but no functional activation. By contrast, C5L2 is a functional receptor for ASP and in adipocytes plays a role in lipid metabolism. In this study, we used adipose tissue microarray analysis to explore the overlap between metabolism and immunology. Subcutaneous (SC AT) and omental (OM AT) adipose tissue were collected from 14 subjects (normal + obese) and divided into two groups based on plasma ASP level (assayed by ELISA): high ASP group (&#8593;ASP) (ASP&gt;40nM) and low ASP group (&#8595;ASP ) (ASP&lt;40 nM).
C5L2 (p=0.06), C5aR (p=0.03), IL-6 (p=0.01), and IL-6R (p=0.03) expression were increased significantly in &#8593; ASP vs. &#8595;ASP in OM AT, with no change in SC AT. Plasma ASP correlated with lipid metabolic factors GPAT-1, FABP1, FABP5, FABP7 and adipophilin, and inflammatory factors CRP, C5aR and IL-6R in OM AT. ASP was mainly correlated with lipid metabolic factors, such as LPL, CD36, FABP3, and FABP6 in SC AT.
ASP-C5L2 may play a role in mediating metabolic- immune interactions in adipose tissue.
Funding: CIHR
Profiling Cytokine Levels Using a Multiplex Human Cytokine ELISA Array (94.26)
Authors: You Min , Lo Jing-Yi , Wang Yanqiu , Yu Xiang 
Published: January 2023

Abstract
The induction of multiple cytokines is a common phenomenon in many immunological responses; therefore, a method to quickly and simultaneously screen in vitro or in vivo samples for multiple cytokine levels is needed. We developed a “multiplex” sandwich ELISA Array on a 96-well plate allowing researchers to screen several human serum, plasma or cell culture supernatant samples for multiple cytokines in one experiment. The ELISA Array is easy to perform and only requires a standard ELISA plate reader. It can be also used in conjunction with corresponding single analyte ELISA kits to perform validation and absolute quantification. As an illustrative example, we monitored the time-dependent (0, 6, 24, and 48 hours) patterns of Th1/Th2 cytokine induction by human peripheral blood mononuclear cells (PBMC) in response to PMA (50 ng/ml) and ionomycin (1 μg/ml). Large amounts of IL-2, IFN-gamma and TNF-alpha are induced after the 6-h stimulation when only more moderate amounts of IL-4, IL-5, IL-10 and IL-13 are secreted. IFN-gamma levels are the highest among the eight cytokines tested after 6 hours, while IL-2 has the highest level after 48 hours. The amount of IL-12 p70 is not detectable even after 48 hours. The ELISA Array thus provides a powerful, easy-to-use solution for profiling the relative levels of several cytokines across multiple experimental conditions at the same time.
Sensitivity of TLR4- and 7-induced NFkappaB1 p105-ERK pathway to TNF-receptor-associated factor 6 (TRAF6) revealed by RNA interference in mouse macrophages (94.6)
Authors: Parameswaran Narayanan , Patial Sonika , Loniewski Katie J 
Published: January 2023

Abstract
TLR4- and -7-induced ERK activation in macrophages (MAC) involves the IκB kinase complex (IKK)-mediated phosphorylation of NFκB1 p105, a member of the IκB family. Major goal of this study was to examine the sensitivity of TLR4- and -7-induced IKK-p105-ERK pathway to TRAF6 in MAC. Using an RNAi approach we reduced the cellular levels of TRAF6 by ~80% in MAC. Although TLR4-stimulated ERK pathway, including p105 phosphorylation by IKK, and ERK phosphorylation, were not affected with TRAF6 knockdown (KD), TLR7-stimulated ERK pathway was completely blocked. In contrast, both TLR4- and 7- induced IKK-mediated IκBα phosphorylation was completely blocked by TRAF6 KD. These results demonstrate that TLR4 activates a TRAF6-sensitive and -insensitive IKK pathways in MAC. Surprisingly, compared to the role of TRAF6 in ERK activation, TLR4- and 7-induced JNK phosphorylation were completely blocked by TRAF6 KD, suggesting differential regulation of MAPK pathways by TRAF6. TLR4-induced TNFα mRNA in MAC is mediated in part via the ERK pathway. Consistent with signaling data, TLR4-induced TNFα mRNA was only partially blocked with TRAF6 KD. Taken together, these results indicate the differential roles of TRAF6 not only between TLRs but also demonstrate differential utilization of TRAF6 in IKK and ERK/JNK activation with in TLR4 signaling. This has important implications for drug development against TRAF6 in inflammatory diseases.
Type I Inteferons Differentially Stimulate Interferon-Gamma Secretion in Human Natural Killer Cells. (95.10)
Authors: Lavoie Thomas B. , Hung Finn , Crisafulli Sara , Reynolds Della , Scarzello Anthony , Moolchan Karlene , Skawinski Michael , Koltchev Doranelly , Pestka Sidney , Young Howard A , Clarke William A 
Published: January 2023

Abstract
The human lymphoma cell line NK-92 has many characteristics of human natural killer cells. We have examined NK-92 activation by a full panel of human alpha Interferons (IFNs) by measuring IFN gamma secretion. We confirmed that NK-92 cells secrete IFN gamma in response to IFN alpha 2 in a dose-dependent manner. The IFN gamma response is enhanced by addition of IL-2, IL-18 or PMA, as has been seen with human donor NK cells. The EC50 for IFN alpha 2 ranges between 700–3000 pg/ml. Inclusion of IL-18 exhibited little effect on IFN potency but significant enhancement of IFN efficacy. EC50 values differ by &gt;1000 fold between the IFN subtypes. Alpha 1 demonstrated the weakest activity while alpha 10 and beta 1a were the most potent. The relative potency of the IFNs correlates well with ability to phosphorylate STAT1 and 2. When NK-92 potency is compared with antiviral activity on A549 cells and antiproliferative activity on OVCAR3 cells, there are subtle but significant differences in the rank orders of potency and potencies relative to IFN alpha 2. These studies will be extended to examination of donor NK cells, additional signal transduction pathways, and the secretion of other cyto/chemokines
Rapid Generation of a Functional NK Cell Compartment (95.12)
Authors: Prlic Martin , Bevan Michael J 
Published: January 2023

Abstract
Anti-IL-2 antibodies have been used with the premise that they block IL-2 signaling in vitro and in vivo, but recent findings demonstrate that anti-IL-2 antibodies form a complex with IL-2 in vivo that actually enhances IL-2 activity. NK cells are one cell type affected by these IL-2 complexes. We asked at what stage IL-2 complexes act on NK cells and whether they could be utilized to generate functional NK cells with potential therapeutic value.
We found that the administration of IL-2 complexes resulted in the rapid generation of NK cells in IL-15−/− animals. IL-15 is required for the development and survival of NK cells. Mature, transferred NK cells survived in IL-15−/− animals treated with IL-2 complexes, but proliferated only modestly. These data suggest that the IL-2 complex preferentially expands an immature subset of NK cells. To determine whether this expansion results in the generation of functional NK cells, we treated lethally irradiated, bone marrow reconstituted mice with IL-2 complexes and assessed the activity of NK cells by an in vivo killing assay. Bone marrow chimeras that were treated with IL-2 complexes displayed greatly enhanced NK lytic activity compared to control chimeras 4 days after initiation of treatment. We conclude that IL-2 complex has the potential to rapidly generate functional NK cells that may reduce the risk of viral infections and influence GVHD processes.
Characterization of foam cell formation in human macrophage subsets (95.17)
Authors: Montoya Dennis , Cruz Daniel , Modlin Robert 
Published: January 2023

Abstract
The hallmark of the human atherosclerotic plaque is the presence of lipid-laden macrophages, or foam cells. However, many macrophage subsets are found within atherosclerotic lesions and it is not well understood how monocytes differentiate into these subsets. We focused on characterizing macrophages derived in vitro from human peripheral blood monocytes treated with IL-15, IL-4 or IL-10. We show these macrophages to have differing phenotypes: CD209+CD64+, CD209+CD23+, or CD209+CD163+ for macrophages derived from IL-15, IL-4, or IL-10 respectively. To characterize the macrophage subsets ability to become foam cells we measured their uptake of fluorescently-labeled oxidized LDL (oxLDL). IL-10 derived macrophages had the greatest amount of oxLDL uptake. We then investigated the mechanism of uptake and found that fucoidan, a class-A scavenger receptor competitor, significantly inhibited uptake of oxLDL in IL-10 cells. On the other hand a blocking antibody against the class B scavenger receptor, CD36, did not inhibit uptake. Furthermore, we found IL-10 macrophages to express higher mRNA levels of the class A scavenger receptors macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor (MSR) expression. These data suggests that IL-10 may promote foam cell formation and is dependent on class-A, but not class B scavenger receptors.