bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of CD40 Ligand (CD40L) mRNA stability by Protein Kinase C (109.15)
Authors: Castro Moraima , Matus-Nicodemos Rodrigo , Covey Lori 
Published: January 2023

Abstract
The expression of the CD40 ligand (CD40L) is controlled at the posttranscriptional level by an activation-induced program of regulated mRNA stability whereby at early times of activation the mRNA is highly unstable and at later times (24-48 hr) the transcript becomes significantly stabilized. Regulated stability is dependent on the formation of a cytoplasmic polypyrimidine tract binding protein (PTB)-containing-complex (Complex I) that binds to the 3’UTR of the CD40L mRNA. Here we report that the broad-spectrum PKC inhibitor H7 destabilizes the CD40L mRNA in both a Jurkat subclone (Jurkat/D1.1) that recapitulates the “stability” phase of the CD40L regulated decay program and in primary human CD4+ T cells activated with PMA and ionomycin. In both cell populations, treatment with H7 also resulted in a corresponding decrease in surface CD40L expression. Analysis of RNA binding complexes by electrophoretic mobility shift assays (R-EMSA) following H7 treatment of Jurkat/D1.1 cells corresponded to an increase in Complex I as well as the formation of a novel PTB-independent complex that bound to the CD40L stability element. These results demonstrate the relationship between PKC-regulated pathways and the control of CD40L expression through mRNA stability. Future work is underway to identify the protein component(s) of this new complex, as well as to identify PKC isoforms that are involved in regulating PTB-mediated decay.
Increased kon of TCR-pMHC interaction influences activation and development of CD4+ T cells (109.18)
Authors: Lynch Jennifer , Donermeyer David , Weber K. Scott , Allen Paul 
Published: January 2023

Abstract
While T cell activation has been correlated with half-life of the TCR-pMHC interaction, the importance of on-rate is only now being realized. In contrast to other studies, our lab has generated a system that separates the kon component from koff by comparing the response of two T cells, 3L2 and M2. 3L2 responds specifically to Hb(64-76) peptide presented on I-Ek. M2 differs from 3L2 by two point mutations in the CDR1α loop, resulting in a 4-fold higher affinity for Hb(64-76) due entirely to a faster kon. We generated an M2 TCR transgenic mouse to compare the affect of kon on T cell responses in vivo and in vitro to 3L2 T cells. M2 expressing T cells are positively selected and detected with a clonotypic antibody in the periphery though at a reduced number in comparison to 3L2. This could be due to decreased positive selection and/or increased negative selection of M2 T cells by alteration of the kinetics of M2 interactions with endogenous pMHC. M2 responds to 12 Hb(64-76) altered peptides (APLs) that are null peptides for 3L2 and has increased reactivity to APLs that are agonists for 3L2. M2 cells have a greater proliferative response at day 3 than 3L2 when exposed to Hb(64-76). While both M2 and 3L2 proliferate by day 3, a larger number of M2 cells than 3L2 cells have divided. This suggests that a fast kon increases early T cell responses. By selectively altering kon, we have revealed the importance of kon for TCR binding pMHC in T cell development and activation.
T cell triggering thresholds are modulated by the number of antigen within individual T cell receptor clusters (109.20)
Authors: Manz Boryana , Jackson Bryan , Petit Rebecca , Dustin Michael , Groves Jay 
Published: January 2023

Abstract
T cells are known to react to extremely small numbers of activating agonist peptides. Spatial organization of T cell receptors (TCR) and their peptide-MHC (pMHC) ligands into microclusters is correlated with T cell activation. Here we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Experiments in which the overall agonist density and the number of agonist per TCR cluster are independently varied in primary T cells indicate that single pMHCs are unlikely to be able to trigger calcium signals. Analysis of activating pMHC distribution among clusters suggests the most probable minimal triggering unit for calcium signaling is at least 4 pMHC per cluster in this system. This is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.
Regulation of T-cell receptor signaling by activation-induced zinc influx (109.21)
Authors: Lee Won-Woo , Yu Mingcan , Tomar Deepak , Li Guangjin , Czesnikiewicz-Guzik Marta , Pryshchep Sergey , Lamar David , Singh Karnail , Weyand Cornelia , Goronzy Jörg 
Published: January 2023

Abstract
Zinc is a trace element that is essential for innate and adaptive immune responses. In addition to being a structural element of many proteins, zinc also functions as a neurotransmitter and an intracellular messenger. Although cytoplasmic zinc concentrations are strictly controlled, temporal or spatial changes in bioavailable zinc may influence the activity of several enzymes, including kinases and phosphatases. In this study, we provide evidence that zinc functions as an ionic signaling molecule after T-cell activation. Cytoplasmic zinc concentrations increased within one minute after TCR triggering by super antigen presented by myeloid dendritic cells. In contrast to activation-induced calcium influx, increased zinc concentrations were compartmentalized in the subsynaptic area. The zinc increase was dependent on the extracellular zinc concentrations and was inhibited by silencing the plasma membrane zinc transporter Zip6 consistent with influx from extracellular compartments. Increased zinc availability augmented ZAP70 phosphorylation, inhibited SHP-1 recruitment to the T cell signaling complex, slowed down Lck rephosphorylation at position 505 and sustained calcium influx. As a result, increased extracellular zinc bioavailability facilitated the induction of T cell proliferative responses to suboptimal stimuli. We propose that zinc calibrates TCR activation thresholds by inhibiting negative feedback mechanisms in TCR signaling.
Drak2 and PKD coordinate early T cell activation events to maintain survival (109.24)
Authors: Newton Ryan , Walsh Craig 
Published: January 2023

Abstract
T cells deficient in Drak2 hyper-respond to suboptimal stimulation through the antigen receptor, exhibiting enhanced calcium mobilization and high Il-2 production. Despite this lack of negative regulation, mice deficient in Drak2 are resistant to models of induced organ-specific autoimmune disease. This has been attributed in part to a defect in survival following activation, and can be rescued in vitro with exogenous co-stimulation and in vivo through expression of a Bcl-xL transgene. Drak2 kinase activity is crucial to maintain normal calcium responses required for productive activation of T cells, and has recently been shown to require the kinase activity of protein kinase D (PKD). PKD and Drak2 associate at mitochondria, and downstream events are dependent on calcium-induced mitochondrial reactive oxygen generation. PKD and Drak2 are thus part of a critical negative feedback loop whereby calcium influx is both required and targeted by Drak2 activity. Recent work suggests that specific ion channels may be directly targeted by Drak2, with consequences on both the activity of these channels and downstream metabolic events that contribute to proliferative capacity. This implicates both PKD and Drak2 as part of a formerly unappreciated signaling complex that coordinates immediate T cell activation events to limit unproductive activation of T cells and maintain survival.
Decoupling age from cytomegalovirus-associated changes in the immune system (109.28)
Authors: Furman David , Shen-Orr Shai , Kidd Brian , Maecker Holden , Dekker Cornelia , Davis Mark 
Published: January 2023

Abstract
CMV infection has been associated with premature immunosenescence and shorter lifespan. To date, no systems-wide analysis for decoupling age from CMV associated changes have been conducted. To address the differences between an immune system in naïve versus CMV infected humans and to decouple the age-related from CMV-related changes, we studied a comprehensive set of assays comprising serum cytokines, cell subset frequencies, whole genome gene expression and various stimulations of immune cells in 30 young and 60 older, CMV seropositive or seronegative individuals. For each measurement we performed a multiple regression analysis on all samples by age, CMV serostatus and the interaction between both variables (age × cmv). We found dramatic differences in multiple measurements of the immune system by age - mostly affecting T cell signaling responses, inflammatory cytokines and many immune-related genes, by CMV - which seems to preferentially modulate the CD4 and B cell compartments at different levels - or when both factors are present with changes mostly restricted to B cell functions. We present, for the first time, a systems immunology view of CMV- and age-associated changes in the immune system.
LAT-independent triggering of granule-mediated cytotoxicity of CD8 T cells (109.32)
Authors: Ouyang Chihwen (Claudia) , Zhu Minghua , Li Qi-jing , Zhang Weiguo 
Published: January 2023

Abstract
LAT (Linker for activation of T cells) is a transmembrane adaptor protein that is integral to the TCR-mediated signaling. LAT has been shown to play an essential role in thymocyte development, T cell activation, and cytokine production by CD4 T cells. Conventional thinking assumed that LAT is also required for the effector function of CD8 T cell. In this study, we used LAT conditional knockout mice to study the function of LAT in the cytotoxicity of CD8 T cells. Surprisingly, although CTL cytotoxicity is reduced in the absence of LAT, it is not completely abrogated. This cytotoxicity appears to be mediated through a granule-dependent, Fas-independent pathway. We further dissected the effect of the LAT deletion within each step of granule exocytosis. LAT-deficiency led to the formation of unstable synapses, subsequently causing unstable T:APC conjugates. Moreover, MTOC polarization and granule reorientation were impaired by LAT-deficiency. Further investigation revealed that, even though TCR-mediated signaling was severely impaired, the residual calcium flux and PLC activity in LAT-deficient CTLs were sufficient to support cytotoxicity. Our study reveals a novel finding that, in contrast to helper T cells, CTL function is not entirely dependent on LAT and, perhaps is not governed by the overall TCR signaling strength.
Discs Large Homolog 1 splice variants couple T cell receptor engagement to distinct T cell effector functions (109.34)
Authors: Silva Oscar , Humphries Lisa , Crocetti Jillian , Round June , Miceli M. 
Published: January 2023

Abstract
The recognition of antigen by the TCR triggers the polarized recruitment of receptors, signal transducers and cytoskeletal components to the T-cell:APC interface, providing a molecular platform for selectively instructing T effector transcription and polarized secretion. How TCR signals are specified to regulate distinct downstream signaling and functional outputs remains poorly understood. We report that T cells utilize distinct protein variants of Dlgh1, a scaffold that regulates synaptic polarity and signaling, to independently regulate pathways controlling TCR-induced transcriptional activation and secretion of effector molecules. T cells express two Dlgh1 protein variants, Dlgh1 i1A-i1B and Dlgh1 i1B, which differ by inclusion of the alternatively spliced i1A-domain, hypothesized to control binding of the TCR proximal kinase, Lck. Over-expression of Dlgh1 i1A-i1B in T cells selectively enhanced TCR-induced TNFα and NFATc1, but not IκBα or IL-2 mRNA expression, while over-expression of Dlgh1 i1B did not. In contrast, knockdown of the Dlgh1 i1B variant in CD8+ T cells attenuated TCR-induced granzyme B secretion, but did not alter TCR induced granzyme B mRNA upregulation. Knockdown of Dlgh1 i1A-i1B reduced TCR-induced TNFα and NFATc1 mRNA upregulation, but not granzyme B secretion. Therefore, alternative splicing specializes Dlgh1 variants to orchestrate TCR signal specificity, selectively coupling TCR engagement to transcriptional activation or secretion of effectors.
The emerging role of Mg2+ availability on B cell receptor signaling (109.4)
Authors: Towne Francina , Perraud Anne-Laure , Schmitz Carsten 
Published: January 2023

Abstract
Hypomagnesemia in patients, as well as in animal models, results in a weaker humoral immune response. The mechanism underlying this Mg2+ dependent modulation of the humoral response remains undefined. The master regulator of Mg2+ homeostasis, TRPM7, is a unique cation channel with a fused serine/threonine kinase domain. Interestingly, TRPM7 interacts with several phospholipase isoforms including PLCγ2, an essential component of B cell receptor (BCR) signaling. Here we address our hypothesis that in response to environmental availability of Mg2+, TRPM7 acts as a homeostatic sensor to adjust BCR signaling strength by serine/threonine phosphorylation of PLCγ2. We show in the DT40 avian B cell line that PLCγ2-dependent BCR-mediated Ca2+-response is reduced under low Mg2+ conditions. Using in vitro phosphorylation assays and a complementation approach in PLCγ2 deficient DT40 cells, we show that TRPM7 kinase is phosphorylating PLCγ2 resulting in the Mg2+ sensitive modulation of BCR-elicited Ca2+ responses. These findings suggest that TRPM7 actively responds to the availability of environmental Mg2+, in turn regulating downstream BCR signaling responses through serine/threonine phosphorylation of PLCγ2. This novel interaction, between an ion channel kinase and a BCR signaling molecule, could be a key piece of the puzzle in explaining why animal models of nutritional hypomagnesemia have weaker humoral immune responses.
Stim1, PKCδ and RasGRP proteins set a threshold for pro-apoptotic Erk signaling during B cell development (109.6)
Authors: Limnander Andre , Depeille Philippe , Freedman Tanya , Liou Jen , Leitges Michael , Kurosaki Tomohiro , Roose Jeroen , Weiss Arthur 
Published: January 2023

Abstract
Antigen encounter induces proliferation in mature B cells but triggers apoptosis in developing B cells. Thus, developing B cells that strongly recognize auto-antigens undergo apoptosis, one mechanism that contributes to prevent autoimmunity. Developing B cells display increased Ca2+ entry in response to antigen as compared to mature B cells, and Ca2+ is required for antigen-induced apoptosis. However, the Ca2+-driven signals involved are poorly defined. Here we identify and characterize a previously unrecognized Ca2+-driven Erk activation pathway, which is pro-apoptotic and biochemically distinct from diacylglycerol (DAG)-induced Erk activation. This pathway requires PKCδ and RasGRP proteins and depends on Stim1 levels, which control the magnitude of Ca2+ entry. We identify a novel phosphorylation site on RasGRP1 as a putative target of PKCδ that is absolutely required for Ca2+-dependent Erk activation but is dispensable for DAG-mediated Erk activation. Developmental regulation of these proteins is associated with selective activation of the pathway in bone marrow B cells prone to negative selection. This checkpoint is impaired in PKCδ-/- mice, which develop B cell autoimmunity. Conversely, Stim1 overexpression confers a competitive disadvantage to developing B cells, an effect that depends on PKCδ. Our findings demonstrate that DAG and Ca2+ can mediate the activation of functionally distinct Erk pathways to determine whether B cells proliferate or die upon antigen encounter.
Prohibitins and the cytoplasmic domain of CD86 are necessary to mediate signaling in B lymphocytes (109.8)
Authors: Lucas Christopher , Campbell Kerry , Bhatia Sumeena , Hodes Richard , Sanders Virginia 
Published: January 2023

Abstract
CD86 engagement on a CD40L/IL-4-primed murine B cell activates signaling intermediates that lead to an increase in Oct-2 and mature IgG1 mRNA and protein expression, without affecting class switch recombination. The most proximal signaling intermediates identified thus far are Lyn and phospholipase Cγ2α/β, which are usually activated after recruitment and binding to phospho-tyrosine residues on receptors, but the cytoplasmic domain of CD86 lacks tyrosines. Using a proteomics-based identification approach, we show that the tyrosine-containing transmembrane adaptor proteins, prohibitin-1 (Phb1) and Phb2, bind to CD86. The basal association is low in resting cells, but increased after priming with CD40L/IL-4. The level of total Phb1/2 expression increased primarily via a CD40-dependent mechanism. Phb1 protein expression and binding to CD86 were consistently greater than that for Phb2 in CD40L/IL-4-primed B cells. The CD86-induced increase in Oct-2 and IgG1 was reduced when either Phb1/2 expression was silenced by shRNA or when the cytoplasmic domain of CD86 was truncated or mutated at the serine/threonine protein kinase C-phosphorylation sites. Truncation of the CD86 cytoplasmic domain did not affect the binding of Phb1/2 to CD86, or the level of T cell activation and class switch recombination. These findings suggested that Phb1/2 and the CD86 cytoplasmic domain are each necessary to mediate the CD86-induced signaling in B cells.
Regulation of interleukin-27 production by Mycobacterium tuberculosis (110.2)
Authors: Liu Jianguo , Zhang Jidong , Qian Xuesong , Ning Huan , Hoft Daniel 
Published: January 2023

Abstract
Mycobacterium tuberculosis (Mtb) remains a major global challenge to human healthcare and the mechanisms of how Mtb evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that BCG and Mtb could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and Mtb stimulation of macrophages induces activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after Mtb stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to Mtb. Furthermore, blockade of p38 by SB203580 enhanced Mtb-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhances the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective TB immunity.
Recognition and functional activation of human FcαRI by C-reactive protein (110.22)
Authors: Marjon Kristopher , Lu Jinghua , Marnell Lorraine , Wang Ruipeng , Mold Carolyn , Sun Peter , Du Clos Terry 
Published: January 2023

Abstract
C-Reactive Protein (CRP) is a member of the pentraxin family and an acute phase serum protein. It has been established that CRP and other pentraxins bind FcγRs and induce phagocytosis, activation of signaling, and cytokine synthesis. We report a novel structural and functional interaction between CRP and FcαRI with μM affinity and the binding site for CRP on FcαRI is distinct from that of IgA. Although FcγR and FcαRI bind their immunologlobulin ligands differently, the binding between CRP and FcαRI or FcγRII was inhibited by C1q indicating a similar binding orientation. However, the CRP mutant T173A, which binds poorly to FcγR, showed enhanced binding to FcαRI compared to wild type CRP. CRP crosslinking of FcαRI induced degranulation, ERK phosphorylation and IL-4 production in rat basophilic leukemia cells expressing FcαRI. CRP increased phagocytosis, surface expression of FcαRI, and the production of TNF-α in human neutrophils. The finding that CRP binds to an additional FcR on leukocytes has broad implications for the roles of CRP in host defense, autoimmunity and regulation of the immune response.
Heme oxygenase-1 induction stimulates host defense mechanism against infections (110.3)
Authors: Dhawan Subhash 
Published: January 2023

Abstract
Innate immune response is pivotal for host defense against pathogenic infections. It is, however, often ineffective to provide protection against infections and fails to efficiently respond to the existing conditions. Induction of host response or direct targeting of the invading pathogens then becomes desirable to reduce the disease progression. Activation of HO-1 by a natural substrate hemin effectively enhanced the ability of human macrophage to resist infections by several pathogens, including HIV-1, dengue virus, West Nile virus, poxvirus, and Leishmania donovani. HO-1 expression in a prostate cancer cell line susceptible to infection by XMRV resulted in substantial reduction in virus production by infected cells. Reduction of intracellular pathogens in HO-1-activated cells was attenuated by pretreatment of cells with SnPP IX, a specific inhibitor of HO-1 activity, indicating a key role of this endogenous enzyme in the host defense mechanism. Considering effective treatment of these infections is a continuous daunting challenge due to the emergence of drug resistant mutants, unlocking of HO-1-dependent regulatory genes may provide novel biomarkers and therapeutic targets for treating multiple infections and disease conditions.
Significant role of IL-1 signaling, but limited role of inflammasome activation in oviduct pathology during Chlamydia muridarum genital infection (110.6)
Authors: Nagarajan Uma , SIkes James , Yeruva Laxmi , Prantner Daniel 
Published: January 2023

Abstract
IL-1β has been implicated in the development of oviduct pathology during Chlamydia muridarum genital infection in the mouse model. The goal of this study was to characterize the role of IL-1 signaling and the inflammasome activation pathways during C. muridarum genital infection. Compared to control mice, IL-1 receptor KO (knockout) mice display delayed clearance and increased infection burden, but very few incidences of oviduct hydrosalpinx. Consistent with this observation, IL-1R antagonist KO mice display an opposite phenotype of rapid infection clearance. Flow cytometry analysis at day 10 and day 20 post infection reveal significantly more macrophages in the oviducts of IL-1R KO mice in comparison to control mice. Despite increased infection and recruited macrophages, IL-1R-KO mice have less tissue fibrosis and dilation, suggesting an anti-inflammatory role for these cells in the absence of IL-1 signaling. To investigate the role of inflammasome components during in vivo genital infection, mice lacking NLRP3, NLRC4 and ASC were tested. Although IL-1β secretion is dependent on caspase-1 during in vitro infection of primed macrophages and requires ASC, it is independent of NLRP3 and NLRC4 activation. Surprisingly, during in vivo genital infection, mice lacking NLRP3, NLRC4, as well as ASC display no reduction in oviduct pathology. These data suggest a major role for IL-1 signaling but a limited role for inflammasome pathway in development of oviduct pathology.