bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of CD154 release from T cells and subsequent biological activities (P3375)
Authors: Yacoub Daniel , Benslimane Nadir , Al-Zoobi Loubna , Mourad Walid 
Published: January 2023

Abstract
CD154 is a type II transmembrane protein critically involved in humoral immune responses. The soluble form of CD154 (sCD154) has been linked to various autoimmune and vascular disorders. Therefore, elucidating the mechanisms by which CD154 is release from the cell surface is of primordial importance. Using co-culture experiments, we show that CD154 is predominantly shed upon its engagement with CD40. Indeed, only CD40 and not α5β1 or αMβ2 is implicated in the cleavage of CD154 from Jurkat E6.1 T-cells. Interestingly, CD154 is cleaved independently of the formation of cell surface CD40 homodimers, a process previously shown to be involved in specific elements of CD154-induced signaling. We previously reported that the translocation of CD154 into lipid rafts is required for critical elements of CD154 signaling. Here, we show that CD154 is shed from the cell surface independently of this phenomenon. In contrast, we found that the protein kinase C signaling family and the metalloproteinases ADAM10 and ADAM17 are intimately involved in this process. Moreover, released sCD154 remains biologically active and can regulate critical biological responses such as activation of key survival signaling pathways and inhibition of cell death in α5β1 positive T-cells. In conclusion, our data add significant insights into the mechanisms by which CD154 is released and may explain the link between enhanced levels of sCD154 and T-cell survival and persistence during chronic inflammatory diseases.
IL-17 gene transfer in vivo induces bone loss and epidermal hyperplasia with implications for psoriatic arthritis (P3377)
Authors: Adamopoulos Iannis , Suzuki Erika , Chao Cheng-Chi , Maverakis Emanual , Bowman Eddie 
Published: January 2023

Abstract
The IL-23/IL-17 axis has been linked with pathologic features of psoriatic arthritis, however the direct role of IL-17 on innate immune cells has not been fully elucidated. We have previously demonstrated that IL-17 in vitro contributes to arthritis pathogenesis by inducing osteoclast differentiation and bone resorption. To validate these findings we overexpressed IL-17A in vivo by combining mini-circle DNA technology with hydrodynamic delivery and performed phenotypic analysis of cell populations by FACS, qPCR, histology and in vivo imaging using nanoprobes. We identified that IL-17 induced two distinct IL-17R+ populations, CD11b+Gr1low and CD11b+Gr1high that differentially contribute to bone erosion and epidermal hyperplasia respectively. Our data show that systemic high levels of IL-17 in the serum induce RANK+/c-fms+ osteoclast precursors within the CD11b+Gr1low fraction. To address the functional importance of these precursors we performed RANKL gene transfer to IL-17 overexpressing mice and noticed a significant increase of serum TRAP that correlated with increased bone erosion by microCT. Accordingly, the increase in the CD11b+Gr1high population correlated with an induction of epidermal hyperplasia, Munro’s microabscesses and hyperproliferative keratinocytes in psoriatic lesions. Collectively our data demonstrate mechanistically the role of innate immune precursor cells in the contribution to bone loss and epidermal hyperplasia, hallmarks of PsA.
The lard legacy: parental dietary fat intake alters offspring microbiome and immunity (P3359)
Authors: Myles Ian , Fontecella Natalia , Janelsins Brian , Vithayathil Paul , Segre Julia , Datta Sandip 
Published: January 2023

Abstract
Mechanisms underlying modern increases in prevalence of human inflammatory diseases remain unclear. The hygiene hypothesis postulates that decreased microbial exposure has, at least in part, driven this immune dysregulation. However, dietary fatty acids may also influence immunity, partially through modulation of host responses to microbes. To test the hypothesis that parental dietary fat consumption during gestation and lactation influences offspring immunity, we compared pups of mice fed either a Western diet fatty acid profile or a standard low fat diet. We found that pups from Western diet breeders were not obese or diabetic, but still had worse outcomes in models of infection, autoimmunity, and allergic sensitization. They had heightened colonic inflammatory responses, with increased circulating bacterial lipopolysaccharide (LPS) and muted systemic LPS responsiveness. These deleterious impacts of the Western diet were associated with alterations of the offspring gut microbiome. These results indicate that parental fat consumption can leave a “lard legacy” impacting offspring immunity.
Toll-like receptor B cell stimulation upregulates FcRL4 expression and FcRL4 amplification of TLR signaling is associated with hematopoietic cell kinase expression (P1374)
Authors: Siewe Basile , Plesac Melissa , Stapleton Jack , Landay Alan 
Published: January 2023

Abstract
Fc receptor-like 4 (FcRL4) attenuates B-cell receptor signaling (BCR) while enhancing TLR-responsiveness. FcRL4-mediated BCR dampening is dependent on tyrosine phosphatases SHP1/2. However, regulation of FcRL4 expression and mechanisms underlying FcRL4 amplification of TLR-signaling remain unknown. We find that exposure of human B cells to TLR-2-7 -9 ligands leads to upregulated FcRL4 expression. In FcRL4 transfected B-cell lines, TLR9-induced FcRL4 upregulation was associated with increased HcK expression. Aditionally, p38 inhibition suppressed upregulation of FcRL4 and CD23 but not HcK expression. HcK is an enhancer of TLR-signaling, likely activated by SHP-1/2 and we found that HcK or SHP1/2 inhibition, significantly suppressed TLR9-induced upregulation of CD23. To determine an in vivo relevance for our findings we assessed TLR-stimulated purified FcRL4+ B cells and compared to FcRL4- B cells, FcRL4+ B cells expressed significantly higher levels of the pro-inflammatory cytokine IL-6. Evaluating B cells of HIV+ viremic subjects showed that ex vivo unstimulated FcRL4hi B cell subsets expressed high levels of IL-6. Further, FcRL4+ B cells exhibited high levels of HcK and phosphorylated MAPkinases p38 and Erk .Our data indicate that TLR-activation upregulates FcRL4 expression, and FcRL4 recruited SHP1/2 likely activates HcK thereby amplifying TLR-signaling. Finally, in HIV+ viremic subjects TLR-activated FcRL4+ B cells likely contribute to inflammation via IL-6 expression.
Sphingosine 1-phosphate receptor-3 enhances pro-inflammatory activity in human monocytes (P1384)
Authors: Hou JinChao , Fang XiangMing 
Published: January 2023

Abstract
Objective: Sphingosine 1-phosphate receptor-3(S1P3) is a G protein-coupled receptor constitutively expressed in human monocytes and plays important roles in the innate immune response. This study is aimed to determine the level of S1P3 in septic patients and explore its role in signaling pro-inflammation activity. Method: Patients with sepsis, non-sepsis and trauma were enrolled, and S1P3 levels in monocytes were determined by real-time PCR and flow cytometry. The S1P3 levels were also measured in primary monocytes from healthy donors and THP-1 cell line after 100 ng/ml LPS or 10 ng/ml TNF-α stimulation for 24 hours. Furthermore, the S1P3 receptor was interfered by agonist (GPS-725.017), antagonist (suramin) or S1P3-specific shRNA to explore the relative intracellular signaling pathway. Results: The levels of S1P3 in septic patients were higher than those in non-septic controls and traumatic patients. LPS or TNF-α stimulation upregulated both mRNA and protein levels of S1P3. Furthermore, the LPS-induced MAPK activity and NF-κB activity in THP-1 cells could be enhanced by GPS-725.017 while reduced by suramin. Knock-down of S1P3 expression in THP-1 monocytes also reduced LPS-induced IL-1β and TNF-α production. Conclusion: The levels of S1P3 in human monocytes could be up-regulated by LPS and TNF-α. S1P3 could enhance LPS-induced pro-inflammatory activity, which involved MAPK and NF-κB pathways.
CD80 dimensions are important for T cell co-stimulation (P1396)
Authors: Lim Hong-Sheng , Cordoba Shaun-Paul , Goyette Jesse , van der Merwe Anton 
Published: January 2023

Abstract
Successful T cell activation depends on the recognition of antigenic peptides by the T cell receptor (TCR) together with additional signals from co-stimulatory receptors such as CD28. To determine the requirement of matching TCR and CD28 ligand complexes in optimal co-stimulation, the length of CD80 was elongated by insertion of 2 or 4 immunoglobulin domains. Despite similar binding to soluble CD28 molecules, elongated CD80 molecules were much less effective at mediating co-stimulation to primary T cells. By splitting the TCR and CD28 ligands into 2 separate APCs, the trans-costimulation assay subsequently suggests that elongated CD80s are incapable of transducing co-stimulation signals into the T cell. In addition, confocal studies using CD80-GFP fusion protein indicated that large phosphatases such as CD45 co-localised with elongated CD80 in the T cell-APC interface. Together, these results demonstrated the importance of ligand dimensions in optimal T cell co-stimulation.
The role of palmitoyl-protein thioesterases in T cell activation (P1398)
Authors: Jehmlich Ulrike , Alahmad Adnan , Biedenweg Doreen , Hundt Matthias 
Published: January 2023

Abstract
Reversible protein palmitoylation is a posttranslational modification important for transport, localization and function of proteins. The acyl-protein thioesterase 1 (APT1) also known as lysophospholipase 1 (LYPLA1) is a cytosolic depalmitoylating enzyme. Another thioesterase APT2 (LYPLA2) is highly homologous to APT1. Jurkat T cells were chosen to analyze the role of APT1 and APT2 in T cell activation. APT1/2 mRNA expression was measured by qPCR and knockdown of APT1/2 gene expression was accomplished by RNA interference, which consisted of transient siRNA expression by electroporation or by stable expression of shRNA by lentiviral infection. Palmostatin B was used to inhibit the APT thioesterase activity pharmacologically. The functional effects of APT1/2 knockdown or inhibition were measured by IL-2 secretion (ELISA) and CD69 upregulation (flow cytometry) after CD3/CD28 stimulation. Jurkat cells expressed APT2 mRNA 3.5-fold higher than APT1 mRNA. Even greater differences were found in primary human and murine T cells. Both, transient and stable knockdown of each of the APTs alone reduced IL-2 secretion and CD69 upregulation following CD3/CD28 stimulation. The effect of APT2 knockdown was always stronger than that of APT1. Inhibition with palmostatin B blocked cell activation in Jurkat cells and in human peripheral T cells. Thus, both palmitoyl-protein thioesterases APT1 and APT2 are involved in T cell activation, but APT2 seems to be the predominant enzyme in T cells.
Toxoplasma gondii protein fraction as a potent activator of dendritic cells (P4383)
Authors: Boghozian Roobina , Saei Azad , Mirzaei Reza , Hajati Jamshid 
Published: January 2023

Abstract
Dendritic cells (DCs) are professional antigen presenting cells that both initiate and modulate the immune responses. DCs activation leads to their maturation and production of cytokines such as IL-12. Therefore, their use in immunotherapy against cancers has been studied. We have investigated the potency of Toxoplasma gondii (TG) proteins for DC maturation to induce IL-12 production and mouse spleen cell proliferation. Methods: DCs were generated from Balb/c mouse bone marrow in the presence of GM-CSF and IL-4. Four protein fractions were obtained from sonically fragmented TG cells by precipitation with ammonium sulfate and were added as maturation factors on the last day of the DCs culture. The phenotype analysis was performed using anti-CD80, anti-CD86, anti-CD-40 and anti-MHC II monoclonal antibodies. IL-12p70 concentration in DCs supernatant was also determined. Ultimately, proliferation assay, phagocytic activity and cytokine production (IL-10 and IFNγ) in supernatants of DCs and T cells co-culture were done. Results: Total extract of TG and fraction 1 are more potent in activating DCs than other fractions and matured DCs (mDC) produced significantly higher levels IL-12p70. Expression of CD40 and CD86 was also significantly higher on these mDCs. Moreover, T cells co-cultured with these mDCs secreted higher level IL-10 and IFNγ. Conclusion: The selected fraction can be used in DC cell based immunotherapy approach to generate competent vaccines against tumor cells.
Targeting chlamydial pathogenesis: a novel vaccine design (P4387)
Authors: O'Meara Connor , Armitage Charles , Harvie Marina , Timms Peter , Lycke Nils , Beagley Kenneth 
Published: January 2023

Abstract
Chlamydial vaccine design is focused on eradicating infection. However, infection-associated infertility is the direct consequence of the same pro-inflammatory immune response required to eliminate the infection, which complicates vaccine design. Using the mouse model of Chlamydia pathogenesis, we immunized animals with the chlamydial Major Outer Membrane Protein (MOMP), via various routes of administration, with a number of adjuvant combinations (CTA1-DD, CT and CpG). This approach identified two contrasting vaccine candidates. One vaccine (sublingual delivered MOMP/CTA1-DD) induced a 70% reduction in infertility, without altering the course of infection. Another vaccine (intranasal delivered MOMP/CT/CpG) eradicated the infection in the upper reproductive tract, yet lacked any beneficial influence on incidence of infertility. This presented a unique opportunity to study how immunity against pathology can develop independently from immunity against infection. Comparing the gene expression in disease susceptible oviduct tissues, between infertility- and infection-protected vaccinated animals, identified a major difference in IL-17 signaling. While both vaccines expressed high levels of IL-17 cytokines, the infertility-protected group displayed reduced expression of corresponding IL-17 receptors. Therefore, the inhibition of IL-17 signaling in infertility-protected mice suggests a role for IL-17 not only in the resolution of infection, but also the development of infertility.
In vivo augmentation of NK cell activity by methanol extract of cultured cambial meristematic cells of mountain wild ginseng (P4393)
Authors: Jang A Yeung , Song Eun-Jung , Shin Sung-Hye , Hwang Pyung Han , Kim Sun Young , Yi Ho Keun , Lee Young Hee , Lee Eun-Kyong , Nam Sang-Yun 
Published: January 2023

Abstract
Although mountain wild ginseng (MWG, Panax ginseng C.A.Meyer) has been regarded to be more effective than cultivated ginseng, very few data have been accumulated until now because of its limited availability and expensiveness. Some of us established a technique of isolation and culture of innately undifferentiated cambial meristematic cells from MWG as an attractive alternative. Using methanol extract of cultured MWG cambium-derived cells (MEGC), we here investigated in vivo immunomodulatory effect in a mouse model. Mice were intraperitoneally given MEGC (200 mg/Kg) and adaptive immunity was unaffected by MEGC treatment. Without Ag immunization, splenic NK cell activity in MEGC-treated mice was significantly higher when compared to controls. However, number of NK1.1+ cells and surface expression of activation/ inhibitory receptors and activation markers were unaltered by MEGC. Instead, RT-PCR data clearly demonstrated that MEGC treatment increases constitutive expression of Granzyme B and perforin in fresh spleen cells. When spleen cells were cocultured with Yac-1 in the presence of IL-2, Granzyme B and IFNγ levels were also higher in MEGC-treated group. However, these effects were achieved solely at early phase of culture (&lt; day 1) and lower concentration of IL-2. Collectively, MEGC augments in vivo NK cell activity through up-regulated constitutive expression of cytolytic mediators and data suggest that ginseng may act at early phase of NK cell response.
The co-expression of TLR4 and CD40 homodimer enhance B cell activation (P4447)
Authors: Nadiri Amal , Jundi Malek , Yacoub Daniel , Mourad Walid 
Published: January 2023

Abstract
CD40 is a critical regulator of the immune response and a key player in several autoimmune diseases. Accumulating evidence indicates that CD40 is essential for both innate and adaptive immunity. Moreover, it has been reported that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers, albeit the role of this response in B-cell function remains elusive. Here, we report that oxidative stress induces CD40 self-association through cysteine-238 residues independently of ligand binding. However, our results show that the production of ROS downstream of CD40 is independent of its dimerization. Notably, CD40 homodimers are required for full B-cell activation and TLR4-induced CD23 and CD80 expression. In addition, these responses were dependent upon Jak3 association to CD40 and PI-3 kinase activation. Our results highlight the role of CD40 homodimers in innate and adaptive immunity and may lead to the generation of novel biological targets for the treatment of CD40-assciated inflammatory disorders.
Axl signaling abrogated HBV clearance in a HBV persistence mouse model (P1360)
Authors: Huang Miao-Tzu , Huang Jian-Jhih , Chiang Bor-Luen 
Published: January 2023

Abstract
Persistent HBV infection is a major etiology for chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Whilst most HBV infection in the adult resolves after the acute phase, over ninety percent of HBV infection in neonates and children leads to chronic HBV persistence. By using hydrodynamic injection of the replication-competent HBV plasmid, pAAV-HBV, we established a mouse model of HBV persistence in which a state of HBV persistence can be generated in 4-6 w/o C57BL/6 young mice, but not in adult mice of older than 10 w/o. We found that there is higher hepatic interferon (IFN)-a/b expression in the young mice after pAAV-HBV injection compared to the adult mice. In addition, we found that excessive IFN-a/b expression of the young mice was associated with the induction of the Axl negative regulatory pathway. We hypothesize that IFN-a/b-induced Tyr3-Axl-MerTk (TAM) feedback regulation hampered the generation of effective anti-HBV immunity, which in turn facilitated the establishment of HBV persistence in the young mice. In accordance with this hypothesis, we found that HBV persistence in the adult mice can be increased by augmented Axl expression. IFN-a/b are well-known for their antiviral effects; however, accumulating studies also demonstrated an immune regulatory function. In line with the previous studies, our findings suggest that Axl signaling suppresses the mounting of anti-HBV immunity, possibly due to the feedback regulatory pathway of IFN-a/b.
Differential antigen-dependency of CD4+ and CD8+ T cells (P1338)
Authors: Obst Reinhard , Rabenstein Hannah , Behrendt Anne , Ellwart Joachim , Horsch Marion , Beckers Johannes 
Published: January 2023

Abstract
Though T cell expansion and effector differentiation are triggered and, perhaps, maintained by antigen, the proliferative behaviors of CD4+ and CD8+ T cells responding to timed antigen presentation have rarely been compared side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal T cells of different backgrounds were analyzed following transient and continuous TCR signals. We found CD4+ T cell proliferation to be dependent on prolonged antigen presence, whereas CD8+ T cells were able to divide and differentiate into effector cells in the absence of it. This proliferation of CD8+ T cells is truly autonomous and independent of any self-MHC derived signals. The discontinued proliferation of CD4+ T cells was not caused by coinhibitory signals like or the lack of inflammatory stimuli and also found in memory cells. Gene expression analyses illustrated differences in global gene transcription between the two subsets following stimulation periods of different lenghts. These T cell data reflect the MHC class difference in that class II molecules, unlike class I, were stabilized on activated DCs in vivo.
T-bet supports differentiation of B-effectors into antibody secreting cells (P1316)
Authors: Stone Sara , Mousseau Betty , Lund Frances 
Published: January 2023

Abstract
Memory B cells (Bmem) and long-lived plasma cells (LLPC) arise from germinal center B cells (GCB). The transcription factor Bcl6 is required for GCB cell survival and the development of Bmem. By contrast, Bcl6 inhibits LLPC development by repressing the transcription factor Blimp1. To date, it is not clear how these opposing transcription factors are regulated in GCB cells. In T cells, T-bet modulates the balance between Blimp1 and Bcl6 and controls their subsequent differentiation into memory and effector cells. We recently defined a B-cell subset, referred to as B-effector 1 cells (Be-1 cells), that express high levels of T-bet and secrete IFNγ. We showed that IFNγ or T-bet deficient Be-1 cells inefficiently seed the LLPC compartment, leading us to suspect that the Be-1 cells may be precursors to LLPCs and Bmem. We hypothesized that T-bet expression by Be-1 cells promotes cytokine production and supports their differential development into antibody secreting cells (ASCs). We found that the IFNγ producing CXCR3+ CCR6+ Be-1 cells expressed higher levels of Bcl6 and T-bet but lower levels of Blimp1 and XBP1 compared to the non-Be-1 cells. T-bet-/- Be-1 cells expressed lower levels of Blimp1 and XBP1, but higher levels of Bcl6. Further, T-bet-/- and IFNγ-/- Be-1 cells had attenuated IFNγ production and antibody secretion. These results support a role for T-bet in determining Blimp1 and Bcl6 levels in B-effectors and modulating their development into ASCs rather than Bmem cells.
Cytokine signaling responses differ between FoxP3+ and T-bet+ T cell subsets in both mouse and human (P1331)
Authors: Wu Xiaowei , Boyce Christopher , O'Donnell Erika , Ernst David , Wang Xiao , Gao Guo-Jian 
Published: January 2023

Abstract
Networks of transcription factors (TF) and cytokines are critical for defining the lineage and function of T cell subsets. The transcription factors FoxP3 and Stat5 play key roles in regulatory T cell differentiation and function, while T-bet, Stat1, and Stat4 are important for T helper 1 (Th1) cells. While assessments of differential cytokine-induced STAT activation in FoxP3+ and T-bet+ T cell subsets are valuable, disparate buffer compatibility characteristics create challenges for these analyses. To enable robust, simultaneous analysis of TF expression and phosphorylation in mouse and human samples, a new experimental buffer system was developed. The buffer allowed successful analysis of phospho-signaling responses to several stimuli in T cell subsets defined by surface CD markers and TF. These studies revealed that FoxP3+ and T-bet+ T cell subsets exhibited similar signaling responses to some cytokines, while responding quite differently to others. For instance, FoxP3+ subsets activated Stat5 more robustly in response to IL-2, while FoxP3- T-bet+ T cells activated Stat4 more robustly in response to IFN-a. Interestingly, the small subset of CD4 T cells co-expressing T-bet and FoxP3 shared many signaling responses with FoxP3+ T-bet- CD4 T cells. The simultaneous analysis of surface markers, TF, and individual phosphorylated signaling proteins provides a powerful tool for the comparison of signaling responses in rare cell subsets.