bluuebunny/crossref_metadata_2025 huggingface dataset:
IL21 is a critical regulator of chronic/persistent auto-immune and allo-immune responses (P5162)
Authors: Khattar Mithun , Schroder Paul , Baum Caitlin , Deng Ronghai , Chen Wenhao , Stepkowski Stanislaw
Published: January 2023

IL21 is critical for T1D development in NOD mice, but its role in sustaining T cell responses against islets during T1D progression remains unexplored. Our studies show that diabetic NOD mice have high levels of serum IL21. We thus hypothesized that IL21 produced by activated CD4 T cells in spontaneously diabetic NOD mice sustains T cell responses to islets. Indeed, IL21 is required for maintaining the function of auto-reactive immune cells during T1D progression as transient therapy with IL21 receptor fusion protein (IL21RFc) was effective in reversing the onset of T1D in NOD mice (n=11) and inducing long-term T1D remission for up to 20 weeks in at least 3 mice. Pancreatic infiltrates of IL21RFc-treated NOD mice contain mostly CD4 T cells that lost surface ICOS expression while containing fewer CD8 T cells, suggesting that IL21RFc inhibited CD4 T cell help to CD8 T cells. In a parallel study, we have shown that IL21 deficient mice were protected from chronic allograft vasculopathy in a MHC class-II mismatched cardiac allograft model. However, IL21 deficient mice acutely rejected fully mismatched cardiac allografts similar to wild type. Thus, we have demonstrated a novel and critical role for IL21 signaling in driving chronic immune responses against auto and allo-antigens. Based on our findings, IL21 has the potential to act as a serum biomarker for early T1D detection and a therapeutic target to abrogate persistent T cell responses in T1D and chronic allograft rejection.
Evaluation of SOCS1 mimetic and antagonist peptides as therapeutic agents (P5152)
Authors: Bedoya Simone , Young Ashley , Wilson Tenisha , Johnson Howard , Larkin III Joseph
Published: January 2023

The intracellular protein Suppressors of Cytokine Signaling1 (SOCS1) plays a critical role in moderating inflammatory cytokine signaling, thereby limiting immune responsiveness. We have shown that a peptide with the kinase inhibitory region of SOCS1 (SOCS1-KIR) inhibits Experimental Autoimmune Encephalomyelitis and rescues SOCS1-deficient mice from perinatal lethality. In contrast, a SOCS1antagonist pJAK2 enhances antiviral immunity to poxviruses. Therefore it is likely that peptides that modulate SOCS1 signaling possess the potential to modulate immune responses, and restore immune homeostasis. To evaluate the translational potential of SOCS1 mimetic peptides, we have assessed their pharmacodynamics by fluorescently labeling the peptides followed by injection into C57BL/6 mice. After 2 hours mice receiving Alexa 647 fluorochrome-conjugated SOCS1-KIR or pJAK2 were sacrificed, and subsequently whole organs and tissue histology were examined for the presence of the labeled peptides. We observed that pJAK2 and SOCS1-KIR were present in the brain, liver, lymph nodes, spleen, kidneys, lungs, and peritoneal cells. Notably, pJAK2 was selectively present within the thymus and heart. On a cellular level, SOCS1-KIR and pJAK2 were differentially present within CD4+ T cells, CD8+ T cells, CD11b+ macrophages, and B cells. These results help to lay the groundwork for the translational use of peptides that modulate SOCS signaling to regulate and/or maintain immune system homeostasis.
Genome-wide DNA methylation study suggests epigenetic accessibility and transcriptional poising of interferon-regulated genes in naïve CD4+ T cells from lupus patients (P4539)
Authors: Sawalha Amr , Jeffries Matlock , Altorok Nezam , Dozmorov Mikhail , Koelsch Kristi , Wren Jonathan , Merrill Joan , McCune W. , Coit Patrick
Published: January 2023

Abnormal T cell DNA methylation and type-1 interferon play an important role in the pathogenesis of systemic lupus erythematosus. We performed a genome-wide DNA methylation study in two independent sets of lupus patients and matched healthy controls to characterize the DNA methylome in naïve CD4+ T cells in lupus. DNA methylation was quantified for over 485,000 methylation sites across the genome, and differentially methylated sites between lupus patients and controls were identified and then independently replicated. Gene expression analysis was also performed from the same cells to investigate the relationship between the DNA methylation changes observed and mRNA expression levels. We identified and replicated 86 differentially methylated CG sites between patients and controls in 47 genes. Importantly, we observed significant hypomethylation in interferon-regulated genes in naïve T cells from lupus patients, suggesting epigenetic transcriptional accessibility of these genetic loci. The hypomethylation in interferon-regulated genes was not related to lupus disease activity. Gene expression analysis showed overexpression of these genes in total but not naïve CD4+ T cells from lupus patients. Our data suggest epigenetic “poising” of interferon-regulated genes in lupus naïve CD4+ T cells, argue for a novel pathogenic implication for abnormal T cell DNA methylation in lupus, and suggest a mechanism for type-1 interferon hyper-responsiveness in lupus T cells.
CD8+ T cells in a normal host limit autoimmune attack by CD-8+ effector T cells (P4541)
Authors: Girtman Megan , Pavelko Kevin , Pease Larry
Published: January 2023

We have developed a mouse model of diabetes by adoptively transferring small numbers of OT1 cells into animals expressing OVA under the rat insulin promoter (RIP-OVA). These cells are activated in vivo with a Picornavirus (TMEV) expressing OVA antigen. Titrating the dose of adoptively transferred cells, changes disease outcome. Mice receiving low amounts of cells develop hyperglycemia, and recover homeostasis. Investigation of recovered animals revealed normal blood glucose regulation. Pancreatic histology of recovered mice showed extensive pancreatic destruction with no or very few remaining insulin-positive islets. While adoptively transferred OT1 cells were present, pancreas infiltrating T cells were largely endogenous. To evaluate the functional importance of the host T cells, we tested disease progression using RAG KO, CD4 KO, and CD8 KO RIP-OVA hosts. Without RAG, CD4, or CD8 we observed increased disease severity. This change is not attributed to homeostatic proliferation in the CD4 and CD8 KO animals, as adoptively transferred OT1 cells behaved similarly in the KO hosts in comparison to RIP-OVA mice. However there is significant homeostatic proliferation occurring in the RIP-OVA RAG KO animals. Our studies reveal the importance of CD4 and CD8 T cells in limiting autoimmune attack in the pancreas. Although populations of CD4 T regulatory cells are well known, the importance of CD8 T cells in limiting inflammation are less well characterized.
The nuclear receptor PPARγ regulates T cell tolerance and germinal center formation via generation of follicular helper T cells (P4545)
Authors: Choi Je-Min , Park Hong-Jai , Choi Jin-Young , Senejani Alireza , Tobiasova Zuzana , Kim Do-Hyun , Bothwell Alfred
Published: January 2023

Peroxisome proliferator-activated receptor gamma (PPARγ) acts as a transcription factor to regulate lipid and glucose metabolism. An emerging role of PPARγ has been suggested that it has important regulatory functions in inflammation and adaptive immunity. However, the intrinsic role of PPARγ in T cells is not fully understood. Here we used CD4-PPARγKO mice to understand the function of PPARγ in T cells. PPARγ deficient T cells produced higher levels of cytokines including IFN-γ, IL-17, or IL-2, and proliferated more than wild type T cells following TcR stimulation with increased phosphorylation of ERK and AKT. Interestingly, PPARγ deficient T cells were prone to differentiate into various helper T cell subsets such as Th1, Th2, Th17, and Th9 cells. Furthermore, aged mice with PPARγ deficient T cells showed increased numbers of follicular helper T cells (TFH cells) and spontaneously develop a moderate autoimmune phenotype with kidney inflammation, enhanced autoantibody production and increased numbers of germinal centers. The abrogation of T cell tolerance was further confirmed with the diminished the expression of IkBα, Sirt1, and Foxo1 to inhibit NF-κB. These results suggest that PPARγ is crucial for maintaining T cell tolerance and germinal center formation and TFH generation to prevent autoimmunity.
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

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

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

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

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

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

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

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

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

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

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 (< 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

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

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

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

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

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.
Intra-epithelial T lymphocytes from the cervical-vaginal mucosa of healthy women contain a majority population of CD4+ cells and express high levels of the HIV-1 co-receptor CCR5. (P3341)
Authors: Swaims Alison , Haaland Richard , Lupo Davis , Evans-Strickfaden Tammy , Kohlmeier Jacob , Haddad Lisa , Hart Clyde
Published: January 2023

Current HIV sexual acquisition models for women posit that cell-free virus is carried into genital tissues by antigen presenting cells in the mucosa and that virus also penetrates the mucosa through microabrasions. The potential for genital mucosal intra-epithelial lymphocytes (IELs) to act as primary targets for HIV acquisition in women has not been well-characterized due to the difficulty of isolating and characterizing these cells free of blood contamination. We developed a method to enhance recovery of IELs from the lower genital tract mucosa of women using an atraumatic cervical-vaginal lavage procedure. This procedure was effective for collecting IELs with a T cell resident memory (T¬RM) phenotype from 10 healthy women without the blood contamination that often occurs using cytobrush or vaginal biopsy methods. We detected a high frequency of mucosal CD103+ expression on the CD8+ TRM cells, confirming their identity as IEL cells. Conversely, little to no CD103 expression was detected on CD4+ TRM cells. Moreover, CD4+ cells comprised up to 80% of the T cell population collected from CVL compared to CD8+ T cells (10-20%), with greater CCR5 expression compared to T cells in the peripheral blood. These results show that our atraumatic isolation method is effective for characterizing IELs in the female genital mucosa and that the abundance of CD4+ CCR5hi TRM cells in this population may likely be a highly vulnerable target for HIV-1 sexual transmission.
TLR4-based IL-8 reporter cell line as a tool for screening of the LPS-mediated immunomodulators to control TLR4 signaling pathway. (P3305)
Authors: Lee Hyun-Ku , Luo Yi Feng , Chitta Sriram , Singh Gita , Rosenberg Jonathan , Singh Sujay
Published: January 2023

The lipopolysaccharide (LPS) receptor complex of TLR4, MD-2 and CD14 plays an important role in sensing LPS-mediated immune signals, which provide immediate host protection and stimulate the host immune responses. Despite the fact that TLR4-mediated innate immune signals are required to combat invading pathogens, when signaling is not well regulated, uncontrolled activation can result in the disruption of host homeostasis leading to chronic inflammation and septic shock. Hence it is crucial to develop an assay system that can identify immunomodulators to control TLR4 signaling pathway. Since IL-8 is a major cytokine induced by TLR4, we developed the reporter cell line which stably expresses TLR4, MD-2 and CD14 proteins as well as an optimized Renilla luciferase reporter gene under the transcriptional control of IL-8 promoter. Functional activity of the cell line was evaluated by LPS dose response, TLR4 antagonist test and IL-8 induction suppressor test. The LPS EC50 of 4.3 ng/ml, TLR4 inhibitory peptide IC50 of 14.85 uM and an IL-8 induction suppressor, curcumin IC50 of 15.29 uM against the cell line were assessed. Using this cell line, a series of natural compounds was successfully screened for TLR4 signaling modulators, among which several compounds were identified as potential TLR4 signaling modulators. Taken together, it is proposed that this reporter cell line can be used as a tool for analyzing TLR4 signaling pathway as well as screening of TLR4 signaling modulators.
Development of a conditional NIK knockout mouse (P3293)
Authors: Brightbill Hans , Jackman Janet , Wu Lawren
Published: January 2023

NF-κB Inducing Kinase (NIK) is a kinase that is critical for the induction of non-canonical NF-κB signaling downstream of BAFF, CD40L, lymphotoxin, and RANK. Mice that are deficient in NIK or that have the ALY mutation that disrupts NIK function display defects in secondary lymphoid organ development, B cell populations and responses, and other immune cell phenotypes. We have developed a conditional allele of NIK and have utilized it to study the effects of NIK deletion in adult mice upon tamoxifen-induced deletion. Our conditional NIK KO mice may have broad utility for the study of the effects of NIK deficiency in adult mice and in specific cell types and tissues.
Preservation of allogeneic aortic grafts in anhydric sodium chloride attenuates host immune reaction (P2227)
Authors: Gewartowska Magdalena , Fronczak-Baniewicz Malgorzata , Buyanovskaya Olga , Moscicka-Wesolowska Maria , Maksymowicz Michal , Olszewski Waldemar
Published: January 2023

In case of infection of artificial arterial grafts, replacement by allografts becomes a method of choice. Unfortunately, allografts evoke strong immune reaction. For this reason, a method for successful long-term preservation of arterial allografts with unchanged morphology and low antigenicity is needed. We preserved the fragments of rat or pig aorta in anhydric NaCl and stored them in 4oC for up to 24 months, and subsequently transplanted them into rat or pig recipients for up to 24 or 6 months respectively. Routine hematoxyline/eosine and trichrome azan stainings, as well as electron microscopic studies showed the preserved anatomical structure of transplanted aortae and revealed normal structure of elastin fibers, the presence of fibroblasts between elastin bundles and single endothelial-like cells. No significant differences between preserved transplanted and non-preserved aortae stained for CD31, CD54, RECA-1 were observed. We also observed only slight infiltration of ED1 (CD 68), OX6 (MHC clas II) and W3/13 (CD43) positive cells around the allografts and this immunological reaction was only weakly expressed. No regional lymph nodes reaction was noticed. These observations suggest that preservation in anhydric NaCl may serve as a novel method to obtain low immunogenicity arterial allografts.
Correlations between cytokines, depression and anxiety in patients with colorectal cancer (P2000)
Authors: Miranda Diego , Azevedo Lucas , de LIma Tais , Veronez Luciana , Feres Omar , da Rocha José , Bisson Gabriela
Published: January 2023

Colorectal cancer (CRC) is a leading cause of cancer mortality in the world. Depression and anxiety are the most prevalent psychological disorders in patients with CRC. This study aims to investigate whether there is a correlation between anxiety, depression and serum cytokines in CRC patients. The study group consisted of 19 patients hospitalized for surgical resection of CRC and 20 healthy volunteers comprised the control group. Depression and anxiety were analyzed using the Hospital Anxiety and Depression Scale and serum levels of IL-1, IL-6, IL-12, TNF-α, TGF-β and IL-10 were measured by ELISA. We found that among CRC patients, 47.4% and 52.6% manifested clinically significant levels of anxiety and depression, respectively and 57.9% manifested a combination of severe anxiety and depression. Furthermore, these patients had increased serum levels of IL-1, IL-6 and TNF-α, but lower concentrations of IL-12. Analysis of correlation between HADS scores and serum levels of cytokines in CRC patients revealed a positive association of anxiety and/or depression with IL-1, IL-6 and TNF-α and a negative correlation with IL-12. These initial results indicate a correlation between serum levels of pro-inflammatory cytokines, anxiety and depression in CRC patients and suggest that cytokines are involved in the pathophysiology of these comorbidities in this sample.
Tumor-expressing CCR6 plays a critical role in human colorectal cancer metastasis (P2018)
Authors: Wang Honglin
Published: January 2023

The chemokine receptor CCR6 has been recently shown to be closely associated with liver metastases of colorectal cancer (CRC). However, the direct evidence for whether tumor-expressing CCR6 is a prognostic marker for the survival of CRC patients, and whether it plays a critical role in CRC progression in vivo is lacking. Here we found that levels of CCR6 were markedly increased in CRC cell lines, particularly un-regulated in metastatic cell lines and primary CRC clinical samples. Up-regulated CCR6 expression by CRC cells predicted poor survival in patients. Overexpression of CCR6 in HCT116 cells increased their proliferation, migration, and colony formation activity in vitro and promoted metastatic potential in vivo. We further demonstrated that CCR6 activated Akt signaling, up-regulated metastasis genes and down-regulated metastasis suppressor genes. Finally, we showed that selective targeting tumor-expressing CCR6 dramatically inhibited the growth of CRC in mice. Thus, our data indicate that CCR6 could be used as a novel prognostic marker for the survival of CRC patients, and targeting tumor-expressing CCR6 may represent a promising therapeutic strategy for CRC.
High-mobility group box 1 promotes NK and DC function as well as crosstalk: studies of selective knockout in immune cells (P2008)
Authors: Lotze Michael , Vernon Philip , Li Guanqiao , Liang Xiaoyan
Published: January 2023

High-mobility group box 1 (HMGB1) is an evolutionarily ancient, highly abundant nuclear protein present in all eukaryotes that has distinct compartmental roles in the nucleus, in the cytosol, and within the extracellular space. Within the nucleus it regulates VDJ recombination in B and T cells as well as promotes access to nuclear hormone/nuclear hormone receptors and p53 and p73 transcriptional complexes. Within the cytosol it promotes and sustains autophagy by displacing Beclin 1 from BCL2 and is critical for mitochondrial quality control. When secreted or released by stressed or necrotic cells, HMGB1 serves as the prototypic Damage Associated Molecular Pattern Molecule (DAMP). We created homozygous floxed HMGB1 mice which deleted exons 2 and 3 in back crossed NKp46-cre or CD11c-cre mice to allow specific deletion in NK or cDC respectively. Loss of HMGB1 in NK cells (NKH-) was associated with diminished number in the spleen, reduced proliferative capacity in response to IL-2 and enhanced tumor growth in a liver metastases model. Conversely loss of HMGB1 in cDC was associated with marked decrease in maturation in response to LPS or CD40 ligation but diminished tumor growth in the same liver model. Markedly decreased maturation of DC by NKH- with decreased CD80, CD86, Class II and CD83 was noted. These studies convincingly demonstrate the critical and central role of HMGB1 in immune regulation.
Differential migration of myeloid-derived suppressor cell subsets to tumor-draining lymph nodes in a murine model of breast cancer (P2014)
Authors: Matson Vyara , Loppnow Jessica , Steeber Douglas
Published: January 2023

Myeloid-derived suppressor cells (MDSC) expand in cancer and suppress the anti-tumor immune response. MDSC are morphologically and functionally divided into monocytic (MO-MDSC) and polymorphonuclear (PMN-MDSC) subsets, identified as CD11b+Ly-6G-Ly-6Chigh and CD11b+Ly-6G+Ly-6Clow, respectively. MDSC accumulate primarily in the blood and spleen, but their suppressive function is limited to the tumor and tumor-draining lymph nodes (TDLN). We sought to determine the involvement of adhesion molecules in MDSC migration to TDLN using the 4T1 murine breast cancer model. Both MDSC subsets expressed L-selectin and LFA-1, although L-selectin levels were higher on MO-MDSC. To determine whether this differential L-selectin expression affected MDSC migration to TDLN, we analyzed tissue distribution and in vivo migration of wild type and L-selectin-deficient MDSC. In the blood and spleen of wild type mice, the PMN-MDSC subset predominated (MO-MDSC/PMN-MDSC ratio of 0.1), while the MO-MDSC subset predominated in the TDLN (ratio of 1.5). By contrast, in L-selectin-deficient mice, the MO-MDSC/PMN-MDSC ratios in the TDLN (0.3) and in the blood and spleen (0.1) were similar. This differential tissue distribution was consistent with increased TDLN entry of wild type, but not L-selectin-deficient, MO-MDSC relative to PMN-MDSC in adoptive transfer experiments. Taken together, these results suggest a migratory preference of MO-MDSC to the TDLN dependent on L-selectin.
Cytokine response and morphologic characterization of chicken bone-marrow derived dendritic cells during infection with high and low pathogenic avian influenza viruses. (P6355)
Authors: Kapczynski Darrell , Jiang Hai Jun
Published: January 2023

Dendritic cells are professional antigen-presenting cells of the immune system that function to initiate primary immune responses. They are specialized at capturing and processing antigen for immune presentation. Infection of chickens with avian influenza can result in a wide range of clinical disease, depending if the virus is of low or high pathogenicity. Low pathogenic avian influenza viruses typically induce mild clinical signs, whereas highly pathogenic avian influenza can result in death within days. In these studies, chicken dendritic cells were co-cultured with high and low pathogenic avian influenza viruses of H5 and H7 subtypes to determine the innate immune response, effect on cell morphology, and examine viral growth. A strong proinflammatory response, including interleukin-1β, and stimulation of the interferon response pathway was observed at 8 hours post infection. Microscopically, the dendritic cells altered their appearance after influenza infection to a rounded shape, and eventually died, most likely through apoptosis. Differences in onset of morphologic changes were observed between H5 and H7 influenza subtypes. Increases in viral titers demonstrated that both pathotypes of influenza could replicate in dendritic cells. The increased activation following infection may be indicative of dysregulation of the immune response typically seen with high pathogenic avian influenza infection.
Co-infection with Streptococcus pneumoniae and influenza A virus leads to increased disease and a decrease in the size and functional capacity of the influenza specific CD8+ T cell response (P6363)
Authors: Blevins Lance , Wren Jack , Holbrook Beth , Johnson John , Pang Bing , Swords Edward , Parks Griffith , Alexander-Miller Martha
Published: January 2023

Most of our understanding of influenza A virus and its interaction with the adaptive immune response has been generated in the context of infection with a single pathogen. It is clear however that infection with influenza virus can lead to increased susceptibility to subsequent infection, often Streptococcus pneumoniae. The potential for a second infection to regulate the ongoing anti-influenza response is not clear. To address this important question we have developed an in vivo co-infection model to test the effects of pneumococcus on the ongoing CD8+ influenza specific T cell response. We show that co-infection with A/PR 8/34 (H1N1) and the non-invasive EF3030 strain of S. pneumoniae leads to increased disease. Within the lungs of the co-infected animals we observe a significant decrease in the size of the flu specific CD8+ T cell response. This decrease is not present in the lung draining lymph nodes, indicating selective regulation at the site of bacterial/viral co-infection. In addition to the decrease in size, the effector function of these CD8+ T cells is qualitatively diminished as measured by their ability to co-produce interferon γ and tissue necrosis factor α. These results demonstrate a dynamic interplay between the two concurrent pathogens and the immune system and emphasize the importance of increasing our understanding of how polymicrobial infections modulate the immune response to individual pathogens.