bluuebunny/crossref_metadata_2025_split huggingface dataset:
The use of zebrafish as a model system for development of new adjuvant strategies in finfish DNA vaccination against viruses (P6072)
Authors: Arnemo Marianne , Kavaliausikis Arturas , Esteba Amparo , Sonia Dios , Beatriz Novoa , Gjøen Tor 
Published: January 2023

Abstract
Zebrafish (Danio rerio) has emerged as an important experimental system to study both vertebrate immunity and as a model system for fish immunology. Studies in this species have already revealed important knowledge about fish-pathogen interactions and fish vaccinology. Although more than 18 different Toll-like receptors (TLRs) have been genomically identified in fish, little is known about their agonists and functional role in fish immunity. We have established a NFkB-luciferase reporter system to analyse agonist profiles for several fish TLRs. In addition, after adaption of zebrafish to 15°C the protective effects of various TLR ligands alone and as adjuvants for DNA vaccination against surface proteins from cold water pathogens like viral hemorrhagic septicemia virus (VHSV) have been investigated. We also investigated the activation of zebrafish leucocytes by TLR ligands and recombinant G protein from VHSV (VHSV-G) in vitro. Our results demonstrate that poly I:C (TLR3 agonist) induce inflammatory transcripts in vitro. In vivo, one injection of poly I:C, could partially protect zebrafish against subsequent VHSV infection. The onset of mortalities was delayed by ten days (day 20 compared to day 10 in control) in the poly I:C treated fish. We will also present interesting results showing the effects of formulating plasmid DNA vaccines against VHSV-G with various TLR agonists included as adjuvants (on-going studies).
Investigating regulatory T cell responses to MAP antigen stimulation using a monocyte-derived macrophage antigen presentation system (P6078)
Authors: Roussey Jonathan , Coussens Paul , Coussens Merrick 
Published: January 2023

Abstract
Johne’s disease is a chronic wasting disease of wild and domestic ruminants caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Previously, we proposed that the chronic nature of Johne’s disease may result in development of a regulatory T cell (Treg) population in the host that functions to shift the immune balance away from a pro-inflammatory Th1-like response to an ineffective Th2-like immune response. However, studying the function and specificity of Tregs is difficult due to low abundance of Tregs in the periphery. Furthering this obstacle, Tregs are usually anergic and the proportion of Tregs that develop in response to any one infectious agent is small. To face these challenges, we developed an in vitro method using a combination of TGF-β, interleukin 2, and rapamycin to stimulate expansion of MAP-reactive Tregs from CD4+CD25+ peripheral blood mononuclear cells in contact with MAP-infected monocyte-derived macrophages. Enriched Tregs may subsequently be used in Treg-mediated suppression assays to explore differences in naïve T cell responses to MAP antigen stimulation, including induction of FoxP3 expression. Following PBMC stimulation, qPCR and flow cytometry are used to analyze expression of FoxP3 as well as various relevant cytokines including: IL-1, IFNγ, IL-10, and TGF-β. Results are compared between cattle that are test-negative and test-positive for Johne’s disease.
M1 monocyte induction from advanced HCC-derived monocytes by CCL1 antisense oligodeoxynucleotides (P2129)
Authors: Tsuchimoto Yusuke , Asai Akira , Ohama Hideko , Fukunishi Shinya , Tsuda Yasuhiro , Fukuda Akira , Kobayashi Makiko , Higuchi Kazuhide , Suzuki Fujio 
Published: January 2023

Abstract
We have reported that patients with advanced stages of HCC were shown to be carriers of M2b monocytes and these monocytes did not have a tumorcidal activity. In this study, we tried to induce M1 monocytes from M2b monocytes in advanced HCC patients. Five advanced HCC patients admitted to Osaka Medical Collage were enrolled. CD14+monocytes were isolated from peripheral blood from patients by Ficoll-Hyperque sedimentation and magnetic beads. These monocytes (1 x 106 cells/ml) were cultured for 48 hrs with 6 μg/ml of CCL1 antisense oligodeoxynucleotides (ODN). Cells harvested were re-cultured with fresh medium supplemented with CpG DNA (10 μg/ml) for 24 hrs and Culture fluids were assayed for IL-12. These cells were co-cultured with syngeneic HCC cells (2 x 10 6 cells/ml) for 48 hrs. In the results, M2b monocytes treated with CCL1 antisense ODN were produced IL-12 after stimulation with CpG DNA. And these cells have a tumorcidal activity against syngeneic HCC cells. These results indicate that M2b monocytes treated with CCL1 antisense ODN were converted to M1 monocytes after stimulation with CpG DNA. And M2b monocytes detected in advanced HCC patients play a role of the suppressing host antitumor surveillance. M2b monocytes/Mϕ may be intervened by the elimination of CCL1, a product of M2b monocytes themselves.
Prolonged survival in mice with advanced tumors treated with intra-bone marrow-BMT plus fetal thymus transplantation with restoration of T cell functions (P2177)
Authors: Hosaka Naoki 
Published: January 2023

Abstract
Thymic function decreases in line with tumor progression in patients with cancer, resulting in immunodeficiency and a poor prognosis. In the present study, we attempted to restore thymic function by BALB/c (H-2d) syngeneic (Syn) or B6 (H-2b) allogeneic (Allo) bone marrow transplantation (BMT) using intra-bone marrow-bone marrow transplantation (IBM-BMT) plus Syn-, Allo- or C3H (H-2k) 3rd-party fetal thymus transplantation (TT). Although the BALB/c mice with advanced tumors (Meth-A sarcoma; H-2d, &gt;4 cm2) treated with either Syn- or Allo-BMT alone showed a slight improvement in survival compared with non-treated controls, the mice treated with BMT + TT showed a longer survival. The mice treated with Allo-BMT + Allo-TT or 3rd-party TT showed the longest survival. Interestingly, although there was no difference in main tumor size among the BMT groups, lung metastasis was significantly inhibited by Allo-BMT + Allo-TT or 3rd-party TT. Although host thymus was still atrophied, transplanted thymus was well-engrafted. T cell functions increased significantly, whereas number of myeloid suppressor cells and regulatory T cells significantly decreased in these mice. These results suggest that BMT + TT, particularly Allo-BMT + Allo-TT or 3rd-party TT, is most effective in prolonging survival as a result of the restoration of T cell function in hosts with advanced tumors.
Targeting antigen and adjuvants to DC-SIGN+ skin dendritic cells with glycosylated liposomes in immunotherapy against cancer (P2101)
Authors: Boks Martine , van Beelen Astrid , Fehres Cynthia , Bruijns Sven , Ambrosini Martino , Kalay Hakan , Unger Wendy , van Kooyk Yvette 
Published: January 2023

Abstract
Cancer immunotherapy requires potent tumor-specific T cell responses, initiated by dendritic cells (DCs). Our aim is to develop a therapy that specifically targets DCs in vivo in human skin using glycosylated liposomes that contain tumor antigen and Toll-like receptor (TLR) ligand. The use of glycans specific for certain C-type-lectin receptors (CLRs) allows directing of the liposomes to specific DC subsets. Targeting the CLR DC-SIGN, which is a potent uptake receptor specifically expressed on human dermal DCs, by using Lewis (Le)X-modified liposomes, resulted in increased internalization of liposomes by human monocyte-derived (mo)DCs. Targeting moDCs with Melanoma antigen MART-1-encapsulated liposomes modified with LeX led to enhanced antigen presentation to CD8+ T cells. For good CD8+ T cell responses, maturation of DCs with LPS was necessary. Incorporation of TLR ligands MPLA, Poly I:C, R848, R837 or Pam3CSK4 into LeX-modified liposomes resulted in maturation of moDCs. Moreover, injection of TLR ligand-glycoliposomes in human skin led to their enhanced uptake by skin DCs and DC maturation. The enhanced uptake of glycoliposomes by skin DCs resulted in enhanced antigen presentation to CD8+ T cells. Our data demonstrate the potency of glycoliposomes as anti-cancer vaccine targeting DC-SIGN+ skin DCs in vivo and inducing tumor-specific T cell responses.
CD8+ T cells are not sufficient for induction of tolerance in a murine model of ovalbumin-induced allergic airway diease (P3209)
Authors: Natarajan Prabitha , Guernsey Linda , Thrall Roger , McNamara Jefferey , Shah Sonali , Adami Alexander , Szczepanek Steven , Secor Eric 
Published: January 2023

Abstract
In our biphasic ovalbumin (OVA)-induced murine model of allergic airway disease (AAD), short-term daily OVA aerosol exposure for 7 to 14 days results in AAD whereas continued exposure for 42 days leads to resolution of disease and local inhalational tolerance (LIT). We have previously demonstrated that OVA-specific CD8+ T cells exhibit plasticity from a pro-inflammatory phenotype at AAD (increased granzyme B and IFN γ) to a suppressive phenotype (NKG2A+, decreased granzyme B and IFN γ) at LIT. Since the role of CD8+ T cells in asthma remains unclear we used our biphasic model to determine whether CD8+ T cells can induce suppression of AAD. We isolated CD8+ T cells from HLN of LIT mice and adoptively transferred them into sensitized mice and challenged with OVA aerosols to induce AAD. We observed no differences in lung histology and total eosinophils in bronchoalveolar lavage of mice that were transferred with CD8+ T cells as compared to mice transferred with naïve T cells (n=5, p &gt; 0.05). We further investigated localization of OVA-specific CD8+ T cells in HLN (confocal microscopy) and showed that these cells do not localize in follicular regions of HLN at LIT unlike what we have previously described with the co-localization of Foxp3+ Tregs and Bregs. Thus, we have demonstrated that CD8+ T cells are not sufficient for suppression of AAD and do not preferentially localize in follicles of HLN at LIT.
Respiratory airway memory cells pre-arm the immune system against viral reinfection (P3215)
Authors: Wilson Jarad , McMaster Sean , Wang Hong , Kohlmeier Jacob 
Published: January 2023

Abstract
Vaccines have long served as a boon for the general population, yet recent inadequacies and global outbreaks of novel subtypes of influenza have given pause to the vaccine community, highlighting the need for new vaccine strategies. Several studies have demonstrated the protective capacity afforded by cellular immunity within the respiratory airway, but exactly how these antiviral T cells mediate protective immunity, and through what pathways these cells utilize to arrive in the airways, remains unexplored. Using our intratracheal transfer approach, we have confirmed the protective capacity of airway resident memory T cells (Tarm). Upon direct ex vivo stimulation, Tarm cells rapidly expressed a unique milieu of inflammatory molecules, including CXCL9 and 10, and CCL3 and 4; molecules not produced by non-respiratory localized antiviral T cells. Along with the production of these inflammatory molecules, we also noted an increased and rapid acquisition of IFN-γ and TNF-α, molecules with both inflammatory and antiviral properties. How viral load and/or immunopathology is moderated by the increased inflammatory molecules and how the unique inflammatory milieu may alter ensuing cellularity changes in the lung microenvironment is a focus of ongoing studies. New studies to determine how memory T cell responses actually control subsequent viral rechallenge will provide crucial insight and correlates of protection when evaluating future vaccine efficacy.
mTOR-dependent regulation of mucosal CD8 T cell immunity (P3221)
Authors: Sowell Ryan , Rogozinska Magdalena , Nelson Christine , Vezys Vaiva , Marzo Amanda 
Published: January 2023

Abstract
Mucosal tissues are sites of frequent pathogen exposure and major routes for the transmission of infectious diseases. Migration of virus-specific memory CD8 T cells into mucosal tissues such as the small intestine and vaginal mucosa is highly restricted to non-circulating tissue resident memory CD8 T cells. Resident mucosal memory CD8 T cells are sufficient to protect against the establishment of mucosa-acquired viral infections, and thus it is essential to develop vaccine strategies that elicit long-lived mucosal CD8 T cells. CD8 T cells residing in mucosal tissues are phenotypically distinct from those in secondary lymphoid tissues and factors critical for their formation are poorly defined. It is also unclear whether mucosal CD8 T cells possess a unique requirement for their generation and maintenance from that of circulating memory CD8 T cells. We report that unlike in the secondary lymphoid tissues, the formation of virus-specific memory CD8 T cells in the small intestine and vaginal mucosa is dependent on mTOR signaling. In addition, we demonstrate using a CD8 T cell mediated model of intestinal autoimmune disease that inhibiting mTOR results in a loss of CD8 T cells in the small intestine and protects mice from lethality. Furthermore, we will discuss data that targets mTOR signaling intrinsically within the CD8 T cells to provide mechanistic insight into how the mTOR pathway specifically controls the generation of mucosal CD8 T cells in response to viral infections.
Persistent exposure to endogenous LPS programs colonic lamina propria dendritic cells to impair CD4+ Th17 immunity in vivo (P3225)
Authors: Janelsins Brian , Valdez Patricia , Munford Robert , Lu Mingfang , Datta Sandip 
Published: January 2023

Abstract
Gram-negative bacteria drive CD4+ T helper type-17 (Th17) immunity via lipopolysaccharide (LPS) activation of Toll-like receptor (TLR)4. However, excessive TLR4 stimulation can lead to a period of LPS tolerance, during which antigen-presenting cells (APCs) become non-responsive to LPS. Acyloxyacyl hydrolase (AOAH) is a host enzyme mainly expressed by APCs that inactivates LPS, preventing LPS tolerance. Since the effects of AOAH function on T cell immunity remains unexplored, we sought to investigate whether AOAH deficiency modulates the ability of dendritic cells (DCs) to generate CD4+ T cell immunity. We demonstrate that DCs isolated from the colonic lamina propria (cLP), specifically the CD103+CD11b+ subset, exhibited high functional levels of AOAH and preferentially polarized Ag-specific Th17 immunity. The absence of TLR4, or the depletion of gram-negative flora, diminished the ability of cLP DCs to express AOAH and promote Ag-specific Th17 immunity. AOAH-deficient cLP DCs reduced the generation of Ag-specific Th17 cells ex vivo and in vivo, while increasing CD4+ T regulatory cell production. These data demonstrate that AOAH deficiency, which increases exposure to LPS, conditions cLP DCs to impair Ag-specific Th17 immunity. Furthermore, these results show that host pathways to handle microflora-derived products can influence T cell polarization, and suggest that these pathways may be involved in susceptibility to infectious and immune-mediated diseases.
Memory γδ T cells are activated by APCs in the mLN following oral Listeria monocytogenes infection (P3189)
Authors: Romagnoli Pablo , Sheridan Brian , Dickinson Mary , Pham Quynh-Mai , Lefrançois Leo 
Published: January 2023

Abstract
Infection with bacterial pathogens can disrupt the intestinal mucosa that maintains a healthy immune system while keeping bacteria at bay. Using a model of oral infection with a murinized form of Listeria monocytogenes (Lm), our lab has identified a γδ T cell population that forms bona-fide memory and provides protection from re-infection. Since the precise mechanism of activation of most γδ T cells has remained elusive, we investigated the requirements for antigen presenting cells (APCs) in the activation of Lm-induced memory γδ T cells. Here, we show that several cells in the mesenteric lymph node (mLN), including dendritic cells (DCs), inflammatory monocytes and granulocytes, can act in conjunction to activate γδ T cells to express IL-17A, IFN-γ or both and induced their proliferation. This activation was largely cell contact dependent, but was augmented by soluble factors at the peak of primary and secondary γδ T cell responses, mainly by IL-1β but partially by IL-2 and IL-23. Moreover, using confocal microscopy we were able to image Lm-induced γδ T cells interacting with DCs and inflammatory monocytes in the interfollicular area of the mLNs soon after Lm challenge infection. We believe these results not only advance the understanding of γδ T cell biology, but also provide novel strategies to design protective vaccines for the intestinal mucosa.
Role for miR-223 and Roquin in IL-10 mediated regulation of IL-17 (P3193)
Authors: Schaefer Jeremy , Nakra Niyati , Montufar-Solis Dina , Vigneswaran Nadarajah , Klein John 
Published: January 2023

Abstract
Interleukin 10 (IL-10) is an immunosuppressive cytokine critical to the regulation of the inflammatory response. IL-10 knockout (IL-10-/-) mice develop chronic and progressive intestinal inflammation similar to that observed in Crohn’s disease (CD) and is characterized by excessive production of pro-inflammatory molecules, particularly IL-17. Our previous studies identified Roquin, a RING finger E3 ligase with immune modulatory functions, as having decreased expression in IL-10-/- mice, and miR-223 as a microRNA involved in Roquin regulation. To further elucidate this pathway, we conducted luciferase reporter assays to identify transcriptional regulators of Roquin and miR-223. STAT1, STAT3, and GATA2 positively regulated the expression of Roquin, while STAT1, STAT3, and Ikaros positively regulated miR-223 expression. Importantly, our data demonstrate that miR-223 and Roquin expression is responsive to IL-10 and IL-10-induced transcription factors via in vitro promoter studies. Thus, Roquin and miR-223 may provide a possible link between IL-10 and the regulatory control of IL-17. These studies provide insight regarding the regulatory control of miRNAs and their target genes.
Tertiary lymphoid tissue with germinal centers and T follicular helper cells in immunologically privileged retinas of mice with chronic autoimmune uveitis (P4172)
Authors: Kielczewski Jennifer , Horai Reiko , Caspi Rachel 
Published: January 2023

Abstract
Mice transgenic for a retina-specific T cell receptor develop spontaneous autoimmune uveitis. In contrast to induced models of uveitis the disease is prolonged and the mice display distinct retinal lesions that resemble tertiary lymphoid tissue. Here we investigate these lesions for presence of germinal center (GC) and T follicular helper cell (TfH) markers. Retinas were immunohistochemically stained for CD4+ T cells, CD8+ T cells, and B cells, as well as the GC markers PNA and GL-7. The retinal lesions stained positively for CD4+ T cells and B cells and also for PNA and GL-7. Laser capture microdissection was then used to isolate the retinal lesions and microarray analysis was performed to assess expression of GC- and TfH-associated genes. The microdissected lesions consistently showed upregulation of germinal center markers and T follicular helper cell markers with a 4 fold increase in Btla, Fas, and CXCL13, a 3.5 fold increase in CXCR5, and a doubling in CXCR4 compared to control retina. Heat map analysis revealed a similar gene expression profile of the retinal lesions to that of germinal centers present in 5 day-immunized spleen and lymph nodes. We conclude that despite the immunologically privileged status enjoyed by the eye, chronic autoimmune uveitis precipitates development of tertiary lymphoid tissue in the retina, expressing genes and immunological markers characteristic of GC and TfH.
IL-23 controls autoimmunity by maintaining the integrity of marginal zone barrier that clears apoptotic bodies (P4178)
Authors: Li Hao , Hsu Hui-Chen , Wu Qi , Yang PingAr , Cua Daniel , Mountz John 
Published: January 2023

Abstract
Th17 cells, which are maintained by IL-23, are important for the spontaneous germinal center (GC) response in autoimmune BXD2 mice. Here, we unexpectedly found that BXD2-Il23-/- mice exhibit accelerated development of spontaneous GCs and production of pathogenic autoantibodies compared with wild-type (WT) BXD2 mice. Administration of adenovirus expressing IL-23 to BXD2-Il23-/- mice at early age delayed the disease onset. Despite a decline of Th17 cells in the spleen of BXD2-Il23-/- mice, there was a severe impairment in clearance of apoptotic bodies and an accelerated age-related loss of marginal zone macrophages (MZMs). In the spleens of both B6 and BXD2 mice, IL-23R was expressed primarily by MZMs which is the major cell that clears apoptotic debris in the spleens. Absence of IL-23 and loss of MZMs in BXD2-Il23-/- mice are associated with elevated production of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Selective depletion of MZMs by repeated injection of clondronate liposome to BXD2-WT, but not BXD2-Ifnar-/- mice, mimicked the phenotypes of BXD2-Il23-/-. Our results suggest a novel mechanism that IL-23 can act as a double edge sword to control autoimmunity in that over-expression of IL-23 can provoke autoimmunity through the maintenance of Th17 yet a complete deficiency of IL-23 induced autoimmunity through the gradual loss of marginal zone barrier, defective clearance of apopototic debris and elevated induction of type I IFNs.
Ceramide analogue displays stimulatory activity on dendritic cells to promote T cell responses upon virus infection (P6115)
Authors: Pritzl Curtis , Seo Young-Jin , Stokes Zach , Hahm Bumsuk 
Published: January 2023

Abstract
The ceramide family of lipids has been shown to play various important roles in both cell structure and signaling in a diverse array of cell types, including immune cells. However, very little is known regarding how ceramide affects the activation of dendritic cells (DCs) in response to viral infection. In this study, we demonstrate that a synthetic ceramide analogue (C8) stimulates DCs to increase the expansion of virus-specific CD8 T cells upon virus infection. Exogenously supplied C8 ceramide elevated the expression of DC maturation markers such as MHC class I and the co-stimulatory molecule B7-2 following infection with the immune suppressive Clone 13 strain of lymphocytic choriomeningitis virus (LCMV) in vitro. In addition, the ceramide analogue enhanced the production of IL-12 from DCs infected by the virus. Importantly, ceramide-conditioned, LCMV-infected DCs displayed a heightened activity to promote proliferation of virus-specific, CD8 T cells when compared to virus-infected DCs. Furthermore, locally instilled, but not systemically administered, ceramide analogue via intranasal inoculation significantly increased virus-reactive CD8 T cell responses in vivo. Collectively, these findings provide new insights into ceramide-mediated regulation of DC responses against virus infection and help us establish a foundation for novel immune-stimulatory therapeutics.