bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking ADP-ribosylation of cell surface proteins during cell preparation with a Nanobody against ART2 enhances chemotaxis of T cells during in vitro migration assays. (P3388)
Authors: Moyron-Quiroz Juan , Rissiek Björn , Soto Hortensia , Pahuja Anil , Wiethe Carsten , Zlotnik Albert , Yang Xifeng , Koch-Nolte Friedrich , Ransom John
Published: January 2023

ADP-ribosylation is the enzymatic transfer of the ADP-ribose moiety from NAD+ to a protein, a posttranslational modification that regulates the function of the targeted proteins. Although in normal conditions there is minimal extracellular NAD+, it is released from cells in inflammation and during preparation of primary lymphocytes. ART2 (CD296) is an ADP-ribosyltransferase expressed by most mature T cells, including NKT, and regulatory T cells. A major substrate for ART2 is P2X7, an ion channel that mediates shedding of CD62L, and NAD+-induced cell death (NICD). In this manner, NAD+ released during cell preparation affects the phenotype and survival of cells expressing ART2. In this study, we used a recombinant variable domain of the llama heavy-chain antibody S+16a to block the ADP-ribosyltransferase activity of ART2. The recombinant antibody was administered i.v. to mice and 1-2 h later, the CD4+ T cells from spleen and lymph nodes were isolated. The cell phenotype and functional state was analyzed by flow cytometry and chemotaxis assay respectively. A single dose of S+16a blocked the shedding of CD62L and reduced the number of apoptotic cells about 80%. The higher cell viability resulted in a 50-100% increase in the cell number responding to the chemokines CCL22 and CXCL12. We conclude that S+16a is a useful tool to protect mature T cells from NICD induced by the released NAD+ from cells during purification, thereby enhancing the efficiency of in vitro migration assays.
In silico high throughput pre-clinical determination of monoclonal antibody immunogenicity (P3391)
Authors: Cousens Leslie , Terry Frances , Ardito Matthew , Martin William , De Groot Anne
Published: January 2023

One of the great surprises of the biologics revolution has been the discovery that recombinant human proteins, including humanized and fully-human monoclonal antibodies (MAb), can be immunogenic when administered to immune-competent subjects. Preclinical and clinical evaluations of the immunogenic potential for biologic drugs primarily focus on humoral immune responses; as a result, the critical contribution of T cells to the development of anti-drug antibodies (ADA) has been somewhat overlooked. Using the EpiMatrix T cell epitope mapping system, we have developed an interactive in silico screening and optimization platform that evaluates the overall immunogenic potential of a biologic as well as identifies individual T cell epitope clusters contributing to its immunogenicity. In contrast to other immunogenicity prediction tools, our platform considers the contribution of regulatory T cell epitopes (Tregitopes) to immunogenic potential. Tregitopes are highly conserved T cell epitopes derived from IgG that we and others have shown activate regulatory T cells and promote tolerance induction to associated antigens. Here we demonstrate the correlation of available clinical immunogenicity data with Tregitope-adjusted immunogenicity scoring for twenty approved MAbs. Further, we present a high-throughput platform from which these scores can be used to triage large pools of candidate MAbs during the discovery phase of antibody development.
A longitudinal flow cytometry based study on phenotypic changes of cryopreserved murine T cells using the BD FACSVerse™ system (P3358)
Authors: Wang Yibing , Cai Shang , Feng Weiguo , Jana Rinku
Published: January 2023

Cryopreservation is commonly used to preserve fresh lymphocytes. However, it is not clear whether the phenotype-distinct populations can be cryopreserved equally effectively, nor how cryopreservation contributes to variation in samples analyzed by flow cytometry. Elucidation of the question has been limited by the issue of instrument-related variations over time. Laboratories have been attempting to establish standardization methods to eliminate such variations. The BD FACSVerse™ system is a high-performance flow cytometer designed to address the need for instrument standardization, specifically when conducting longitudinal and cross-site studies. The flow cytometer provides an easy-to-use setup system designed to maintain daily, consistent instrument settings for different applications, including fluorescence compensation. To take advantage of this new technical capability, we conducted a study to compare cryopreserved murine lymphocytes to freshly prepared ones, looking at the variability between the data sets. Freshly prepared murine cells were initially phenotyped and then frozen down. Afterwards, the cryopreserved cells were thawed on various days and examined using the same phenotyping method. We found that cryopreservation does have an impact on different populations of lymphocytes in a longitudinal study, which demonstrated the utility of the BD FACSVerse system’s built-in standardization in detecting phenotypic changes of cell populations.
Master chaperone gp96 for TLRs and integrins regulates canonical Wnt pathway and gut homeostasis (P3278)
Authors: Liu Bei , Staron Matthew , Hong Feng , Sun Shaoli , Li Zihai
Published: January 2023

gp96 is a master protein chaperone for Toll-like receptors and integrins in the endoplasmic reticulum (ER) and plays critical roles in innate immunity (Yang et al., Immunity, 2007 26:215-226). Using a conditional and lineage-specific knockout strategy, we have unexpectedly observed that gp96 is required for maintaining intestinal integrity. Loss of gp96 in adult mice causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear beta-catenin translocation, loss of crypt-villus structure and impaired barrier function. Mechanistic study revealed that the observed intestinal pathology in gp96 KO mice is unlikely due to compromise in the gut defense or dysregulated unfolded protein response. Rather, gp96 was found to be essential for maturation of Wnt co-receptor LRP6 and thus canonical Wnt signaling. Our study places Wnt pathway under the direct regulation of a TLR/integrin-master chaperone, suggesting that innate immunity and organ development converge on gp96.
Intestinal mucus-derived nanoparticles carry prostaglandin E2 and suppress liver inflammation through Wnt/β-catenin signaling (P3294)
Authors: Deng Zhong-bin , Zhuang Xiaoying , Xiang Xiaoyu , Jiang Hong , Wang Qilong , Miller Donald , Zhang Huang-ge
Published: January 2023

The Wnt/β-catenin pathway has been known to play a role in induction of immune tolerance, but its role in the induction and maintenance of NKT cell anergy is unknown. We found that activation of the Wnt pathway(s) in the liver microenvironment is important for induction of NKT cell anergy. We identified Glycolipid α-GalCer treatment of mice induced the expression of wnt3a and wnt5a in the liver, and subsequently resulted in a liver microenvironment that induced NKT cell anergy to α-GalCer restimulation. Mice treated with PGE(2) associated with intestinal mucus-derived exosome-like nanoparticles (IDEN) induced NKT cell anergy. PGE2 treatment leads to activation of the Wnt/β-catenin pathway by inactivation of GSK-3β of NKT cells. Pro-inflammatory conditions enhanced the migration of IDEN to the liver where both α-GalCer and PGE2 induced activation of the Wnt pathway thereby inducing NKT anergy in response to subsequent α-GalCer stimulation. IDEN-associated PGE2 also induces NKT cell anergy through modification of the ability of DCs to induce IL-12 and IFN-β in the context of both glycolipid presentation and TLR-mediated pathways. These findings demonstrate that IDEN associated PGE2 serves as an endogenous immune modulator between the liver and intestines and maintains liver NKT cell homeostasis and this finding has implications for development of NKT cell-based immunotherapies.
The anti-oxidative role of glutathione s-transferase mu 2 in anti-GBM induced glomerulonephritis by inhibiting inflammation and oxidative stress (P5121)
Authors: Li Yajuan , Yan Mei , Du Yong , Min Soyoun , Mohan Chandra , Li Quanzhen
Published: January 2023

Oxidative stress is a common manifestation in chronic inflammation and closely associated with autoimmune diseases. Impaired balance between oxidative stress and antioxidant systems exhibits an important impact on pathogenesis of immune-mediated nephritis. Using anti-GBM induced nephritis mouse model, we have identified a group of redox related genes dysregulated in mouse kidney upon anti-GBM antibody challenge. In this study, we investigated the anti-oxidative effect of glutathione s-transferase mu 2 (Gstm2) on immune-mediated nephritis. Human GSTM2 gene was transduced into mesenchymal stem cell (MSC) and hGSTM2-MSCs were injected to anti-GBM antibody challenged 129/svj mice. Mouse blood urea nitrogen (BUN) and proteinuria were measured to evaluate renal function change and renal histopathological changes were appraised. We found the Gstm2 could alleviate the renal inflammatory damage by suppressing expression of inflammatory chemokines, CCL2, IL1β and IL6 to reduce macrophage and T lymphocyte infiltration. Gstm2 improved renal function by reducing BUN and proteinuria. Moreover, hGSTM2-MSCs significantly reduced the apoptosis of tubular cell by regulating the expression of anti-apoptotic genes (BCL2 and CD40LG) and hGSTM2-MSCs resisted oxidative stress by increasing the expression of catalase and glutathione peroxidase 1. In summary, Gstm2 exhibits protective effect on immune-mediated nephritis by inhibiting inflammatory damage and oxidative stress during inflammatory process.
Toll-like receptor 10: its expression and role in chemotaxis in human neutrophils (P1234)
Authors: Singh Baljit , Balachandran Yadu , Knaus Steven
Published: January 2023

TLR10 is a member of TLRs that recognize pathogen-associated molecules and play critical roles in host defense. However, very little is known about the expression and function of TLR10. We found TLR10 expression in vascular endothelium of normal and inflamed lungs from chickens and humans. Western blots showed increased TLR10 protein in lungs of chicken infected with E. coli or Adenovirus. Human neutrophils challenged with E. coli lipopolysaccharide (LPS) showed decreased total TLR10 protein and surface expression in 90 minutes. Confocal microscopy detected cytosolic and nuclear TLR10 in normal neutrophils. Upon activation with LPS, TLR10 colocalized with TLR4, flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, to suggest the cycling to endocytic compartments. TLR4 neutralization reduced cytoplasmic localization of TLR10 in neutrophils. Depletion of reactive oxygen species (ROS) with FCCP in LPS-treated neutrophils reduced p65 nuclear translocation and TLR10 expression. Single cell video imaging of live LPS-activated human neutrophils showed the translocation of TLR10 to the leading edge and siRNA-mediated silencing of TLR10 in HL-60 cell line reduced their chemotaxis towards fMLP. We conclude that TLR10 expression is modulated via TLR4 signalling and ROS production, and that TLR10 participates in neutrophil chemotaxis. Altered TLR10 expression in activated neutrophils and inflamed lungs suggests a role for this receptor in lung inflammation.
Differential requirements of CD14 and Lipopolysaccharide Binding Protein in Monophosphoryl lipid A-mediated TLR4 activation (P1209)
Authors: Tanimura Natsuko , Miyake Kensuke
Published: January 2023

Toll-like receptor 4 (TLR4) ligation causes multiple immune responses to host, including inflammations, type-I interferon productions and acquired immune responses. There are a variety of ligands against TLR4, and TLR4 senses those impalpable differences with its co-receptor MD-2. Since TLR4 agonist has highly immunomodulatory functions, it would be useful as an immunostimulator when and if we can harness the responses’ balance. Monophosphoryl lipid A (MPL), one of the artificial derivatives, lacks 1- phosphate and is well-known detoxified agonist. Because of its low inflammatory aspect, MPL has been used for a vaccine adjuvant ingredient. Previous studies reveal that MPL-activated TLR4 transduces its signal by p38-dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-biased signaling, though the mechanisms how TLR4 discriminates of MPL has been unclear. Here we focused on the phases of ligand coming into TLR4 and found specific requirement of CD14 and Lipopolysaccharide binding protein (LBP). This unique TLR4 activation induced impaired receptor dimerization and subsequent low inflammatory responses. Our data suggested that the modification of TLR4 activation manners changed responses’ balance.
Simple and robust whole blood staining procedure using PerFix-nc reagents generates unprecedented FOXP3 signal/noise ratio. (P1053)
Authors: Malergue Fabrice , Khemici Laeticia , Montero-Julian Felix A.
Published: January 2023

FOXP3 is a nuclear antigen expressed specifically in regulatory T cells (Tregs). Tregs play critical role in the maintenance of the dominant self-tolerance and have been subject of extensive research effort for the last two decades. Today the detection of FOXP3 is laborious, time consuming (3-4 hours) and poorly reproducible. A new commercially available method, named PerFix-nc, allows simultaneous intra- and extra-cellular staining without any wash step. This new kit allows for the reduction of the workflow to 45 min. In this study we aimed at the optimization of the PerFix-nc protocol for the detection of FOXP3. We evaluated various clones from various vendors, optimizing titration and incubation time, and adding some extra washing steps. Signal to noise ratio at least 40% better than the one obtained for the same samples with reference procedure. Improved cell scattered separation compared to reference method was revealed. Purity and recovery were also significantly increased. Furthermore the method was very robust and demonstrated excellent repeatability with respect to percentage of FOXP3-positive cells and signal to noise. Total workflow was reduced from 3-4 hours in reference method to about 95 min. In conclusion, the novel PerFix-nc reagent significantly improves FOXP3 detection in several different ways: it simplifies FOXP3 intracellular staining procedure, generates robust and highly reproducible results, and provides unprecedented signal to noise ratio.
Ubiquitous MHC class II peptides shape regulatory T cell development (P1039)
Authors: LeGuern Christian , Germana Sharon
Published: January 2023

T cell auto- and allo- reactivity is primarily tamed in the periphery by thymus-derived CD4 regulatory T cells (Tregs) that mature in the medullar compartment. We have achieved Treg-dependent tolerance of fully allogeneic transplants via the transfer of donor MHC class II genes (MHCII) that induced newly made Tregs specific of donor MHCII peptides. Given that gene therapy for tolerance was effective with MHCII but not MHCI genes, the goal of the study was to identify natural MHCII peptides able to impact Treg development and function. Using experimental models that either recapitulated MHCII peptide presentation - the IEα 52-68 peptide presented on I-Ab molecules - or promoted Treg-mediated tolerance to heart grafts via MHCII gene transfer, we confirmed that high amounts of IEα peptide were presented by MHCII from most if not all I-Ab+ CD11c+ dendritic cells of the thymic medulla, suggesting its role in Treg maturation. This hypothesis was confirmed by demonstrating that the introduction of the IEα peptide through hematopoietic chimerism converted host IEα-specific CD4 T cells into CD25hiFoxp3+ suppressive Tregs. Treg suppression for tolerance to transplants required prior activation by cognate pMHCII complexes as MHCII-treated recipients rejected MHCII-deficient while accepting MHCII-sufficient grafts. These data are consistent with a prominent role of “MHCII self-presentation” in shaping thymic Treg TCR specificities and controlling Treg function in the periphery.
STING ligand c-di-GMP improves vaccine efficacy in metastatic breast cancer model (P4265)
Authors: Chandra Dinesh , Asafu-Adjei Denise , Quispe Wilber , Jahangir Arthee , Ramos Ilyssa , Karaolis David , Gravekamp Claudia
Published: January 2023

Cancer vaccination or immunotherapy may be our best and most benign option for preventing or curing metastatic cancer. However, a real breakthrough is hampered by immune suppression in the tumor microenvironment (TME). Myeloid- derived suppressor cells (MDSC) are one of the most important players in immune suppression in the TME. In the study presented here we found that c-di-GMP (ligand for STING), an ubiquitous bacterial signaling molecule and promising vaccine adjuvant, improved the therapeutic efficacy of an attenuated L. monocytogenes-based vaccine expressing tumor-associated antigen (TAA) Mage-b, in a mouse model of metastatic breast cancer (4T1), by targeting MDSC. An extreme low dose of c-di-GMP (0.01 nmol) combined with a low dose of Listeria-Mage-b (104 CFU), dramatically reduced the number of metastases, in correlation with a significant reduction of MDSC and a significant increase in CD8 T cell responses to Mage-b. Moreover, low dose of c-di-GMP increased the production of IL-12, while high dose killed tumor cells directly. Administration of one high dose c-di-GMP to kill the tumor cells, followed by repeated administrations of low dose c-di-GMP to improve CD8 T cell responses, provided similar efficacy against the metastatic breast cancer as Listeria-Mage-b and c-di- GMP in the 4T1 model. These results demonstrate that c-di-GMP is an attractive adjuvant for cancer therapy.
In situ immune modulation therapy with a novel chimeric chaperone generates a potent antitumor response (P4271)
Authors: Yu Xiaofei , Guo Chunqing , Yi Huanfa , Fisher Paul , Subjeck John , Wang Xiang-Yang
Published: January 2023

The immunosuppressive tumor microenvironment (TME) represents a major hurdle for cancer immunotherapy. In the present study, we evaluate the feasibility of conditioning the immune environment in the tumor sites for induction of effective antitumor immunity using a novel immune modulator Flagrp170. This chaperone-based immune modulating agent is created by integrating a pathogen (i.e., flagellin)-derived ‘danger’ signal into Grp170, the largest chaperone resident in the endoplasmic reticulum. This chimeric chaperone molecule is highly efficient in inducing functional activation of dendritic cells via engaging the NF-κB and MAPK signaling pathways, and concurrently facilitating cross-presentation of tumor antigen. Intratumoral delivery of adenoviruses encoding Flagrp170 provokes a strong tumor-specific cytotoxic T lymphocyte response, leading to profound inhibition of tumor growth and metastasis. In situ Flagrp170 therapy preferentially drives the Th1 polarization of the TME, as evidenced by marked elevation of IL-12 and IFN-γ as well as increased tumor infiltration by CD8+ cells. Antibody depletion and blocking assays reveal that CD8+ cells and IFN-γ are essential for enhanced antitumor activity of Flagrp170. Our data demonstrate that this modified chaperone represents a promising immune modulating agent that may be utilized to potentially reverse immunosuppression in the TME and improve immunotherapeutic response to metastatic malignancies.
Use of HSP90 inhibitors in the combination immunotherapy of melanoma (P4272)
Authors: Raveendran Subhara , Rao Aparna , Storkus Walter
Published: January 2023

The molecular chaperone, HSP90 is commonly overexpressed in human melanomas, where it serves to stabilize a broad range of client proteins providing growth, survival and migratory advantages to tumor cells and conditions a pro-angiogenic tumor microenvironment (TME). HSP90-mediated stabilization also limits the proteasome-dependent processing of client proteins and the subsequent presentation of derivative peptides in tumor cell-associated MHC class I complexes which is required for effective host CD8+ T cell recognition of melanoma cells. As a consequence, we have analyzed the anti-tumor impacts of clinical-grade HSP90 inhibitors (i.e. STA9090, 17-DMAG, Novobiocin) on client protein expression in vitro (human melanoma cell line Mel526, murine melanoma cell line B16) and in vivo (s.c. B16 tumors in C57BL/6 mice). We observed that HSP90 inhibitors promote the proteasome-dependent (i.e. blocked by inhibitors PS-341 and MG-132) degradation of client proteins such as c-MET, EphA2, HER2/neu, PDGFRβ and Survivin, with STA9090 appearing to be most efficient on a molar basis. We are currently investigating the capacity of STA9090 to therapeutically sensitize established melanomas to client protein-specific CD8+ T cells (induced by active vaccination or provided via adoptive cell transfer; ACT) in combination therapy approaches. We believe such protocols may hold translational promise for the treatment of patients with advanced-stage melanoma.
IL-9 strategy to perturb Treg function and enhance anti-tumor immunity (P4273)
Authors: Hoelzinger Dominique , Dominguez Ana , Pollock Kevin , Lustgarten Joseph , Cohen Peter , Gendler Sandra
Published: January 2023

IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.
Molecular characterization and antimicrobial susceptibility of canine Staphylococcus pseudintermedius isolates from the United States (P6063)
Authors: Videla Ricardo , Bemis David , Kania Stephen
Published: January 2023

Staphylococcus pseudintermedius, the primary cause of canine pyoderma, is therapeutically challenging due to a high, and increasing, rate of resistance to antibiotics. The objectives of this study were: to investigate the genetic diversity within canine S. pseudintermedius isolates in the United States and to characterize their antimicrobial susceptibility profile. Samples from 221 dogs were obtained from veterinary diagnostic facilities located in different regions of the United States. Genetic groups were generated based on Pulsed-field gel electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) results. Conventional or real-time PCR was performed to detect presence of the mecA gene as an indicator of methicillin resistance, and the Kirby-Bauer disk diffusion method was used to test the susceptibility to 13 antimicrobials. An association between the largest clonal group and the most commonly encountered susceptibility pattern was found indicating the presence of one major clonal population. These findings represent a significant contribution for the better understanding of S. pseudintermedius genetic diversity in the United States and also reflect the high degree of antimicrobial resistance. Our findings emphasize the need for the development of novel therapeutic alternatives, such as vaccines, to address the increasing number of S. pseudintermedius isolates resistant to the most clinically useful antimicrobials that are available to veterinarians.
Remarkable conservation of natural killer receptor, NKp30 (P6089)
Authors: Ota Yuko , Flajnik Martin
Published: January 2023

Comparative analysis of natural killer receptor (NKR) among vertebrates have revealed that they are remarkably plastic; some receptor families have expanded greatly, while others have been lost in a species-specific manner. Thus, NKR are extraordinarily rapidly evolving receptors that must adapt to rapidly changing pathogens. Therefore we were surprised to identify the activating NKR, NKp30 in the amphibian Xenopus and cartilaginous fish (shark, skate) databases. NKp30 is MHC-linked in humans and possesses a unique V-type immunoglobulin superfamily (IgSF) domain, the so-called “VJ”-type, which resembles the precursor of antigen receptors (AgR) and is assumed to have predated the emergence of AgR. Further database searches for NKR in jawless vertebrates and lower deuterostomes revealed no NKR orthologs, implying that NKp30 is the most conserved and oldest NKR in jawed vertebrates. While mammalian NKp30 is encoded by a single copy gene, with a cytoplasmic region that interacts with an ITAM-containing adaptor molecule, multiple NKp30 genes were found in Xenopus and sharks, some with ITIM-containing cytoplasmic regions, indicating inhibitory activity characteristic of other NKRs like KIR/CD94-NKG2. We are testing whether the MHC association of NKp30 is primordial, which would further suggest its coevolution with other MHC-linked proteins such as class I/II or B7 family members.
Comparative cancer immunology: Inducing myeloid derived suppressor cells in vitro (P6092)
Authors: Papenfuss Tracey , VanGundy Zachary , Sherger Matthew , Wasserman Jacob
Published: January 2023

Comparative oncology has broad applications in the development and optimization of novel therapeutics for cancer in both humans and animals. In this study, we developed in vitro tools to study canine myeloid derived suppressor cells (MDSCs). Previously, we have shown that tumor derived soluble factors (TDSFs) modified the function of a dendritic cell line. In this study, we investigated TDSF’s ability to induce MDSCs. TDSFs were generated from canine tumor cell lines and canine bone marrow precursors were differentiated in the presence of GM-CSF with or without TDSFs. We found that TDSFs dramatically increased the expression of CD11b+CADO48A+ (a canine MDSC marker akin to CD11b+Gr1+ MDSCs in mice) in cells between days 3-7 compared to controls with the highest percentage (40%) occurring on day 5 of differentiation. Day 5 TDSF-exposed cells suppressed the proliferation of responder immune cells. Similar results were found in companion studies using immunocompetent mice and murine in vitro TDSF-generated MDSCs. Taken together, these findings support that TDSFs can generate canine MDSCs in vitro that are similar to mouse MDSCs and that the in vitro generation of MDSCs mimics the immunosuppression seen with ex vivo derived MDSCs in humans and mice. These data have important implications both in understanding canine cancer immunology and for translational and therapeutic applications utilizing the dog as a model for human cancer.
Identifying the cellular interactions required for diversification of the primary antibody repertoire in rabbit GALT (P6101)
Authors: Lanning Dennis , Knight Katherine
Published: January 2023

Rabbits generate an initial antibody repertoire of limited diversity due to preferential use of the 3’-most VH gene segment during V-D-J gene rearrangement. Around one week of age, rabbit B cells begin diversifying this antibody repertoire in gut-associated lymphoid tissue (GALT), by mutating their V-D-J genes in response to antigen-independent signals acquired from intestinal commensals. To identify the cell-cell and cell-microbe interactions required for V-D-J gene diversification, we examined B cell expression levels of four chemokine receptors that mediate homing to important sites within GALT. We stained the chemokine receptors CXCR4, CCR6, CXCR5 and CCR7 on appendix B cells from two- to eight-day-old rabbits with fluorescence-labeled antibodies or chemokine-Ig fusion proteins and analyzed them by flow cytometry. The changes in chemokine receptor expression we observed during the first week of life suggest that, after entering appendix follicles, B cells first home to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells then home sequentially to the follicular dendritic cell network, the T cell area and finally to the basal region of the follicle, where they proliferate and diversify their V-D-J genes to generate the primary antibody repertoire.
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

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

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

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

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, >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

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

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 > 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

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

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

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

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

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.