bluuebunny/crossref_metadata_2025 huggingface dataset:
Granzyme B plays a differential role in CD4+ T cell subsets versus CD8+ T cells in mediating the graft-versus-tumor effect and graft-versus-host disease (TRAN3P.888)
Authors: Du Wei , Leigh Nicholas , Bian Guanglin , Qiu Jingxin , Chen George , Liu Hong , Hahn Theresa , McCarthy Philip. , Cao Xuefang
Published: January 2023

After allogeneic hematopoietic cell transplantation, donor-derived T cells can attack host tumor cells, producing a favorable graft-vs.-tumor (GVT) effect. However, graft-vs.-host disease (GVHD) may develop when donor T cells attack normal host tissue. We previously reported that while granzyme B (GzmB) is required for CD8+ T cells to cause GVHD, GzmB-mediated damage of CD8+ T cells impairs the GVT effect. In this study, we examined the role of GzmB in GVT and GVHD mediated by CD4+ T cell subsets, using an established A20 tumor model and bioluminescence imaging to monitor tumor growth in BALB/c hosts. We found that CD4+CD25+ regulatory T cells (Tregs) derived from C57BL/6 donors significantly suppressed GVT activity mediated by both CD8+ T cells and CD4+CD25- effector T cells (Teffs). However, GzmB-/- Tregs were as equally suppressive as WT Tregs, consistent with our previous report that GzmB is not required for Treg-mediated suppression of GVHD. Nonetheless, GzmB-/- Teffs were less effective in controlling tumor growth than WT Teffs; but surprisingly, GzmB-/- Teffs caused more severe GVHD than WT Teffs. Our data reveal that GzmB is essential for CD4+CD25- Teff-mediated optimal GVT effect but diminishes the ability of Teffs to cause GVHD, which contradicts its role in CD8+ T cells where GzmB is essential to cause GVHD but impairs GVT effect. These differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to separate GVT from GVHD.
Ethyl pyruvate, an inhibitor of high-mobility group box 1 (HMGB1) release, modulates dendritic cell activation and survival (TRAN3P.897)
Authors: Chakhtoura Marita , Chain Robert , Varghese Linda , Gallucci Stefania
Published: January 2023

Dendritic cells (DCs) activated by stimulatory danger signals initiate the detrimental anti-graft immune responses that lead to transplant rejection. High-mobility group box1 (HMGB1) is a nuclear DNA-binding protein that signals danger if released, contributing to DC activation and acute allograft rejection. Preventing HMGB1 release is a way to block its effects, promoting less active DCs. Here we assessed the effects of ethyl pyruvate (EP), proposed to potentially prevent HMGB1 release from cells, on DC responses. We found that EP (10mM) abolished pro-inflammatory cytokine (IL-12, IL-6 and TNFa) and IL-10 induction by the strong DC stimulus LPS in myeloid bone marrow-derived dendritic cells (BMDCs). EP also significantly decreased BMDC surface MHC expression, suggesting a possible role in inhibiting antigen presentation. We also determined EP effects on BMDC viability and found that EP strongly inhibited in vitro DC activation without inducing death. Furthermore, EP could prevent LPS-induced apoptosis that normally occurs 72h post-stimulation. Moreover, EP suppressed DC responses to other TLR stimuli including CpGs and R848. Our results indicate that EP inhibits most of the biological responses of DCs to LPS and other TLR stimuli. We are investigating the link between EP and HMGB1 release prevention from BMDCs. Testing the effect of HMGB1 blockade on DC activation will highlight a novel target to induce tolerogenic DCs and prolong solid organ graft survival.
Pancreatic islets engineered to display on their surface an apoptotic form of SA-FasL induce localized tolerance via phagocytes/TGF-β/Treg axes (TRAN3P.866)
Authors: Woodward Kyle , Yolcu Esma , Zhao Hong , Bandura-Morgan Laura , Askenasy Nadir , Shirwan Haval
Published: January 2023

Islets engineered to display on their surface a FasL protein chimeric with streptavidin (SA-FasL) induce robust localized allotolerance under the transient cover of rapamycin. We hypothesized that immunomodulation with SA-FasL sets in action a regulatory circuit that involves apoptosis of alloreactive T cells within the graft microenvironment, clearance of apoptotic bodies by phagocytes, secretion of TGF-β, and generation/expansion of Tregs. This study was designed to test this hypothesis. SA-FasL-engineered islets resulted in alloreactive T cell depletion within the graft-draining lymph nodes with minimal impact on the cells in the spleen. Depletion of phagocytes one day prior to transplantation resulted in prompt rejection of SA-FasL-engineered islets (n=7). Similarly, treatment of graft recipients with a blocking Ab to TGF-β early (day 0, n=7), but not late (> 100 days, n=5), post-Tx resulted in prompt rejection of long-term allogeneic SA-FasL-islet grafts. In marked contrast treatment with a blocking Ab to IL-10 had no effect on tolerance induction. F4/80+ cells in the graft were shown to co-localize with TGF-β 5 days post-Tx, suggesting that TGF-β in the graft produced by macrophages may induce tolerance. Taken together, these studies demonstrate that immunomodulation with SA-FasL-engineered pancreatic islets results in a localized immunoregulatory cascade driven by SA-FasL-mediated apoptosis of alloreactive T cells, phagocytes, and TGF-β that confers tolerance.
Friend or foe? How an invertebrate distinguishes self from non-self in a natural histocompatibility interaction. (TRAN3P.912)
Authors: Karadge Uma , Gosto Minja , Nicotra Matthew
Published: January 2023

The existence of sophisticated self/non-self systems is not limited to vertebrates. Several colonial marine invertebrates have evolved systems that distinguish between conspecifics via cell-cell contact. This phenomenon, known as allorecognition, occurs when colonies encounter each other as they grow across their substrate. Compatible colonies fuse or co-exist, while incompatible colonies reject and often aggressively compete for space. In all taxa studied to date, allorecognition phenotypes are determined by highly polymorphic loci, which ensure that colonies are only compatible with themselves or close kin. How allorecognition molecules achieve this specificity remains unknown. Allorecognition in Hydractinia is controlled by at least two genes, alr1 and alr2, which encode highly polymorphic transmembrane proteins similar to immunoglobulin superfamily molecules. Colonies with matching alleles at alr1 and alr2 fuse, while colonies with no matching alleles reject. Using in vitro assays with recombinant proteins, we demonstrate that alr1 alleles bind to themselves but not to other alr1 alleles. We suggest that, in vivo, compatibility between colonies is also determined by allele-specific homophilic binding of alr proteins. Since fusion is rare in nature and single populations contain hundreds of unique alr alleles, the Hydractinia allorecognition system appears to be based on a biophysical mechanism with unprecedented allelic diversity and specificity.
A bacterial protein supresses direct allorreactivity through induction of MARCH-I and downregulation of MHC class II in skin migratory dendritic cells (TRAN3P.913)
Authors: Bonorino Cristina , Borges Thiago
Published: January 2023

We have elucidated a major innate mechanism leading to the immunosuppressive actions of a member of the Hsp70 family, the DnaK of Mycobacterium tuberculosis. Treatment of skin allografts with Hsp70 lead to delayed acute rejection. This was mediated by skin CD103+ DCs, which migrated from the skin graft to the host DLN, resulting in decreased CD8+ and CD4+ T cell proliferation. Hsp70 modulated direct alloreactivity, signalling on donor DCs through TLR2 for the induction of IL-10, which resulted in MHC class II downregulation. Membrane-Associated Ring-CH I (MARCH-I), an ubiquitinator molecule responsible for downregulation of MHC II, was induced in DCs by Hsp70 via ERK, STAT3 and IL-10. This is a potent immunosuppressive effect that probably plays a role in immune evasion in vivo, and yet can be harnessed to manage graft rejection, without the burden of systemic immunosuppression for the host.
SA-FasL-engineered rat pancreatic islets induces robust localized tolerance through TGF-β/CD4+CD25+FoxP3+ Treg cells axis in mice (TRAN3P.867)
Authors: Zhao Hong , Yolcu Esma , Woodward Kyle , Ahamad Niloufar , Morgan Laura , Askenasy Nadir
Published: January 2023

Islets engineered to display on their surface an apoptotic form of FasL protein chimeric with streptavidin (SA-FasL) induce tolerance in the BALB/c-to-C57BL/6 allogeneic model. This study assessed the efficacy of this approach in a xenogeneic setting. WF rat islets were modified with biotin and engineered with SA-FasL protein. SA-FasL-islets survived indefinitely (n=10, >200 days) in chemically diabetic C57BL/6 mice under transient cover of rapamycin. SA-engineered (n=5) or unmanipulated control (n=6) islet grafts were rejected acutely with MST=34.6±5.0 and 13.8±4.0, respectively. Tolerance was localized to the graft as long-term (> 100 days) mice rejected a second set of unmodified graft transplanted under the contralateral kidney capsule without an impact on the survival of original graft. Flow-cytometry analysis and immunohistochemistry demonstrated significantly higher percentage of CD4+CD25+FoxP3+ Treg within the graft and graft-draining lymph nodes, but not spleen, of long-term recipients. Tolerant mice had decreased percentages of T cells expressing IL-2 and IFN-r as compared with rejecting animals. Treg cells depletion resulted in prompt graft rejection. Blocking of TGF-β in vivo during tolerance induction also resulted in the abrogation of graft survival. Testing the efficacy of this approach in a large animal model of xenotransplantation will be an important next step towards clinical translation of this novel tolerance concept.
FWGP activates the immune system (TUM7P.930)
Authors: Abuhay Mastewal , O'Donnell Robert , Ma Yunpeng , Xiong Chengyi , Tuscano Joseph
Published: January 2023

The immune system is an essential defense against malignancy; immunosuppression increases the risk of developing cancer. A better understanding of how to modulate the immune system may lead to new methods to prevent and treat disease. While recent developments in targeted immunomodulation have improved cancer treatments, most therapeutics are toxic, expensive, or have marginal efficacy. We developed a natural product derived from fermented wheat germ extract (FWGE) that acts as an immune modulator. Through further purification we identified protein components, (fermented wheat germ proteins, FWGP), that are more potent than FWGE. In vitro, FWGP was cytotoxic against B cancer cells with IC50s ranging from 10μg/ml to 17.5μg/ml. In vivo studies showed substantial efficacy without toxicity. We found that FWGP was a potent immune modulator. It increased CD4+ T cells, NK-T cells, & activated monocytes by 4, 10, & 50 folds, respectively, while decreasing CD8+ T cells. CD56-bright NK cells treated with FWGP increased compared to the heat-inactivated FWGP treatment, and NK-mediated IFN-γ production rose as [FWGP] increased. Also, FWGP (0.1 mg/ml) activated complement with 2-fold potency compared to either control or FWGE (55mg/ml). It enhanced ADCC as well; PBMCs treated with FWGP showed a dose dependent increase in killing of Ramos. FWGP’s in vivo efficacy could be due to its immune modulating effects and may be the basis for a non-toxic treatment for lymphoma and other malignancies.
Enhancement of neutrophil signaling and effector function by Imprime PGG in response to tumor cells in biomarker positive individuals (TUM7P.937)
Authors: Leonardo Steven , Gorden Keith
Published: January 2023

Imprime PGG is a yeast β-glucan immunomodulator being developed for tumor therapy. Imprime PGG, in combination with tumor-specific monoclonal antibodies (MAb), is more effective in human trials than MAb therapy alone. Imprime PGG’s mechanism of action involves redirecting the innate immune system, in particular neutrophils, to kill MAb-targeted tumors cells. Recent work has identified a subset of individuals with high anti-β-glucan antibodies (ABA) that demonstrate greater efficacy to Imprime PGG treatment in vivo. Using ABA levels, we investigated signaling and functional differences in Imprime PGG treated neutrophils between biomarker positive and negative individuals. In this study we developed a calcium flux assay to assess Imprime PGG’s ability to modulate neutrophil signaling in response to MAb-targeted tumor cells. Our data demonstrates that ABA high individuals show a 3-fold greater enhancement in calcium signaling then ABA low individuals after Imprime PGG treatment. Functional activity was measured by Fc-mediated oxidative burst in response to antibody coated particles; a mechanism mimicking how antibody-coated tumor cells are recognized and eradicated by Imprime PGG treated neutrophils. ABA high individuals demonstrated a 5-fold greater enhancement in Fc-mediated oxidative burst upon Imprime PGG treatment then ABA low individuals. Our results demonstrate that Imprime PGG modulates signaling and functional activities in neutrophils of biomarker positive individuals.
MUC1 modulates NF-kB-dependent gene expression of pro-inflammatory cytokines by altering their epigenetic regulation (TUM7P.944)
Authors: Cascio Sandra , Sciurba Joshua , Finn Olivera
Published: January 2023

MUC1 is a transmembrane glycoprotein overexpressed and hypoglycosylated in human adenocarcinomas as well as sites of chronic epithelial inflammations. The MUC1 extracellular domain plays an important role in cell-cell communication, protein-protein interaction and signaling pathway activation. We investigated the role of MUC1 in transcriptional regulation of pro-inflammatory cytokines, and whether NF-kB signaling pathway is required. Our results showed that under TNFα treatment the entire molecule MUC1 is translocated to the nucleus and binds cytokine promoters. In particular, ChIP and Real-time PCR experiments revealed that MUC1, in association with p65, up-regulated IL6 and TNFα expression by binding to their promoter regions in both mouse and human cancer cells. Because proteomic analysis showed that MUC1 is able to interact directly with histone proteins, we hypothesized that MUC1 might influence the transcriptional activity of cytokines by inducing epigenetic modification. Consistent with other studies revealing the cross-talk between histone acethylation, methylation and ubiquitination, our preliminary data demonstrated that full length form of MUC1 modulates the expression of several factors involved in chromatin remodeling such as CBP/p300, HDAC1, ubH2A and EZH2. We also found that MUC1 controls their interaction with IL6 and TNFα promoters in breast cancer cells. This is an important finding that may explain the mechanism by which MUC1 regulates cancer progression.
Immunization of CD200R1KO mice post surgical resection of EMT6 breast cancer leads to long-term (>1year) cure of metastatic tumor growth (TUM7P.921)
Authors: Gorczynski Reg , Chen Zhiqi , Khatri Ismat , Podnos Anna
Published: January 2023

Attenuated expression of CD200 decreases local tumor growth and metastasis in immunocompetent animals. We explored the ability to cure BALB/c CD200KO or CD200R1KO mice of tumors ≤1cm3 in size by surgical resection of localized tumor, followed by immunization with irradiated EMT6 cells along with CpG as adjuvant. Control animals developed pulmonary and liver metastases within 30 days of surgery. Protection was seen in both CD200KO or CD200R1KO mice, with no macroscopic lung/liver metastases observed in CD200R1KO mice on sacrifice at day 300. After resection and immunization, draining lymph nodes from controls contained tumor cells cloned at limiting dilution in vitro even before pulmonary and hepatic metastasis was seen. Within the limits of detection of the assay used (sensitivity ~1 in 107 cells) no tumor cells were detected at limiting dilution in similarly treated CD200R1KO mice, and significant reductions were seen in CD200KO mice. Infusion of anti-CD4, but less so anti-CD8, mAb into surgically treated and immunized CD200R1KO mice attenuated protection from both macroscopic (liver/lung) and microscopic (assayed by limiting dilution of DLN) metastasis. Adoptive transfer of lymphocytes from treated CD200R1KO mice to surgically-treated control mice also attenuated metastatic growth of tumor, which was abolished by pretreatment of transferred cells with anti-CD4 mAb. Our data suggest CD200:CD200R attenuates a potentially tumor-protective CD4 host response to breast cancer.
Necrosis associated factors (S100A4 and uric acid) promote accumulation of MSCs which induce IL10+/IDO+ lymphocytes (TUM7P.949)
Authors: Lotfi Ramin , Eisenbacher Judith , Jahrsdörfer Bernd , Kaltenmeier Christof , Rojewski Markus , Yildiz Tatjana , Beyer Thamara , Erle Alexander , Wiegmann Denis , Grassl Stefan , Körper Sixten , Wiesneth Markus , Schrezenmeier Hubert
Published: January 2023

Necrosis with subsequent release of damage-associated molecular patterns (DAMPs) is a characteristic feature of advanced solid tumors. DAMPs impact the tumor-microenvironment stimulating tumor-associated cells like mesenchymal stromal cells (MSCs). Tumor-infiltrating-MSCs are associated with tumor progression and metastasis. Oxidized DAMPs lose their stimulatory capacity on MSCs. As a DAMP-member S100A4 is sensitive to oxidation while uric acid (UA) acts as an antioxidant. We tested these two DAMP-members separately and in combination for their biologic activity on MSCs. S100A4 and UA showed a dose-dependent chemotactic activity on MSCs with synergistic effects when both DAMPs were combined. Substituting for UA, alternative antioxidants (Vitamin-C, dithiothreitol, and acetylcystein) had also a comparable synergistic effect on the chemotactic activity of S100A4, emphasizing the reducing potential of UA being responsible for the observed synergy. Yet, S100A4 and UA inhibited MSC proliferation without impacting their viability. Nevertheless, in the presence of S100A4 or UA, MSCs gained immunosuppressive capacities by inducing IL-10 and indoleamine dioxygenase expressing lymphocytes. We characterized S100A4 and UA as necrosis-associated factors playing a crucial role within MSC biology and thus immunoregulation. Our results have implications for therapeutic approaches targeting the tumor microenvironment and addressing the immunosuppressive nature of necrotic tumor.
The role of intestinal epithelial exosomes in the progression of colon cancer (TUM7P.952)
Authors: Deng Zhong-bin , Wang Baomei , Zhuang Xiaoying , Jiang Hong , Wang Qinglong , Zhang Lifeng , Zhang Huang-Ge
Published: January 2023

Accumulating evidence indicates that dysregualtion of host-microbiota interaction in gut is associated with the development of colon cancer and with chronic inflammation. This chronic inflammation results in part from the interaction between inflammatory cells and gut microbiota, but pathway(s) leading to gut microbial mediated inflammation, and promoting colon cancer were heretofore unknown. Here we demonstrate that exosomes that bud from colon tumor cells play a key role in the recruitment of inflammatory CCR6+CD4+Th17+ into tumor site and subsequently promote tumor growth. We found that administered tumor exosomes can induce spontaneous intestinal polyposis in the Apc (Min/+) mouse model, a widely used spontaneous colorectal cancer model for human familial adenomatous polyposis (FAP) in a CCL20 dependent manner. We further demonstrate that bacterial products up taken by tumor cells promote the production of PGE2+CCL20+ tumor exosomes. Our studies identify a key mechanism of tumor exosomes-mediated T cells homing to intestine.
IRF5 is a novel regulator of CXCL13 expression in breast cancer that increases CXCR5+ B and T cell trafficking to the tumor (TUM7P.955)
Authors: Pimenta Erica , De Saurav , Feng Di , Hall Kelly , Ran Sophia , Barnes Betsy
Published: January 2023

Clinical studies using predictive signatures have shown that a high immune signal from whole tumor expression profiles correlate with improved outcome in some cancers, including breast cancer. Factors regulating immune cell trafficking to the tumor, however, are not known. Utilizing MDA-MB-231 breast cancer cells grown in 3D culture, we examined whether intratumoral expression of interferon regulatory factor 5 (IRF5) regulates the profile of cytokines/chemokines expressed. Previous work has shown that IRF5 expression is lost in ~80% of all invasive ductal carcinomas examined. Using focused arrays, we found a number of cytokines/chemokines that were dysregulated between IRF5-positive and -negative MDA-MB-231 cells. CXCL13 was identified as a direct target of IRF5. Using tumor-conditioned media (TCM) from IRF5-positive and -negative cells, we found a significant increase in B and T cell trafficking to TCM from IRF5-positive cells. Subset analysis revealed that primary CXCR5+ B and T cells showed significantly reduced migration to IRF5-negative TCM or IRF5-positive TCM depleted of CXCL13. CXCR5 is the primary receptor for CXCL13. Analysis of human breast tumor tissues revealed a positive correlation between IRF5 and CXCL13 expression providing clinical relevance to this study. Data support that IRF5 directly regulates a network of genes that shapes a tumor immune response and may serve as a novel prognostic marker for chemotherapy and immunotherapy response.
NLRX1 attenuates tumorigenesis through the negative regulation of AKT and NF-κB signaling (TUM7P.924)
Authors: Allen Irving , Simmons Alysha , Capria Vittoria , LeRoith Tanya , Robbins Greg , Heid Bettina , Philipson Casandra , Dervisis Nikolaos , Yuzbasiyan-Gurkan Vilma , Hontecillas-Magarzo Raquel , Bassaganya-Riera Josep , Ting Jenny
Published: January 2023

Members of the NLR family of pattern recognition receptors are essential mediators of the host immune response. Recently, our lab and others identified a novel sub-group of NLRs that function as negative regulators of inflammation. One of the members of this sub-group, NLRX1, is a potent regulator of interferon, NF-κB and autophagy signaling. Each of these signaling pathways has been linked to diverse types of cancer. Thus, we hypothesized that NLRX1 attenuates tumorigenesis through the negative regulation of overzealous innate immune system signaling. To evaluate this hypothesis, we utilized a urethane based model of tumorigenesis and explored disease progression in Nlrx1-/- mice. Mice lacking NLRX1 exhibited dramatically increased morbidity and mortality and were found to develop a rare neoplasia, diagnosed as histiocytic sarcoma. Of the already rare histiocytic disorders in humans, histiocytic sarcoma is exceedingly uncommon and the rarity of this disease has resulted in a paucity of data pertaining to diagnostic and therapeutic options. Mechanistically, our data indicates that NLRX1 attenuates tumorigenesis through the negative regulation of AKT and NF-κB signaling. These pathways were found to be associated with macrophage hyperproliferation in the absence of NLRX1. Our data extends the function of NLRX1 beyond its currently characterized role in host-pathogen defense and reveals a significant contribution for this NLR in cancer pathobiology.
A novel protein from lamprey immune body with selective cytocidal actions to tumor cells (TUM7P.925)
Authors: Li Qingwei , Pang Yue , Wang Shiyue , Ba Wei , Yu Tao , Liang Hongfang , Bi Dan
Published: January 2023

We purified and identified a novel protein, derived from cells of immune body tissues in the adult lamprey, the protein has strong cytocidal activity against various human cells with a markedly divergent target specificity, recognizes and kills human breast adenocarcinoma MCF-7, human caucasian chronic myelogenous leukaemia k562 cancer cells as well as some classes of human tumor cells, and less cytotoxic to the normal tissue cells. The novel protein induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, mitochondrial and endoplasmic reticulum fragmentation. Then the plasma membrane permeability was increased immediately and most of the cytoplasmic proteins and organelle proteins released into medium from tumor cells after the protein treatment. The novel protein shows specific binding to cell membrane and requires the high content of phosphatidylserine for the effective cytolysis. To confirm the novel protein effect on tumor, tumor xenograft experiments were performed, these results confirm that the novel protein inhibit tumor growth in vivo. Taken together, these observations suggest the novel protein play a crucial role in tumor cells survival and growth.
Lymphatic-targeted liposome vaccines promote long-term immunological memory (VAC10P.963)
Authors: Ma Yifan , Wang Ce , Liu Peng , Cai Lintao
Published: January 2023

Lymphatic-targeted vaccine delivery with liposome formulations has been reported to enhance the immunogenicity of vaccines. However, its effect on long-term immunological memory remains unclear. In the present study, OVA vaccines were formulated by cationic liposomes with different surface modification: DOTAP liposomes (LP), PEGylated DOTAP (LP-PEG) liposomes, and mannosylated DOTAP (LP-Man) liposomes. The in vivo imaging showed that LP mostly accumulated near the injection site, indicating its depot effect. In contrast, LP-PEG and LP-Man liposomes effectively accumulated in draining lymph nodes and the spleen, indicating their lymphatic targeting. More importantly, different liposomes differentially regulate antibody response and B-cell memory. Although LP vaccine led to the highest level of anti-OVA IgG among three liposome formulations on day 40 post priming, they failed to establish an effective memory response. In contrast, lymphatic-targeted LP-PEG and LP-Man vaccines elicited sustained antibody response and robust recall responses 3 months after priming. The enhanced long-term immunological memory by lymphatic-targeted liposome vaccines could be attributed to vigorous and persistent germinal center response, as well as increased Tfh cells and central memory CD4+ T cells in the spleen and lymph nodes. Hence, lymphatic-targeted vaccine delivery with surface modified cationic liposomes could be an effective strategy to promote long-lasting immunological memory.
Th1-promoting adjuvanticity of cholera toxin depends on Gsα in CD11b+ dendritic cells (VAC10P.969)
Authors: Lycke Nils , Mattsson Johan , Yrlid Ulf
Published: January 2023

Despite an extensive literature on the mechanisms of action of cholera toxin (CT) we still lack critical information about how CT augments immune responses. Although several studies have identified dendritic cells (DC) as critical target cells for the adjuvant function there is still not a distinct subset of DCs that has been shown to convey the adjuvant effect. Even more confusing is the fact CT has been considered a strong Th2-skewing adjuvant, but many researchers have found potent IFNγ and Th1 promoting function, despite that CT interferes with IRF8 and blocks IL-12 production. We immunized wild-type, Batf3-/- (lacking CD8α+ DCs) and Cre-lox mice with a CD11c -selective defect in Gsα, the membrane target protein for CT’s ADP-ribosylation. While CT exerted extensive global effects on secondary lymphoid tissues with recruitment and redistribution of both innate and adaptive immunocytes leading to a complete loss of CD4 T cell priming ability, this condition was effectively restored by transferring freshly isolated CD11c+ DCs. As Batf3-/- mice exhibited intact adjuvanticity, CD8α+ DCs were not required for the adjuvant effect. However, we found that CD11b+CD11c+ cells were absolutely needed and that Gsα-expression was critical. CD4 T cell priming was unrestricted and Th1, Th2 and Th17 differentiation was observed also in the absence of IL-12, arguing that CT exerts an adjuvant effect independent of IRF8.
Chimeric-antigen receptor engineered human T lymphocytes from induced pluripotent stem cells for cancer therapy. (VAC11P.1003)
Authors: Themeli Maria , Sadelain Michel
Published: January 2023

We hypothesized that genetic engineering of induced pluripotent stem cells (iPSC) with Chimeric Antigen Receptors (CARs) would be an efficient strategy to generate readily available functionally enhanced T cells targeted to a tumor antigen of interest. Since combined CD28 and 4-1BB signaling promote T cell proliferation and survival, we genetically engineered iPSCs from peripheral blood lymphocytes (T-iPSC) to express 1928z, a second-generation CAR specific to the B-cell lineage antigen CD19, and 4-1BBL. Stably transduced 1928z/4-1BBL-T-iPS lines were then directed to differentiate in vitro into CD3+TCRαβ+ T cells that expressed the 1928z CAR and 4-1BBL on the surface (1928zBBL-TiPSC-T). Upon exposure to cells expressing CD19, 1928zBBL-TiPS-T cells rapidly responded by forming clusters, increased the expression of activation markers and secreted type 1 cytokines. We were able to expand 1928zBBL-TiPSC-T cells and expanded 1928zBBL-T-iPSC-T cells displayed high antigen-specific cytotoxic activity in vitro. Treatment of CD19+ tumor-bearing mice with 1928zBBL-TiPSC-T cells delayed tumor progression and resulted in a significant survival advantage. In summary, the combination of iPSC and CAR technologies we describe here offers a tantalizing new source of “off the shelf” T cells of predetermined antigen specificity and enhanced properties.
Hyper-IL15 enhances human immune function and suppresses hepatitis B virus replication in humanized mice with both human liver and immune system (VAC12P.1024)
Authors: Cheng Liang , Li Feng , Bility Moses , Chi Liqun , Su Lishan
Published: January 2023

HBV infection remains a leading cause of death worldwide. However, the lack of robust small animal model has hindered the progress in hepatitis B virus research and the development of new treatment options. Immunodeficient mice reconstituted with both human immune system and liver provide us a powerful in vivo model to study HBV pathogenesis and evaluate potential therapeutics. Cytokines targeting T cells and NK cells are attractive clinical interventions against viruses. Here we tested the therapeutic effect of human hyper-IL15(IL15 fused with IL15Rα sushi domain) on HBV replication in vivo in our humanized mice with both human liver and immune system. We showed that hydrodynamic injection of HBV1.3 expression plasmid led to persistent HBV replication for at least 4 months in humanized mice. Ectopic expression of hyper-IL15 in humanized mice one week after HBV plasmid injection significantly inhibited HBV replication. The HBV inhibition effect was correlated with increasing human T cell and NK cell numbers and activation in humanized mice. To further test the therapeutic potential of hyper-IL15 on chronic phase of hepatitis B, we treated the humanized mice with hyper-IL15 7.5 weeks after HBV persistence in vivo. Our data showed that the HBV genome was undetectable in 75% of hu-mice 3 weeks after hyper-IL15 treatment. Our encouraging results warrant future exploration of the use of hyper-IL15 as an immunotherapy reagent to treat chronic hepatitis B in humans.
Therapeutic vaccination against tumor pericyte-associated antigens DLK1 and DLK2 (VAC12P.1013)
Authors: Fabian Kellsye , Chi-Sabins Nina , Storkus Walter
Published: January 2023

The tumor microenvironment (TME) promotes unique epigenetic programming of stromal cell populations, including vascular endothelial cells (VEC) and pericytes, leading to differential expression of antigens that may be recognized by therapeutic T cells. These antigenic differences may be targeted immunologically via specific vaccination. We show that pericytes in human and murine renal cell carcinoma (RCC) and melanoma differentially overexpress the antigen Delta-like homolog 1 (DLK-1). DLK-1 is an EGF-like protein that functions as a Notch pathway antagonist. We demonstrate that both peptide and genetic vaccination of RENCA-bearing BALB/c mice against DLK-1 results in tumor growth inhibition and removal of DLK-1 within the TME. However, DLK-1 removal also led to a compensatory increase in DLK-2 expression (a DLK-1 homolog and NOTCH antagonist) in the murine RENCA TME. Using recombinant lentiviruses encoding murine DLK-1 and DLK-2, we show that coordinate genetic vaccination against these two antigens leads to superior therapeutic inhibition of RENCA tumor growth compared to vaccination against either DLK-1 or DLK-2 alone. The coordinate vaccination also resulted in the activation of DLK-1- and DLK-2-specific CD8+ T cells, recruitment of T cells into the TME and normalization of tumor blood vessels. We conclude that vaccination against DLK-1 and/or DLK-2 may represent an effective strategy for vascularized, solid forms of cancer, including RCC and melanoma, among others.
NK cell activation by the heat shock protein gp96 (VAC12P.1018)
Authors: Sedlacek Abigail , Binder Robert
Published: January 2023

Heat Shock Proteins (HSPs) are key mediators in the development of immune responses. Following immunization with HSPs chaperoning tumor antigens, anti-tumor immune responses are elicited. The current model of HSP mediated anti-tumor immunity suggests HSPs bind to and are internalized by antigen presenting cells (APCs), which results in the priming of effector T cells that subsequently inhibit tumor growth. In addition to APCs and T cells, NK cells have also been demonstrated to be essential for HSP mediated anti-tumor immunity. However what role NK cells play in eliciting immunity is poorly understood. In these studies, we aim to characterize the activation of NK cells following stimulation with the prototypical HSP, gp96. Our results show that, following incubation with gp96 in vitro, we observe an increase in the frequency of NK cells containing the intracellular effector proteins granzyme B and IFNγ. Although intracellular levels of IFNγ are increased, we do not detect IFNγ secretion and the intracellular levels are less than what is observed following cytokine mediated activation. Due to these responses, we are currently investigating NK cells for the presence of the HSP receptor CD91. Collectively, these data indicate that directly, HSP stimulation results in partial NK cell activation, and other mechanisms, such as indirect activation via HSP activated APCs, may be necessary to observe optimal NK cell effector functions.
Age influences the lymphocyte response to the ellagitannin oenothein B and acai polysaccharides. (VET2P.1040)
Authors: Ramstead Andrew , Schepetkin Igor , Quinn Mark , Jutila Mark
Published: January 2023

The effects of nutritional supplements on immunity are still not well understood. For example, although aging affects immune cell responsiveness, it is not well known if immune stimulation by some nutritional supplements is also affected by aging. We have previously shown that the ellagitannin oenothein B and Acai berry polysaccharides (AcaiPS) enhance IFNγ production by both bovine and human NK cells and T cells. We wanted to examine the effect age had on this activity. Studies comparing immune responses in immature individuals to those in adults are difficult to perform in humans and mice. As such, we utilized the bovine system to examine the responsiveness of T cells and NK cells from newborn calves, older calves, and adult cows to oenothein B and AcaiPS in vitro. Oenothein B induced greater IFNγ production by T cells from adults compared to calves. Within the responsive T cell population, we found that CD45RO+ T cells expressed more IFNγ in response to oenothein B than CD45RO- T cells. Similar responses were seen in a limited analysis with human cells. In contrast, AcaiPS, both alone and in combination with IL-18, induced greater IFNγ production by NK cells from calves, especially newborn calves, compared to adults. Our data suggest that immune stimulation of T cells and NK cells by oenothein B and AcaiPS is influenced by age. It also suggests that the bovine model may be very useful in examining the effect of age on immune stimulation by nutritional supplements.
Anti-fungal immune responses to Pseudogymnoasces destructans in bats affected by white-nose syndrome (VET2P.1041)
Authors: Field Kenneth , Reeder Sophia , Rogers Elizabeth , James McMichael , Sigler Lauren , Vodzak Megan , Moore Marianne , Johnson Joseph , Reeder DeeAnn
Published: January 2023

White-nose syndrome is a devastating disease affecting bats in North America. It is caused by Pseudogymnoasces destructans, a fungal pathogen that colonizes the muzzles and wings of hibernating bats. Our study focuses on the relative differences between the immune responses of various bat species to this pathogen. The strength and type of immune response triggered by an antigen can be measured by determining the gene expression profile of cytokines. We have developed a quantitative PCR panel for bat cytokine genes in order to measure the immune responses involved in susceptibility to white-nose syndrome. We also used a cytokine ELISA to determine serum cytokine levels. In addition, we analyzed the neutrophil:lymphocyte ratios from blood smears collected both during hibernation and post hibernation, as another measure of immune response over time. By comparing the relative expression of certain cytokine genes, we can determine which subsets of T cells are mediating the immune response to the fungal pathogen. Our research will shed light on the mechanisms by which some bat species have a higher survival rate, and this knowledge can be used to further develop strategies for protecting North America’s bat population.
Mucosal correlates of cross-protection for live-attenuated influenza virus vaccines in pigs (VET2P.1031)
Authors: Hughes Holly , Vincent Amy , Loving Crystal
Published: January 2023

Controlling swine influenza A virus (IAV) has become increasingly difficult with the emergence of novel reassorted strains and introduction of human seasonal IAV into pigs. In North American swine there are 6 antigenically distinct H1 subtypes currently circulating. Live-attenuated influenza vaccines (LAIV) provide broader cross-protection than whole-inactivated virus (WIV) vaccine making LAIV a candidate for next-generation swine IAV vaccines. However, there is no standardized assay to predict cross-protection following LAIV vaccination. Hemagglutination-inhibiting (HI) immunoglobulin (Ig) in serum has long been the gold standard correlate of protection following WIV vaccination; however, LAIV does not elicit a robust serum HI Ig titer. Oral fluids (OF) have become a rapidly developing diagnostic specimen for a variety of animal pathogens. We evaluated nasal wash (NW) and OF samples for measuring cross-reactive Ig responses to a panel of H1 viruses following vaccination. Both NW and OF had detectable levels of IAV-specific Ig when measured by ELISA. NW from LAIV vaccinated pigs had significant cross-reactive IgA titers, while NW from WIV vaccinated pigs did not have detectable IgA. Samples of OF from either LAIV or WIV vaccinated pigs had high titers of cross-reactive IAV-specific IgG and measurable IgA. OF samples also displayed IAV specific neutralization. These data suggest OF can serve as a sample for evaluating LAIV immunogenicity and predicting cross-protection.
Progress in using coral as a model to study intrinsic and innate immune responses to herpes-like viruses (VET2P.1032)
Authors: Garrison Keith , Hernandez Austin
Published: January 2023

Corals have been shown to produce herpes-like viruses when triggered by abiotic stressors. In addition, new associations are being discovered between known coral diseases and viral pathogens. We intend to use coral as a model species to study invertebrate anti-viral immune responses in organisms critical to the resilience of the coral reef ecosystem. Our long-term goals include identifying possible immune genes in corals and measuring changes in gene expression in response to virus production. Fragments of Acropora were propagated in controlled aquarium systems mimicking fundamental properties of shallow reef with live rock, live sand, and artificial seawater. We acclimatized corals by providing sufficient photosynthetic active radiation, feeding, and periodic replenishment of macro- and microelements. RNA extractions were performed on coral specimens to synthesize cDNA for qPCR. Known human innate immune pathways such as STING, cGAS, Factor X, and NF-kB are under investigation using primers targeting the coral orthologs of critical human genes in these pathways. We are optimizing qPCR as a tool to amplify and quantify target genes from cDNA. We are also investigating western blot anaylsis of NF-kB to assess levels of immune activation in coral cells. Eventually, we hope to develop minimally-invasive tests to provide leading indicators of coral stress to help identify and manage threats to reef ecosystem health.
Distinct and overlapping roles for cell intrinsic IL-6 and IL-27 signaling in promoting virus specific CD4+ T cell numbers and function during acute and chronic viral infections (VIR7P.1055)
Authors: Harker James , Dolgoter Aleksandr , Lewis Gavin , Zuniga Elina
Published: January 2023

Chronic viral infections such as human immunodeficiency virus 1 and hepatitis C virus represent a significant global health burden. Virus specific CD4+ T cell number and function are critical in determining the outcome of these chronic viral infections, but little is known about the signals that can promote their survival or helper functions in this environment. By studying both acute and chronic variants of lymphocytic choriomeningitis virus infection of mice we found that the IL-6 cytokine family signal transduction molecule gp130 expression on CD4+ T cells was essential for long term viral control in chronic, but not acute infection. It was critical for virus specific CD4+ T cell survival, differentiation and the production of IL-21, factors which are critical in the control of chronic infections. Dissection of the individual IL-6 family of cytokines revealed that IL-6 was critical for the upregulation of the transcriptional regulator Bcl6 and subsequent escalation in T follicular helper cell responses and development of antiviral antibody. Contrastingly IL-27 was uniquely required in the survival of these virus specific CD4+ T cells at late stages of chronic infection. Use of Il6ra-/-;Il27ra-/- ¬mice showed however that IL-6 and IL-27 had an overlapping role in the production of IL-21. Our data situates the IL-6 cytokine family as central orchestrators of anti-viral CD4+ T cell responses and subsequent immune responses vital during established chronic viral infections.
High levels of interleukin-21(IL-21) are produced during primary dengue infections. (VIR8P.1067)
Authors: Maldonado Rentería Mathywz , Sánchez Vargas Luis , Izaguirre Irma , Hernández Flores Karina , Vivanco Cid Héctor
Published: January 2023

Introduction: IL-21 is produced by CD4 T cells, mainly follicular T cells and exerts multiple and pleiotropic effects on innate and adaptive immune as well as on non-immune cells. IL-21 is involved in various viral infections but has not been described in dengue virus (DENV) infection context. Objective: The aim of this study was to investigate the IL-21 level production during the clinical course of primary DENV infections. Material and Methods: We collected 122 serum samples of primary DENV-infected patients at different days after the onset of symptoms and classified according to their phase of disease (acute vs. convalescent phases), and the clinical severity (dengue fever vs. dengue hemorrhagic fever). IL-21 levels were measured using a quantitative capture ELISA. Results: IL-21 levels were significantly elevated in the disease group compared with the control group (P < 0.0001). IL-21 was detected in primary DENV infections with a significantly higher concentration in the convalescent phase. Conclusions: Our results show for the first time the involvement of IL-21 during the clinical course of DENV infections. We speculate that IL-21 may play a protective role in primary infections.
Peptide presentation by different HLA-Class I molecules during viral infection (APP2P.106)
Authors: Kaabinejadian Saghar , Vernon Stephen , McMurtrey Curtis , Mojsilovic Danijela , Bardet Wilfried , Schafer Fredda , Jackson Kenneth , Buchli Rico , Piazza Paolo , Hildebrand William
Published: January 2023

Class I Human Leukocyte Antigens reveal intracellular viruses to the immune system by presenting viral epitopes at the cell surface. In this study we hypothesized that during infection particular HLA class I alleles consistently present more virus-derived peptide ligands to virus-specific CD8+ T cells than do other HLA class I. To test this hypothesis, we infected cells with West Nile virus (WNV) and directly identified and enumerated the virus specific peptides presented by various HLA class I molecules. Cells expressing a number of different HLA-A and HLA-B class I were cultured in bioreactors and infected with WNV. HLA/peptide complexes were harvested from infected and uninfected cells, peptides were eluted from affinity-purified HLA, comparatively mapped by mass spectroscopy, and sequenced. Twenty-four peptides, 20 eluted from HLA-A complexes (HLA-A*02:01, A*01:01, A*11:01 and A*24:02) and 4 from HLA-B complexes (HLA-B*27:05, B*07:02 and B*35:01), were identified as unique to infected cells. Ligands represented different parts of the viral polyprotein demonstrating that peptides sampled by class I HLA are distributed widely throughout the WNV proteome. These data indicate that, in WNV-infected cells, HLA-A present more virus-specific peptides than do HLA-B suggesting a potentially different role for HLA class I loci in developing immune responses that control WNV infection whereby HLA-A is responsible for diverse reactivity and HLA-B leads to more focused immunity.
Detection and validation of HLA Class I presented viral epitopes using T cell receptor mimics (APP2P.107)
Authors: Kaabinejadian Saghar , McMurtrey Curtis , Kim Sojung , Jain Rinki , Bardet Wilfried , Schafer Fredda , Diamond Michael , Hansen Ted , Weidanz Jon , Hildebrand William
Published: January 2023

Class I Human Leukocyte Antigens distinguish healthy cells from infected cells by presenting peptides at the cell surface. Viral epitopes confirmed as unique to infected cells can lead to successful development of vaccines and therapeutics, and a tool to validate viral epitope presentation on a variety of cell lineages is essential. Here, comparative mass spectrometry shows that the West Nile virus peptide epitopes SVGGVFTSV and ILRNPGYAL are presented by HLA-A*02:01 and HLA-B*07:02 of infected cells. To generate monoclonal antibodies against these HLA/WNV peptide epitopes, mice were immunized with peptide/HLA complexes, splenocytes were fused to myeloma cells, and single clones were picked and grown. Hybridoma supernatants were screened for recognition of the appropriate HLA/WNV peptide complex on peptide-pulsed cells with irrelevant HLA/peptides acting as a negative control. The T cell receptor mimic (TCRm) monoclonal antibodies RL15A and RL29A were found to be specific for A*02:01/SVG9 and B*07:02/ILR9, respectively. The RL15A and RL29A T cell receptor mimic mAb were then used to track viral epitope presentation on WNV infected cell lines and primary cells. In summary, we demonstrate the implementation of a mass spectrometry system for the direct discovery of class I HLA-presented viral epitopes from infected cells followed by the complementary use of TCRm mAb as a companion diagnostic for tracking epitope presentation during the course of a viral infection.