bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4+ T cell-derived Interleukin-21 is required for the efficient clearance of Citrobacter rodentium (MUC5P.744)
Authors: Silberger Daniel , Zindl Carlene , Moseley Carson , Maynard Craig , Vallance Bruce , Zajac Allan , Weaver Casey
Published: January 2023

Interleukin-21 (IL-21) is critical for host defense against infections with intracellular and extracellular pathogens. Although most infections occur following an initial colonization at mucosal surfaces, no studies have addressed the role of IL-21 in controlling bacterial colonization and dissemination from the colon. Using a novel Il21fl/fl reporter and CD4-Cre conditional knockout mouse, we show that IL-21+CD4+ T cells are the dominant source of IL-21 required for the efficient clearance of the enteric bacterial pathogen Citrobacter rodentium. Consistent with the complete Il21-/- mice, we found that mice with a conditional deletion of Il21 from CD4+ T cells had a greater incidence and severity of colitis, increased bacterial dissemination to the spleen and liver, reductions in IFN-γ+ IL-17A- CD4+ T cells, and a decreased ratio of CXCR5+PD-1+ T follicular helper (Tfh) to FoxP3+ T follicular regulatory cells (Tfr) following infection. In association with impaired T cell differentiation, we found that conditional deletion of Il21 from CD4+ T cells lead to a reduction in the number of plasma cells and a reduction in C. rodentium-specific IgG. Finally, using bone marrow chimeric mice, we found that B cells and T cells require IL-21R expression to mediate the clearance of C. rodentium. These findings establish that IL-21-producing CD4+ T cells can inhibit the dissemination of enteric bacterial pathogens and enhance their clearance from the site of infection via humoral immunity.
CD47 deficiency ameliorates autoimmune nephritis in Faslpr mice via suppressing IgG autoantibody production (THER5P.912)
Authors: Shi Lei , Bian Zhen , Lv Zhiyuan , Liu Yuan
Published: January 2023

CD47, a self-recognition marker, significantly regulates both innate and adaptive immune response. To explore the potential role of CD47 in activation of autoreactive T and B cells and the production of autoantibodies in diseases such as systemic lupus erythematosus, we generated CD47-knockout in spontaneous lupus mice (Faslpr). In comparison with Faslpr mice, CD47-/--Faslpr mice exhibited a prolonged lifespan, with no apparent signs of autoimmune nephritis including glomerular cell proliferation, acute tubular atrophy and vacuolization. CD47-/--Faslpr mice had lower level of proteinuria, lower deposition of C3 and C1q in the glomeruli and lesser pronounced splenomegaly compared to the age-matched Faslpr mice. Serum levels of antinuclear antibodies and anti-double-stranded DNA antibodies in CD47-/--Faslpr mice were also significantly reduced. The mechanistic studies further suggested that CD47 deficiency might impair the antigenic challenge-induced production of mouse high affinity IgG but not IgM through reducing T follicular cell production, which results in an impaired formation of germinal centers in lymph tissue. In conclusion, our results demonstrate for the first time that CD47 deficiency ameliorates lupus nephritis in Faslpr mice via suppressing IgG autoantibody production.
Inhibition of branched chain amino acid aminotransferase 1 suppresses the severity of collagen-induced arthritis in mice (THER5P.905)
Authors: Papathanassiu Adonia
Published: January 2023

BCAT is the enzyme responsible for the reverse transamination of branched chain amino acids to corresponding α-keto acids. It exists as two distinct isozymes with mutually exclusive tissue distribution: a mitochondrial one (BCAT2) and a cytosolic one (BCAT1). The present study reports on the effect of BCAT1 inhibition on CIA, a murine model of rheumatoid arthritis (RA). Oral administration of ERG240, a small molecular weight BCAT1 inhibitor, alleviated the severity of the clinical development of CIA and exerted a strong protective effect on the integrity of the joints of affected animals. When the drug was given at 1000 mg/kg 5x per week for 4 weeks the clinical score and paw thickness was reduced by 40%. Immunohistochemical (IHC) analysis of preserved tissues revealed that ERG240 treatment reduced inflammation by ~50%, pannus formation by ~90%, cartilage damage by ~75%, and bone erosion by ~90%. Further IHC analysis indicated that the mechanism of action of ERG240 involved a reduction in the number of macrophages and osteoclasts present in the joints and CD45R+ cells in the bone marrow of the animals. Analysis of terminal sera, collected from treated and control animals, suggested that ERG240 administration also suppresses the levels of secreted cytokines such as TNF-α and RANKL. In vitro, ERG240 inhibited serum-driven migration of bone marrow derived macrophages. Collectively, the data indicate that BCAT1 constitutes a new druggable target for the treatment of RA.
A new bioinformatics analysis can reveal intestinal infection as a cause of graft rejection in islet xenotransplantation in non-human primate. (TRAN1P.940)
Authors: Kim Hyun-Je , Moon Ji-Hwan , Shin Jun-Seop , Kim Bongi , Kim Jung-Sik , Min Byong-hoon , Cho Kyo-ri , Kang Sung-Jun , Byun Nari , Phuong Thuy , Kim Jong-Min , Kim Sun , Park Chung-Gyu
Published: January 2023

Pancreatic islet transplantation is one of the most attractive treatment modality for select type 1 diabetes patients suffering from frequent hypoglycemia unawareness. However, because of the shortage of human donor organs, xenotransplantation is becoming more highlighted. Under this circumstance, we have recently achieved reproducible and consistent survivals of adult porcine islet for more than 180 days in the liver of five diabetic monkeys under immunosuppression regimen base on anti-CD154. Interestingly, among five monkeys, one has exhibited graft failure after having experienced severe intestinal infections. Because the porcine islets were exposed to direct blood flow from the intestine, intestinal infection would exert detrimental effect on porcine islets. To test this hypothesis, we performed RNA-seq with peripheral blood from graft maintaining monkey or rejecting monkey after intestinal infections. By using novel bioinformatics tool called Time-series RNA-seq analysis package, we demonstrated highly relevant activated immunologic pathway in graft rejecting monkey, but not in well maintaining monkey. Consistent with this notion, we further confirmed that porcine islets were indeed rejected by CD8+ T cells by immunohistochemistry. Taken together, we for the first time showed that a new bioinformatics analysis using peripheral blood RNA-seq data can pinpoint intestinal infection as a cause of graft rejection in intraportal islet xenotransplantation.
Endogenous graft-specific Tregs expand and differentiate into effector Tregs during the induction of tolerance to allografts (TRAN1P.949)
Authors: Young James , Chen Jianjun , Miller Michelle , Yin Dengping , Vu Vinh , Moon James , Alegre Maria-Luisa , Chong Anita
Published: January 2023

Peripheral transplantation tolerance is donor-specific and requires the presence of Tregs. We hypothesized that allospecific Tregs are critical for the donor specificity of tolerance. To date, studies of Foxp3+ regulatory T cells (Tregs) in transplantation tolerance have examined endogenous Tregs without defining their antigen specificity, or used seeded populations of TCR-transgenic T cells that only allow the study of induced Tregs. To study endogenous alloreactive Tregs in tolerance, we used a novel transgenic donor mouse that expresses the model antigen 2W and 2W:I-Ab tetramers to track 2W-specific (2W+) Tregs and Tconv cells. We used hearts from F1 (BALB/cxC57Bl/6.Act-2W) mice and C57Bl/6 recipients. Tolerance was induced with anti-CD154 (D0,7&14) + donor splenocytes (D0) or with CTLA-4Ig (D0&2). In both models, 2W+ Tconv increased 3-fold whereas the 2W+ Tregs increased 9-fold over naïve mice. 2W+ Tregs differentiated from a naïve, CD44loCD62Lhi to an effector CD44hiCD62Llo phenotype but 2W- Tregs largely did not. Without treatment, 2W+ Tconv cells increased 17-fold at D30 post-transplant whereas the 2W+ Tregs increased 6-fold. These observations unexpectedly show that graft-specific Tregs expand in tolerance and rejection, and that Tconv cells are uniquely constrained during tolerance induction. The ability to track the fate of endogenous donor-reactive Tregs is a powerful approach to gain insight into their fate in tolerance induction and maintenance.
Transient receptor potential melastatin 2 regulates autophagosome-lysosome fusion in macrophages and protect endoxemia (TUM6P.972)
Authors: zhang kai , Zhang ZhanQin
Published: January 2023

Background: Disruption of autophagy in macrophages can lead to inflammation and exacerbation of sepsis. Transient Receptor Potential Melastatin 2 (TRPM2), a nonselective Ca2+-permeable channel, is important for immune function of macrophage. However, whether TRPM2 participates in macrophage autophagy during endoxemia remains unknown. Methods: Endotoxemia was induced via intraperitoneal injection of lipopolysaccharide (LPS) in wild-type and TRPM2-deficiency mice. The mortality was evaluated. The protein levels of Rab7A and LAMP1 were assayed both in peritoneal macrophages from the LPS-challenged mice and LPS-stimulated bone marrow-derived macrophages (BMDMs). mRFP-GFP-Lc3 distribution in BMDMs were also analyzed by confocal microscopy. Results: TRPM2-/- mice challenged with LPS had increased mortality. In peritoneal macrophages from the LPS-challenged TRPM2-deficiency mice and LPS-stimulated TRPM2-deficiency BMDM, the expressions levels of RAB7A and LAMP1 were significantly decreased. Compared with that in wild-type macrophages, most puncta of mRFP-GFP-Lc3 in transfected TRPM2-deficiency cells were yellow (autophagosome) after LPS stimulation. Conclusions: TRPM2 may regulate the fusion of autophagosomes and lysosomes in macrophages after LPS stimulation, which may play important roles during endoxemia.
Tumor released autophagosomes regulate M2-like macrophage polarization (TUM6P.974)
Authors: Wen Zhifa , Liu Hongxiang , Zhou Meng , Wang Li-xin
Published: January 2023

Tumor cells secrete mediators that modulate macrophage activation and polarization into M2 type tumor associated macrophages (TAMs), which contribute to tumor progression. However, the mechanisms that regulate the polarization are poorly defined. We have previously reported that the enrichment of autophagosomes in tumor cell conditioned medium and malignant ascites from patients. The present study investigated the hypothesis that tumor released autophagosomes promoted M2 polarization. Here we isolated autophagosomes released from murine liver cancer cells using affinity purification. In vivo mouse experiments demonstrated that intraperitoneal injected autophagosomes were efficiently internalized by peritoneal macrophages, which correlated with upregulation of M2 markers. In vitro, coculture of autophagosomes with bone marrow-derived macrophages (BMDMs) also induced macrophage polarization into a M2-like phenotype. Furthmore, this effect of autophagosomes was largely abolished in BMDMs from MyD88 KO mice. Taken together, our findings suggest that, under stress conditions, tumor released autophagosomes may promote tumor progression via regulation of TAMs in tumor microenvironment, providing new insight into TAMs polarization. Keywords: Tumor released autophagosomes, Macrophage, Polarization, MyD88.
Plasminogen activator inhibitor-1 increases migration of monocytes to the tumor and skews their differentiation towards M2 macrophage phenotype (TUM6P.1002)
Authors: Kubala Marta , Placencio Veronica , DeClerck Yves
Published: January 2023

Macrophages exhibiting M2 (pro-tumorigenic) phenotype have been linked to cancer progression. Plasminogen activator inhibitor-1 (PAI-1) is overexpressed by a variety of tumors and indicates poor clinical outcome. This pro-tumorigenic role has been linked to pro-angiogenic and anti-apoptotic functions. Here we demonstrate that PAI-1 is an orchestrator of monocyte migration towards tumor cells and of their M2 polarization. We observed that PAI-1 KO mice xenotransplanted with HT1080 PAI-1 KD cells exhibited tumors less infiltrated with macrophages compared to WT mice (p-value: 1.27E-09). In vitro, recombinant PAI-1 (40 nM) increased monocyte migration by an average 4-fold and increased the expression of CD163 (a M2 marker) by 10-fold (Median Fluorescence Intensity 3 to 30). PAI-1 induced monocyte production of IL-6 and IL-10 to 4000 pg/mL and 600 pg/mL, respectively, and activated STAT3 in the cells. Supporting the critical role for IL-6 and STAT3 in PAI-1 effect on M2 polarization in monocytes, we show that both blocking the IL-6 receptor (tocilizumab) and STAT3 activation (ruxolitinib) in monocytes treated with PAI-1 blocked CD163 expression. Our data point to a potential new pro-tumorigenic activity of PAI-1 via an effect on monocyte migration and polarization.
Scavenger receptor A is involved in tumor associated immune suppression (TUM6P.958)
Authors: Guo Chunqing , Wang Xiang-Yang
Published: January 2023

Tumors activate a plethora of immunosuppressive mechanisms to counteract effective immune responses, e.g. establishing T cell tolerance against tumor antigens or inducing the expansion of T-cell suppressive cell populations, in particular myeloid derived suppressor cells (MDSCs). Our previous studies have demonstrated that scavenger receptor A (SRA) dampened the intrinsic immunogenicity of antigen presenting cells. To determine the potential involvement of SRA in tumor-mediated immune suppression, we examine the expression of SRA on MDSCs during tumor progression and its impact on the suppressive activity of MDSCs. We show that expansion of MDSCs in tumors and peripheral lymphoid organs (e.g., spleen) is dependent on tumor size. SRA is predominantly expressed on monocytic MDSCs (not granulocytic population), which shows a positive correlation with tumor development. Regardless of tumor growth stages, SRA expression in tumor infiltrating MDSCs is substantially higher compared with their counterparts in lymphoid tissues. Interestingly, tumor growth-dependent elevation of SRA expression correlates with the T-cell suppressive activity of MDSCs. Additionally, the absence of SRA strongly promotes the therapeutic efficacy of adoptively transferred tumor-reactive T cells. Our findings suggest that SRA may participate in tumor-mediated immunosuppressive mechanisms and contribute to the immune evasion during cancer progression.
Effector TCD8 use different homing receptor/ligand interactions to enter subcutaneous and intraperitoneal B16 melanoma tumors (TUM7P.1022)
Authors: Woods Amber , Srivinasan Nithya , Engelhard Victor
Published: January 2023

Immunotherapies have the potential to control cancer, but are currently only effective in patients with a preexisting TCD8 infiltrate. TCD8 infiltration into tissues relies on interactions between homing receptors on TCD8 and ligands on the vasculature. However, those required for entry into tumors are incompletely characterized. Using B16 melanoma expressing the strong antigen ovalbumin, the homing receptor ligands VCAM-1 and E-selectin were more highly expressed on SC tumor vasculature, while MadCAM-1 was expressed more highly on IP tumor vasculature. B16 parental tumors and tumors grown in Rag1KO animals expressed negligible levels of VCAM-1 and MadCAM-1, suggesting that activated adaptive immune cells that previously infiltrated the tumor drove their expression. Our previous work showed that anatomical site of TCD8 priming results in differential expression of α4β1, α4β7, and E-Selectin Ligand (ESL) on effectors. We showed that α4β1+ effectors enter SC and IP tumors via interaction with VCAM-1, while ESL+ effectors preferentially enter SC tumors via interaction with E-Selectin. Unexpectedly, α4β7 and MadCAM-1 did not mediate entry into IP tumors, apparently because MadCAM-1 was not expressed on the luminal surface. Our results suggest that the commonly used SC vaccination route generates effectors that may not home efficiently to visceral metastases. Vaccination targeting non-skin draining LN may generate effectors that infiltrate visceral metastases more efficiently.
Adoptive transfer of autologous tumor-infiltrating lymphocytes in metastatic human papilloma virus-induced carcinoma (VAC7P.1040)
Authors: Stevanovic Sanja , Campbell Tracy , Draper Lindsey , Langhan Michelle , Somerville Robert , Wunderlich John , Kwong Mei Li , Kerkar Sid , Yang James , Sherry Richard , Kammula Udai , Rosenberg Steven , Hinrichs Christian
Published: January 2023

Given the constitutive tumor-specific expression of high risk human papilloma virus (HPV) E6 and E7 oncoproteins in HPV+ carcinomas, we evaluated whether adoptive transfer of autologous tumor-infiltrating lymphocytes (TIL) selected for HPV E6/E7 reactivity could mediate regression of metastatic HPV+ carcinomas. TIL were selected preferentially for HPV E6/E7 peptide pool reactivity, when possible. Patients received a single TIL infusion and interleukin-2 to tolerance after non-myeloablative chemotherapy. The treatment regimen led to objective clinical responses in 6/23 patients, including 4/15 cervical cancer, 1/5 anal cancer and 1/3 head and neck cancer patients. Two complete responses occured in cervical carcinoma that are ongoing 20 and 26 months after treatment. The other four responses were partial, three lasted from 3 to 5 months, and one is ongoing at 3 months. The frequency of HPV reactive T cells in the TIL infusion of 17 patients analyzed thus far (including five with a clinical response), as measured by IFNg ELISPOT and CD137 upregulation, correlated with clinical response (P=0.0238 and P=0.0485). In addition, the magnitude of HPV reactivity as measured by IFNg production correlated with clinical response (P=0.0003). Regression of metastatic HPV+ carcinomas can occur after TIL therapy in some patients. Exploratory studies suggest a correlation between HPV reactivity of the infusion product and clinical response.
Adoptive therapy of ROR1+ murine solid tumors with chimeric antigen receptor-modified T cells (VAC7P.1045)
Authors: Srivastava Shivani , Riddell Stanley
Published: January 2023

Adoptive therapy with T cells modified to express chimeric antigen receptors (CARs) holds promise for treating epithelial cancers if target molecules can be identified and strategies developed to overcome the immunosuppressive mechanisms of solid tumors. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed in many blood and solid tumors but is absent from vital adult tissues, making it a potential tumor-specific target for therapy. We developed ROR1-specific CARs comprised of an extracellular single chain antibody linked to various intracellular signaling modules. T cells modified to express ROR1-CARs exerted potent anti-tumor effects against ROR1+ leukemia and lymphoma in vivo, but their effectiveness against solid tumors is not known. To test this, we used the orthotopic 4T1 mouse mammary tumor model that recapitulates the immunosuppressive microenvironment and metastatic pattern of human breast cancer. We transduced 4T1 cells with firefly luciferase (ffluc) to allow in vivo imaging of tumor growth, along with mouse or human ROR1. Murine CD8+ T cells modified to express ROR1-CARs lysed ROR1+ but not ROR1- 4T1 cells in vitro, while unmodified T cells were unable to lyse either ROR1+ or ROR1- 4T1 cells. We are currently evaluating the anti-tumor activity of ROR1-CAR T cells against 4T1-ffluc/ROR1 cells in vivo to define optimal CAR constructs and cell compositions and identify what immunosuppressive pathways to target to improve anti-tumor activity.
Retroviral transfer for a chimeric antigen receptor to generate regulatory T-like cells for the suppression of undesired immune responses. (VAC7P.1036)
Authors: Stavrou Maria , Pule Martin , Holler Angelika , Shlomchik Warren , Stauss Hans
Published: January 2023

Adoptive transfer of antigen specific regulatory T (Treg) cells was shown to be beneficial for the suppression of autoimmunity and Graft versus Host Disease (GvHD). To create a cellular regimen for the targeted suppression of unwanted immune responses, we engineered mouse MHC-class II specific Treg-like cells. The restricted expression of MHCII to professional antigen presenting cells and presence at high levels at all inflammatory sites, allow the employment of MHCII specific Treg-like cells for suppression in various inflammatory settings. A mouse MHCII-specific Chimeric Antigen Receptor (CAR) was used to redirect the specificity of T cells via retroviral transduction, while Foxp3 gene transfer into purified CD4+ cells leads to the acquisition of the Treg-like phenotype. Incorporation of a suicide mechanism within the engineered Treg-like cells, for their in vivo selective deletion, when desired, will ensure the short-term nature of suppression. The functionality of the generated CAR and the specificity of responses elicited by CAR bearing T cells were validated in vitro. In subsequent preliminary in vivo experiments, intravenous injection of C57BL/6 mice with syngeneic mouse MHCII-specific CD4+ T cells, led to GvHD-like toxicity, while no signs of toxicity were observed when Treg-like cells of the same specificity were transferred alone or in 1:1 mix with mouse MHCII specific CD4+ T cells. These data suggest the suppressive potential of the engineered Treg-like cells.
A fusion protein containing the adenovirus type 2 fiber trimerizing and knob domains linked to HIV-1 gp120 exhibits increased nasal immunogenicity in rabbits (VAC8P.1046)
Authors: Staats Herman , Jones Dorothy , Maddaloni Massimo , Pickup David , Permar Sallie , Abraham Soman , Pascual David
Published: January 2023

We previously reported that the inclusion of the trimerizing and knob domains from adenovirus type 2 fiber (Ad2F) in protein immunogens enhanced their mucosal binding and immunogenicity when delivered nasally. This study was performed to determine if Ad2F would enhance the nasal immunogenicity of HIV-1 gp120 from the Clade C Env C.1086. A fusion protein with Ad2F at the C-terminus of HIV-1 gp120 was used as gp120-Ad2F. NZW rabbits were nasally immunized with 100, 200 or 300 µg of gp120 or equimolar doses of gp120-Ad2F fusion proteins adjuvanted with the cationic mast cell activating peptide mastoparan 7 on days 0 and boosted on days 28 - 34. Intramuscular immunization with 100 µg gp120 adjuvanted with an MF59-like adjuvant served as the positive control. Serum anti-HIV-1 gp120 IgG titers were measured 2 weeks after the final boost. While only the 200 and 300 µg doses of gp120 delivered intranasally induced serum anti-gp120 IgG similar to those induced by IM immunization, all doses of gp120-Ad2F fusion proteins induced serum anti-gp120 IgG titers similar to those induced by IM immunization. While the 100 µg dose of gp120 delivered nasally induced serum anti-gp120 IgG titers significantly lower than that induced by 300 µg of gp120 delivered nasally, all doses of gp120-Ad2F induced similar anti-gp120 IgG titers. Our results demonstrate that the addition of Ad2F to HIV-1 gp120 enhances its nasal immunogenicity, lessening the amount of gp120 required for vaccination.
Mycobacterium tuberculosis Ag85C, MPT51, HspX, and the fusion recombinant CMX proteins or rBCG CMX vaccine modulate the innate immune response in a pro and anti inflammatory manner (VAC9P.1103)
Authors: da Costa Adeliane , de Rezende Danilo , Santos Bruno , Machado Renato , Kipnis André , Junqueira-Kipnis Ana Paula
Published: January 2023

Introduction: Mycobacterium tuberculosis produces several proteins that have been associated with its virulence and therefore intensively studied as target for subunit vaccine against tuberculosis. Ag85C, MPT51 and HspX were shown to be immunogenic and antigenic proteins. Also, a fusion protein (rCMX) developed using part of these individual molecules induced specific Th1 and Th17 and poly functional Th cells, that were related to tuberculosis protection. Objectives: This study aimed to understand how this immune response was induced. Methodology: Pulmonary, peritoneal, alveolar, and bone marrow derived macrophages from BALB/c mice were stimulated with the recombinant antigens: Ag85C, MPT51, HspX, CMX or with BCG or rBCG CMX vaccines. Results: All macrophage types responded to Ag85C and CMX with elevated NO production when compared with unstimulated macrophages, while the response to MPT51 and CMX produced urea at similar levels as M2 derived macrophages. HspX did not stimulate macrophages. Pulmonary macrophages infected with BCG produced NO similarly to M1 macrophages, whereas rBCG CMX infection induced urea comparable to M2 macrophages. Conclusion: Ag85C, CMX and MPT51 proteins induced M1, mixed and M2 macrophage phenotypes, respectively. However when CMX was produced within rBCG CMX, a predominant M2 phenotype was observed. The results suggest that the used antigens can modulate the immune response similarly to that shown by rBCG CMX in vivo.
Engineering enhanced vaccine cell lines to eradicate vaccine preventable diseases: the polio endgame (VAC9P.1107)
Authors: Tripp Ralph , van der Sanden S. M.G. , Wu W , dybdahl-Sissoko N , Weldon William , Brooks P , O'donnell J , Jones L , Brown C , Tompkins Stephen , Oberste M , Karpilow J
Published: January 2023

Advances in genomic screening and gene editing technologies provide a means to create the next generation of vaccine manufacturing cell lines. A genome-wide RNAi screen was used to identify gene knock-down events that enhance poliovirus replication with the aim of increasing vaccine yield and reducing the costs and time associated with vaccine production. Primary screen hits were validated with several attenuated and wild poliovirus strains in a Vero cell line employed in vaccine production. Multiple genes were identified that upon silencing increased titers >20-fold, and in some instances, knockdown of a gene or gene combination increased virus titers by >50-fold. Importantly, stable knockout of top candidates from the screen and validation studies using CRISPR technology provided equivalent or improved viral production of multiple serotypes. Several of these gene knockdowns also enhanced titer of enterovirus 71, a clinically relevant virus for which vaccines are being targeted. Host gene silencing events that facilitate poliovirus replication mapped to several cell pathways, and in all cases, gene silencing did not affect poliovirus antigenicity. This study suggests that modulating host genes to increase vaccine production can significantly enhance vaccine production at reduced costs, a finding that should greatly facilitate global implementation of inactivated poliovirus vaccine for polio eradication, and change the paradigm in mammalian vaccine cell line production.
Antiviral inducing properties of staphylococcal enterotoxin mimetic peptides (VAC9P.1065)
Authors: Mujtaba Mustafa
Published: January 2023

Superantigens, like the staphylococcal enterotoxins, activate vast numbers of T-cells to produce large amounts of cytokines, including interferon-gamma (IFN-γ), and eventually cause these cells to undergo apoptosis. The objective of this research was to design mimetic peptides of staphylococcal enterotoxins that are not toxic to cells, but can produce antiviral activity in cells via production of IFN-γ. Based on the amino acid sequences of staphylococcal enterotoxins (SE) A, B, C, and toxic shock syndrome toxin, peptides were designed to mimic the antigenic sites of these superantigens. Whole protein superantigens (SEA) at concentrations ranging from 1.0 to 10.0ng/mL stimulated T-cell proliferation and induced IFN-γ production. Superantigen concentrations at or above 100ng/mL showed cellular toxicity. Of the eight mimetic peptides tested, SEA 3 was the only peptide that induced IFN-γ production, as determined by the IFN-γ ELISA kit, in HPBMC but did not induce cellular proliferation. SEA1, SEA2, and TSST peptides induced cellular proliferation but no IFN-γ stimulation. The peptides showed no toxicity directly on HeLa cells or HPBMC at 100 ug/mL or lower. Cell supernatant from the SEA3 peptide treated HPBMC also had antiviral activity against vesicular stomatitis virus. Thus, this study showed that mimetic peptides of superantigens could be developed that can induce T-cells to produce IFN-γ without the cellular toxicity associated with superantigens.
Zebrafish diverse immunoglobulin domain-containing proteins share structural features and ligands with mammalian CD300 and TREM family members and are linked to a MHC class I gene cluster (VET1P.1126)
Authors: Rodriguez Nunez Ivan , Haire Robert , Cannon John , Litman Ronda , Litman Gary , Yoder Jeffrey
Published: January 2023

Bony fish encode multiple multi-gene families of innate-type immune receptors that are comprised of immunoglobulin (Ig) domains and are of unknown function. One of these families, the diverse immunoglobulin (Ig) domain-containing protein (DICP) genes, recently have been identified and characterized from three chromosomal loci in zebrafish. DICPs possess one or two Ig ectodomains and include membrane-bound and secreted forms. The membrane-bound DICPs include putative inhibitory and activating receptors. Recombinant DICP Ig domains bind lipids with varying specificity, a characteristic shared with mammalian CD300 and TREM family members. Here, we employ PCR based strategies to investigate the expression of DICPs and their variability between individuals from three different zebrafish lines. We demonstrate that certain DICPs display restricted expression in adult tissues while others are ubiquitously expressed. In addition, transcripts of a subset of DICPs are detected during embryonic development suggesting roles in innate immunity or other embryonic processes. Finally, the observation that the expression of certain DICPs is linked to their genetic background led to the identification of a new DICP haplotype that is linked to an alternative MHC class I Z lineage haplotype on chromosome 3. These findings highlight the complexity and dynamic nature of the DICP family within zebrafish and it is likely that differences in DICP haplotype may result in altered immune phenotype.
Alterations in MHCII expression on B cells isolated from cattle naturally infected with bovine leukemia virus. (VET1P.1115)
Authors: Frie Meredith , Coussens Paul
Published: January 2023

Bovine leukemia virus (BLV) is a deltaretrovirus that infects B cells and is widespread in US dairy herds. 83% of US dairy herds are infected with BLV and the within-herd infection burden is estimated to be as high as 46%. BLV infection causes reduced milk production and decreased longevity, which leads to economic losses of $520 million every year. However, BLV is predicted to cause immune suppression in infected cattle, which would increase the economic losses incurred as a result of BLV infection in dairy cattle. To characterize cellular mechanisms of immune suppression in BLV-infected cattle, the expression of MHCII in B cells from naturally infected dairy cattle was investigated. PBMCs were isolated from healthy dairy cattle or naturally infected dairy cattle and the MHCII expression on B cells was assessed in unstimulated or pokeweed mitogen-stimulated culture conditions. qRT-PCR and flow cytometry were used to determine the change in MHCII expression on B cells isolated from BLV-infected cattle. BLV-infected cattle have a significantly higher mean relative percentage of B cells expressing MHCII, but significantly reduced mean fluorescence intensity of MHCII. This could suggest that B cells from BLV-infected cattle are less efficient at presenting peptide antigens to CD4+ T cells.
Interaction between feed management systems and omega-3 fatty acid supplementation on bovine immunization parameters (VET1P.1116)
Authors: Clarke Sandra , Fredeen Alan , Glover Kathleen , Fillmore Sherry , MacLaren Leslie , Enright Catherine , Green-Johnson Julia
Published: January 2023

A trial was designed to investigate impacts of omega-3 fatty acid feeding regimes on bovine immunization responses. Dietary supplements contained either fish oil or microalgae as omega-3 fatty acid sources, and cattle were divided into 2 feed management systems; they either grazed pasture or were kept in barn tie stalls and fed total mixed rations (TMR). Cattle were immunized with keyhole limpet hemocyanin (KLH), a standard T cell dependent antigen. Dietary supplements and feeding systems influenced the response of cattle to immunization with KLH. Anti-KLH IgM, IgG and IgA antibody levels in serum were significantly higher in cattle grazing pasture than those fed TMR. Cattle consuming microalgae had consistently higher anti-KLH IgG2 and IgM antibody levels compared to cattle fed fish oil. Effects of omega-3 fatty acid supplements and TMR versus pasture-based diets were also apparent in cytokine profiles from spleen and cecal tissues. Levels of transforming growth factor-beta (TGF-β) were highest in cecal tissue of pasture-grazing cattle (P < 0.0001), and spleen TGF-β levels were highest in pasture-grazing cattle consuming microalgae (P = 0.0073).These findings suggest that effects of omega-3 supplements on bovine immunity are influenced by the feed management system, with heightened effects on antibody production observed with a pasture system relative to a confinement and TMR-based diet.
Influenza infection results in upregulation of inflammatory and atrophy genes in murine skeletal muscle (VIR5P.1146)
Authors: Bartley Jenna , Hopkins Jake , Haynes Laura
Published: January 2023

Though influenza is a respiratory infection, myalgia is a common symptom and associations with acute myositis and rhabdomyolysis have been noted. It is currently unclear if these symptoms are secondary due to the systemic response or if muscle may contribute to inflammation. Further, the effect of influenza on muscle damage and potential repair is not well established. We aimed to determine the expression level of select myokines, atrophy, and myogenic markers in skeletal muscle during influenza infection. Young C57BL/6 mice were inoculated with Pr8 influenza virus (IN, 500 EID50) and sacrificed 7 or 15 days post infection (DPI) or were uninfected (n=4/group). Gastrocnemius gene expression was analyzed via RT-qPCR. Myostatin, IL4, IL6, IL6 receptor alpha, Ubiquitin B, Ubiquitin C, Foxo1, Atrogin-1, and MuRF-1 gene expression was upregulated 7 DPI indicating increased inflammation, protein degradation, and atrophy. Expression of positive regulators of muscle growth and maintenance (Igf-1, MyoD, and Mef2C) was downregulated at this time as well. At 15 DPI Atrogin-1 and MuRF-1 were downregulated though upregulation of myogenic markers did not occur. Our results indicate influenza infection is associated with muscle-localized inflammation, protein degradation, and atrophy that is resolved by day 15 post infection. It is known that influenza infection in the elderly leads to increased disability and loss of independence; it is likely muscle atrophy is a contributing factor.
Mapping the immunostimulatory activity of a Sendai virus defective viral genome (VIR5P.1152)
Authors: Xu Jie , Mercado-Lopez Xiomara , Lopez Carolina
Published: January 2023

Defective viral genomes (DVGs) generated during Sendai virus infection are the primary triggers of the host antiviral response. DVGs induce the expression type-I interferons (IFN) and other cytokines upon binding through the intracellular viral sensors RIG-I and MDA5. The molecular mechanism behind the superior immunostimulatory activity of DVGs is unknown. To identify RNA motifs that provide potent immunostimulatory activity to DVGs, we generated a series of deletion mutants of a prototype DVG derived from Sendai virus. In vitro transcribed RNA from these mutants were tested for their ability to induce type I IFNs upon transfection. In silico single strand RNA modeling of the mutants folding identified an AU-enriched stem loop domain formed by nucleotides 70-114 of the DVG that is essential for type I IFN induction. Consistent with this prediction, we demonstrate that mutants lacking this region lose their stimulatory activity, while mutants that kept intact this region preserved it Thus, a minimal RNA motif at the 5’ but not the 3’ complimentary sequence is critical for maximal DVG activity and oligonucleotides including such region may represent novel alternatives to be harnessed as potent adjuvants for vaccination.
Vitamin A and D deficiencies affect antibody isotype distribution and the respiratory virus-specific immune response (VIR5P.1161)
Authors: Hurwitz Julia , Jones Bart , Sealy Robert , Penkert Rhiannon , Thomas Paul , Surman Sherri
Published: January 2023

Vitamins A and D are highly interactive in their binding of receptor complexes RAR-RXR and VDR-RXR, which regulate genes essential for normal immune function. To determine how vitamins impact the immune response toward respiratory viruses, we employed mice deficient in vitamin A (VAD), vitamin D (VDD), or both. Analysis of virus-specific antibody-forming cells (AFCs) in VAD mice following influenza virus infection revealed a reduction in IgA-producing AFCs in the respiratory tract mucosa, a first line of defense against respiratory viruses. The IgG/IgA ratio was increased in VAD, but not VDD mice. Interestingly, in double-deficient mice, inhibition was exacerbated to the extent that there were virtually no detectable virus-specific AFCs in the lower respiratory tract. To determine if vitamin deficiencies impact antibody isotypes in humans, we examined blood samples from residents of Memphis, TN. A substantial fraction of individuals exhibited low levels of vitamin A, D or both. As in mice, vitamin A and IgA levels were correlated. When vitamin A was low, antibody isotypes encoded by CH genes at the 3’ end of the heavy chain locus (IgA and IgG2) were poorly represented relative to those encoded at the 5’ end (IgM, IgG3 and IgG1), indicating abnormal CH switching or improper maturation or stability of switched AFCs. Results help explain how vitamin deficiencies render humans vulnerable to respiratory virus disease and encourage rapid implementation of corrective measures.
CD8 T cells are sufficient to induce blood-brain barrier disruption as sole perforin expressing cell type during viral infection (VIR6P.1167)
Authors: Willenbring Robin , Johnson Holly , Jin Fang , Manhart Whitney , LaFrance Stephanie , Pirko Istvan , Johnson Aaron
Published: January 2023

Despite numerous neurological disorders characterized by central nervous system (CNS) vascular permeability, the immune factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. To address this, we developed an inducible model of BBB disruption using Theiler’s murine encephalomyelitis virus (TMEV). This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and is perforin dependent. However, the cellular source of perforin has remained unidentified. Various immune cells, including CD8 T cells, express perforin and therefore could each contribute to BBB disruption. In order to investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. Using small animal magnetic resonance imaging (MRI), confocal microscopy, among other techniques, we determined C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with those associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.
Dissociation of beta-2 microglobulin determines the surface quality control of MHC class I molecules (APP5P.109)
Authors: Springer Sebastian , Montealegre Sebastián , Hein Zeynep
Published: January 2023

MHC class I proteins are endocytosed from the cell surface and destroyed rapidly once their peptide ligand has dissociated. The molecular mechanism of this cellular quality control process, which prevents re- binding of extraneous peptides and thus erroneous immune responses, is unknown. To identify the nature of committed step and its location, we have followed the removal of optimally and suboptimally peptide-loaded murine H-2Kb class I proteins from the cell surface. We find that the binding of their light chain, beta-2 microglobulin (β2m), protects them from endocytic destruction. Thus, the extended survival of suboptimally loaded Kb molecules at 25 °C is due to decreased dissociation of β2m. Since all forms of Kb are constantly internalized but little β2m-receptive heavy chain is present at the cell surface, it is likely that β2m dissociation, and recognition of the heavy chain for lysosomal degradation, takes place in an endocytic compartment.
Calculating the efficiency of direct MHC class I antigen presentation of a model antigen in a lymphoblastoid cell line (APP5P.110)
Authors: Simmons Ryan , Palmer Amy , Dolan Brian
Published: January 2023

The direct MHC class I antigen presentation pathway presents endogenous peptides at the cell surface, allowing the body to mount a CD8+ T cell response in order to eliminate diseased or damaged cells. In order to target this pathway for disease treatment, we must first understand the efficiency of antigen presentation, or the number of proteins that must be degraded in order to present one peptide-MHC complex at the cell surface. Previous studies have primarily examined the efficiency of proteins produced by infectious agents with values ranging from 0.03%-25. In this study, we investigated antigen presentation efficiency of different recombinant fusion proteins expressed in a human lymphoblastoid cell line expressed as self-proteins. These proteins are composed of a destabilization domain, an antigenic peptide that is presented on MHC class I HLA-A2 molecules, and a fluorescent reporter. We calculated the efficiency of antigen presentation in JY cells to be 0.35%. This value is considerably lower than what was previously observed with similar constructs in murine EL4 cells. We also found that treatment of the cells with either TLR agonists or IFNg, which have both been shown to increase MHC class I expression, did not alter the efficiency of antigen presentation. These findings suggest that the efficiency of MHC class I antigen presentation may be mechanistically distinct from common immune signaling pathways.
The hierarchy of two different immunodominant epitopes influences CD4+ T cell responses (APP5P.112)
Authors: Kim Ae , Hartman Isamu , Lanar David , Boronina Tatiana , Cole Robert , Sadegh-Nasseri Scheherazade
Published: January 2023

A cell-free antigen processing system for MHC class II was recently reported that has proved to be effective in identifying physiologically relevant CD4+ T cell epitopes from proteins (Hartman/Kim, et al. Nat Med. 16, pp1333-40 (2010)). This system has also provided insights into the mechanism of epitope selection (Kim, et al. Nat Commun. 5:5369 (2014)). We applied this system to a mixture of two different antigens, H5N1-HA1 and LSA-NRC of malaria to examine if there would be a competition for epitope capture. While each dominant epitope from either protein was identified in the system individually, when both proteins were simultaneously present, HA1 dominant epitope was predominantly selected. The competition between different epitopes was verified in vivo by immunizing HLA-DR1 transgenic mice with a mixture of HA1 and LSA-NRC. A recall T cell proliferation showed that T cells responded only to the HA1 and its dominant epitope. These observations did not change even when DR1 mice were immunized with increasing molar ratio of LSA-NRC to HA1 (1:1, 2:1 or 4:1). However, T cell responded to both antigens if mice were immunized first with LSA-NRC dominant epitope followed by HA1 epitope. These findings show a hierarchy for the selection of dominant epitopes from different proteins during antigen processing. Immunodominance hierarchy among different antigens influences the epitope presentation to the same MHC II, which can be important factor for designing combination vaccines.
Use of functional polymorphisms to elucidate the peptide binding site of the transporter associated with antigen processing (TAP) complexes (APP5P.101)
Authors: Geng Jie , Pogozheva Irina , Raghavan Malini
Published: January 2023

The major histocompatibility (MHC) class I antigen presentation pathway plays an important role in alerting the immune system to virally infected and transformed cells. TAP translocates peptides from the cytosol to the endoplasmic reticulum lumen for peptide loading onto MHC class I molecules. The peptide specificity of TAP has been correlated with the antigenic peptide repertoire of MHC class I molecules in several species, but the precise location of the peptide binding site of TAP is unknown. Polymorphisms in rat and chicken TAPs influence peptide transport selectivity. Combining homology modeling of TAP with experimental measurements of peptide binding selectivity, we demonstrate several residues of rat TAP that are critical for the recognition of basic or hydrophobic residues at the peptide C-terminus, as well as of side chains near the peptide N-terminal and central regions. Based on these interaction sites of peptides with rat TAP, we propose models for rat, chicken and human TAPs in complex with selected peptides. The bound peptides adopt bent conformations and occupy a site near the cytosolic membrane surface, which is similar to that for glutathione derivatives in the NaAtm1 transporter, highlighting conserved elements of bacterial detoxification and eukaryotic immunity-related proteins. These experimentally verified models will facilitate the development of improved epitope discovery tools for assessments of CD8+ T cell prevalence and function in diseases.
The ER-resident protease ERAAP generates CD4+ T cell epitopes (APP5P.104)
Authors: Yang Soo Jung , Shastri Nilabh
Published: January 2023

ERAAP, the ER aminopeptidase associated with antigen processing is well established as a critical player in shaping the peptide repertoire presented by MHC class I molecules to CD8+ T cells. Remarkably, we found that ERAAP also influences the repertoire of endogenous peptides presented by MHC class II molecules to CD4+ T cells. ERAAP-knock out mice elicited CD4+ T cells in response to ERAAP-dependent peptides presented by Ab MHC class II molecules. The generation of these peptides was TAP-independent but required endosomal proteolysis. Further supporting its role in MHC class II processing pathway, ERAAP was found in Lamp1+ vesicles in addition to its normal ER location in mature dendritic cells. Thus, a novel ERAAP-dependent endosomal pathway influences the peptide repertoire presented by MHC II molecules and CD4+ T cells responses.
Streamlined protocol for TCR sequencing, cloning, and in vivo functional analysis of islet antigen reactive human T cells (BA11P.131)
Authors: Sprouse Maran , Redondo Maria , Bettini Matthew , Bettini Maria
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of insulin secreting beta cells present in the islets of Langerhans. A large body of evidence has implicated T cells as the predominant mediators of beta cell destruction leading to T1D development. The goal of our research is to develop a system for isolation and characterization of diabetogenic CD4+ T cells from the peripheral blood of early onset T1D patients. We were able to obtain a blood sample from a treatment-naïve T1D patient at the time of diagnosis. Through a novel multiplex-nest PCR protocol, we cloned corresponding alpha and beta TCR chains from individual islet antigen-specific T cells and tested them in vitro for antigenic specificity and HLA restriction. Utilizing a humanized TCR/HLA retrogenic mouse system, we are testing human islet antigen reactive TCRs for their in vivo functionality and diabetogenic potential.