bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.
Lack of TNFR2 expression by CD4+ T cells limits IL-2 production and promotes Th17 differentiation (BA11P.138)
Authors: McKarns Susan , Miller Patrick , Bonn Michael 
Published: January 2023

Abstract
Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.
Identifying the cytokines provided by antigen-presenting cells critical for generating an encephalitogenic T cell (BA11P.126)
Authors: Lee Priscilla , Smith Alan , Yang Yuhong , Racke Michael , Lovett-Racke Amy 
Published: January 2023

Abstract
Multiple sclerosis (MS) is a CD4 T cell-mediated demyelinating disease; however, what determines the encephalitogenicity of T cells remains unclear. Recognizing myelin peptides is not sufficient, as the frequencies of myelin-reactive T cells are similar between MS patients and healthy individuals. In experimental autoimmune encephalomyelitis (EAE), anti-CD3/CD28 antibodies-generated myelin-specific T cells do not transfer disease, but antigen-presenting cells (APC)-generated ones do. This suggests that the cytokines secreted by APC provide critical signals beyond T cell receptor activation and co-stimulation, contributing to an encephalitogenic phenotype. To identify the critical cytokines, myelin-specific T cells were activated with anti-CD3/CD28 in the presence of various cytokines, and the cells were adoptively transferred into naïve recipients. T cells generated with the combination of IL-6/IL-23 or IL-12/IL-23 induced severe EAE. We showed that IL-6 and IL-12 induced the initial expression of IL-23R on naïve T cells, and IL-23 further enhanced the expression of its own receptor. Neutralizing IL-6 or IL-12 in the APC-T cell culture significantly reduced the encephalitogenicity of T cells. Furthermore, IL-23R signaling induced STAT3 and STAT4 phosphorylation in both IL-6/IL-23 and IL-12/IL-23-generated T cells. Taken together, we have identified the minimal signals required to generate an encephalitogenic T cell, which provides potential targets for novel MS therapy.
Mathematical modeling of lymphocyte kinetics reveals true residence times in and preferential migration of lymphocytes via ovine lymph nodes (CAM4P.157)
Authors: Ganusov Vitaly , McDaniel Margaret 
Published: January 2023

Abstract
There are many published values of lymphocyte residence times in lymph nodes of various mammals such as mice, rats, and sheep. Yet, most of these estimates have been obtained semiquantitatively, without the use of mathematical modeling, which raises a question about assumptions underlying such estimates. We developed a novel mathematical model for migration of lymphocytes in the whole body which takes into account the known biological details of how lymphocyte enter and exit major lymphoid tissues. We applied this novel mathematical model to experimental data on cannulation of different ovine lymph nodes (LNs). In particular, we predicted that in sheep, recirculatory lymphocytes spend on average 3 hours in the spleen and 20 hours in LNs with a distribution of residence times in LNs following a skewed gamma (lognormal-like) distribution. This is in contrast which a recent suggestion that the distribution of residence times of naïve T cells in murine LNs is exponential. We also found that lymphocytes isolated from the prescapular LN have a two-fold increased entrance rate into the prescapular LN as opposed to the intestinal LN. Importantly, the data could not be explained by preferential retention of cells in a specific LN. Taking together, our work illustrates the power of mathematical modeling in predicting the kinetics of lymphocyte migration in sheep and provides quantitative estimates of lymphocyte residence times in major ovine lymphoid tissues.
IL-33/ST2 correlates with severity of haemorrhagic fever with renal syndrome and regulates the inflammatory response in Hantaan virus-infected endothelial cells (CCR5P.205)
Authors: Zhang Yusi , Zhang Chunmei , Zhuang Ran , Ma Ying , Zhang Yun , Yi Jing , Jin Boquan 
Published: January 2023

Abstract
Hantaan virus (HTNV) causes a severe lethal haemorrhagic fever with renal syndrome (HFRS) in humans. Despite a limited understanding of the pathogenesis of HFRS, the importance of the abundant production of pro-inflammatory cytokines has been widely recognized. IL-33 has been demonstrated to play an important role in physiological and pathological immune responses. After binding to its receptor ST2L, IL-33 stimulates the Th2-type immune response and promotes cytokine production. Depending on the disease model, IL-33 either protects against infection or exacerbates inflammatory disease, but it is unknown how the IL-33/ST2 axis regulates the immune response during HTNV infection. We found that the levels of IL-33 and sST2 were significantly higher in HFRS patients’plasma than in healthy donors and the elevated IL-33 and sST2 levels were positively correlated with the disease severity. Furthermore, we found that IL-33 enhanced the production of pro-inflammatory cytokines in HTNV-infected endothelial cells through NF-κB pathway and that this process was inhibited by the recombinant sST2. Our results indicate that the IL-33 acts as an initiator of the "cytokine storm" during HTNV infection, while sST2 can inhibit this process. They may exert their pro- and anti- functions in inflammatory response during HTNV infection, respectively. Our findings indicated that the IL-33/ST2 axis may be exploited as a therapeutic target for Hantavirus infections.
Thymocyte requirements for normal thymic medullary epithelial cell development (HEM7P.227)
Authors: Williams Joy , Jeon Hyein , Hodes Richard 
Published: January 2023

Abstract
The thymic medullary compartment has a central role in tolerizing developing TCRαβ+ CD4 and CD8 single positive (SP) thymocytes. In turn, SP thymocytes are required for normal maturation of medullary thymic epithelial cells (mTEC). In the absence of TCRαβ+ SP thymocytes, the thymic medullary compartment is much smaller and numbers of mTEC are dramatically reduced. We recently demonstrated that the requirement for TCRαβ+ SP thymocytes in mTEC development can be overcome when Traf3, an inhibitor of NIK-dependent NF-κB signaling, is absent in TEC. In contrast to TCRα KO mice, TCRα KO/TEC-specific Traf3 KO mice have mTEC numbers comparable to that of wildtype mice. Traf3 inactivation also overcomes the requirements for LTβR and CD40 in mTEC development. Interestingly, however, we find that mTEC cell numbers are not rescued in TCRβ KO/TEC-specific Traf3 KO mice. TCRβ KO mice, like TCRα KO mice, lack SP thymocytes but also lack post-β-checkpoint TCRβ+ DN and DP thymocytes. This result suggests that a cell type(s) other than a TCRαβ+ SP thymocyte is required for normal mTEC development even in the absence of Traf3 and that this requirement is mediated by molecular interactions other than those involving LTβR and CD40. Studies to determine the identity of this thymic cell population and the corresponding molecular pathways involved in mTEC development are underway.