bluuebunny/crossref_metadata_2025 huggingface dataset:
The complex of damage-associated molecular pattern and its inducer, pathogen-associated molecular pattern enhance triggering innate immunity in shrimp (VET1P.1128)
Authors: Chen Jiann Chu
Published: January 2023

Peptidoglycan (PG) derived commonly from Gram-positive bacteria, is one of pathogen-associated molecular pattern (PAMP). Incubating hemocytes of shrimp in PG caused degranulation, changes in cell size, reduction in the percentage of cell viability, necrosis of hemocytes, and released intracellular molecules containing damage-associated molecular pattern (DAMP) that is well known in mammals and teleosts. Incubating shrimp hemocytes in PAMP, DAMP or the mixture of PAMPs plus DAMPs all induced significant increases in phenoloxidase (PO) activity and respiratory burst (RB, release of superoxide anion) in vitro. The PO activity and RB induced by the mixture of DAMP-PAMP were much greater than that induced by DAMP alone or PAMP alone. In conclusion, the mixture of DAMP-PAMP cause enhancement in eliciting innate immunity of shrimp. This is the first study to confirm that cell necrosis caused by PAMPs releases intracellular molecules, DAMPs, and the mixture of DAMP-PAMP elicit the innate immunity in an invertebrate.
The role of mobile elements in the evolution of pathogen-sensing gene clusters in coral (VET1P.1129)
Authors: Garrison Keith , Moore Christine
Published: January 2023

While the molecular mechanisms of coral immune responses are unclear, the Acropora genome reveals complex gene networks associated with innate immunity, apoptosis, and autophagy. We are interested in whether mobile genetic elements help drive the evolution of disease resistance gene clusters in corals. The purpose of this project is to mine the coral genome for pathogen recognition and disease resistance loci in the leucine-rich repeat (LRR) and immunoglobulin (Ig) families to map and analyze regions with an increased density of these genes. Pfam searches in the A. digitifera genome for Ig and LRR loci were performed. Ig- or LRR-containing regions were clustered based on genome location/scaffold position, and the resulting genome sequence region was extracted. The identities of the translated protein sequences within the extracted regions were determined using BLAST. LRR or Ig designations were annotated onto their corresponding open reading frame sequences. CENSOR was used to detect and annotate mobile element sequences within each cluster. We built phylogenetic trees to analyze relationships of the LRR and Ig loci within and between these clusters to determine whether similar mobile element sequences were associated with the amplification and dissemination of disease resistance loci. We found related resistance gene sequences associated with similar mobile element sequences both within and across resistance gene clusters.
Differentially expressed immune related genes in bovine neonatal development (VET1P.1114)
Authors: Pasman Yfke , Merico Daniele , Kaushik Azad
Published: January 2023

A thorough understanding of the developing bovine neonatal immune system is important for the development of novel vaccines and therapeutics, as well as to improve breeding practices and farm management, in order to increase the survival and productivity of calves. In this study blood samples were taken from neonates on day of birth before colostrum feed (day 0), and at 7, 14 and 28 days after birth. RNA was extracted from peripheral blood mononuclear cells present in the samples and processed for next generation sequencing. An average of 27 million 151bp reads per sample was generated, and global analysis of the whole transcriptome was performed, based on Hereford cow genome assembly UMD3.1. Differential expression analysis (DE) of immune response related genes were performed at each time point, as well as gene-set enrichment analysis to identify DE genes in immune related pathways comparing day 0 and 7. The analysis identified several immune response related functional coherent DE gene-sets, for example: immune response, defense response, regulation of defense response, cytokine production, and response to cytokine. Within these clusters, the genes with significantly altered expression rates (False Discovery Rate FDR<0.05) and logFC>1 or logFC<-1) were identified. These studies will help to elucidate the mechanisms that are important in bovine neonatal immune development, which will contribute to the development of targeted strategies for disease prevention.
Deciphering the adaptive immune system of bats (VET2P.1131)
Authors: Martínez Gómez Julia , Wang LinFa , Alonso Sylvie
Published: January 2023

Bats are natural reservoirs for several viruses, many of which cause morbidity and high mortality rates in other mammals, including humans but they generally don’t cause disease in bats. In fact, fruit bats have been found to be the principal suspect that initiated the latest 2014 West-Africa Ebola outbreak. How bats cope with such deadly viruses without getting sick remains unknown. Efforts are being increasingly devoted to dissect the bat’s immune system, however, due to the lack of reagents progress has been limited. Our group is focusing on characterizing bats adaptive immunity. The first step consists of generating a systematic knowledge on the various immune cell populations (T and B cells mainly). Subsequently, functional assays will be optimized to study how bats adaptive immunity reacts to viral infections. Screening of human and mouse antibodies against the main components of the bats adaptive immunity revealed poor cross-reactivity to T and B cell main surface markers (CD3, CD4, CD8, CD19). Combining flow cytometry and real-time PCR approaches, we have been able to characterize different T cell subset populations present in bats spleen and blood. Understanding how bats control viral infection can provide important insights into anti-viral strategies that may result in the development of new therapeutics for other mammals
Generalised inflammation with CD3ε and MHC class II positive cells and dislocated epithelial cells in the mid-intestine in farmed Atlantic salmon (Salmo salar L.) (VET2P.1132)
Authors: Bjørgen Håvard , Koppang Erling , Kaldhusdal Magne , Moldal Torfinn , Dale Ole-Bendik
Published: January 2023

Farmed Atlantic salmon is routinely fed with pellets containing vegetable ingredients. Certain plant proteins may induce an inflammatory environment in the intestinal tract of fish, and carcinogenesis has also been noted. The pathogenesis is similar to that of inflammatory bowel disease (IBD)-related cancer in humans. A feed trial was designed to stimulate to inflammation in experimental fish. Three separate groups were subjected to feeding regimes where two of the groups received feeds containing different plant ingredients (A and B), and a third group received a diet based on marine ingredients. Immunofluorescence targeting CD3ε and MHC class II were used to study the inflammation. Epithelial cells were identified with haematoxylin & eosin staining and immunohistochemistry. A pan cytokeratin antibody was used to identify epithelial cells, and a proliferating cell nuclear antigen (PCNA) antibody was used to mark proliferating cells. Periodic acid Shiff (PAS) staining was used to detect mucins. Immunofluorescence revealed severe generalised inflammation and various amounts of dislocated epithelial cells in the fish receiving experimental diet B. The fish given experimental diet A was less affected. The control group was unaffected. There was a clear association between inflammation and dislocated epithelial cells. The inflammatory response was dominated with T-cell infiltrates in a similar fashion as that seen in IBD-affected human intestine.
Extensive cell-mediated cross-reactivity between diverse influenza strains in the ferret model (VIR5P.1143)
Authors: Reber Adrian , Music Nedzad , York Ian
Published: January 2023

Influenza causes widespread epidemics affecting the world’s population. The virus achieves this on a yearly basis due to its ability to escape prior immunity by rapidly mutating surface proteins, the target of neutralizing antibodies. In contrast, the internal viral proteins, targets of the cell-mediated immune (CMI) response, are highly conserved, and have potential for cross-protection. However, little has been done to comprehensively evaluate the true extent of CMI cross-reactivity between influenza strains. Unvaccinated or vaccinated ferrets were challenged with 2008-2009 pandemic H1N1 influenza, A/Perth/16 (H3N2), or mock challenged. Cellular responses were examined 14d post-infection by stimulation with a diverse array of influenza viruses, and IFN-γ production by CD8+ and CD4+ T cells was assessed by flow cytometry. CMI responses exhibited extensive cross-reactivity across the majority of strains examined, but not to an unrelated virus, canine parainfluenza. Cross-reactivity was observed even between influenza A and B strains. While prior vaccination had little effect on the breadth of cross-reactivity, cross-reactive CD4+, but not CD8+ responses were heightened by prior vaccination. Challenge with pandemic H1N1 drove better cross-reactivity with B strains. These results confirm the widely held belief that CMI responses exhibit extensive cross-reactivity and extend this concept to strains considered antigenically distant such as influenza A and B strains.
Human γδ T cells: diverse orchestrators of the host response to influenza infection. (VIR5P.1145)
Authors: Calverley Matthew , McClaren Jennifer , GUO XIZHI , Dash Pradyot , Thomas Paul
Published: January 2023

While often mild, influenza infection can occasionally progress to severe disease and even death. It is recognized that disease progression and outcome is often worse in the young and the old; however, the reason infection progresses in some individuals and not others is still unclear. Previous findings, reported by our laboratory, from a five-year longitudinal human study have shown that a definable inflammatory cytokine signature correlates with poor clinical outcome. Moreover, we have shown that this cytokine profile correlates with inflammatory monocyte numbers. Here we show a correlation between γδ T cell numbers and inflammatory monocyte numbers during infection within this clinical cohort. Further, we show that a subset of peripheral γδ T cells exhibit a surprisingly high T cell receptor (TCR) variable region gene diversity in infected individuals. Additionally, the CDR3 TCR sequences of this cellular subset show very high diversity, while retaining repetitive VJ gene junction-determined conserved motifs. The data presented here suggest that human peripheral γδ T cells are a more diverse cellular population than previously realized and are capable of playing a pivotal role in influencing the local immune environment during influenza infection with the potential to effect long-term outcome.
Age-related prevalence of serum hemagglutination-inhibition antibodies cross-reactive to 2012 and 2013 newly emerging swine-origin influenza A(H3N2) variant viruses (VIR5P.1134)
Authors: Liu Feng , Veguilla Vic , Gross Felicia , Levine Min , Xu Xiyan , Tumpey Terrence , Katz Jacqueline , Lu Xiuhua
Published: January 2023

Recent outbreaks of swine-origin influenza A(H3N2) variant virus (H3N2v) in the U.S., mainly among young children with swine exposure, have raised concerns. Previous studies demonstrated older children and young adults had the highest hemagglutination-inhibition (HI) antibodies (Ab) to 2011 H3N2v viruses. In 2013, newly emerging antigenic variants of H3N2v viruses, responsible for 18 human infections, acquired N145K/R mutations in hemagglutinin (HA). Here, human and postinfection ferret sera were tested in HI assays against 2012-13 H3N2v viruses possessing 145N, K or R HA mutations, together with nine historical seasonal H3N2 (sH3N2) viruses circulating since 1968. We found the H3N2v viruses were antigenically distinct but retained a low degree of serologic cross-reactivity (CR) (with ferret antisera) to sH3N2 viruses that circulated in 1990s. Across all age groups (6-80+ years), approximately half were seropositive (HI titer≥40) to the H3N2v viruses. The age related seroprevalence among children were likely associated with primary exposure to sH3N2 viruses in 1990s. We observed a single substitution at 145 in HA was sufficient to change an individual’s seropositivity to the virus and significantly influenced the seroprevalence in certain age groups. The susceptibility to 2012-13 H3N2v viruses remains high in young children, and low in older children and young adults. Improved knowledge of age-related CR Ab is critical for influenza risk assessment and pandemic preparedness.
MicroRNA-30c is utilized by PRRSV in immune evasion through targeting JAK1 in the type I interferon signaling pathway (VIR5P.1153)
Authors: Zhang Qiong , huang chen , yang qian , Gao Li , Liu Hsiao-Ching , Tang Jun , Feng Wen-hai
Published: January 2023

Innate immune responses constitute the first line of defense against viral infections, and many viruses are capable of interfering with IFN signaling to escape from the innate immune responses and facilitate their proliferation and spread. Here, we found that a host miRNA, miR-30c, was up-regulated by porcine reproductive and respiratory syndrome virus (PRRSV) via activating NF-κB and benefited its infection. Subsequently, we demonstrated that miR-30c impaired the type I interferon (IFN-I) signaling pathway by targeting JAK1, resulting in the enhancement of PRRSV infection. To our knowledge, this is the first report of a host miRNA that suppresses IFN-I signaling by targeting JAK1, a vital component in the signaling of IFN-I. In addition, we found that miR-30c targeting sequences in JAK1 were conserved throughout mammals, and miR-30c was able to enhance both DNA virus (Herpes simplex virus) and negative-strand RNA virus (influenza virus) replications. Moreover, miR-30c is induced by IFN-I and acts as a negative feed-back modulator. These broad spectrum results suggest that miR-30c sets host IFN-I signaling response a threshold and is a universal regulator of viral replication. Our findings thus improve our understanding of the pathogenesis of PRRSV infection by identifying a new strategy whereby PRRSV to escape IFN-I-mediated antiviral immune responses by engaging miR-30c.
Protective role of neutrophils in respiratory paramyxovirus infection (VIR5P.1157)
Authors: Cheemarla Nagarjuna , Banos-Lara Rocio , Guerrero-Plata Maria
Published: January 2023

Polymorphonuclear leukocytes (PMNs or neutrophils) are the most abundant leukocyte in humans and essential for innate immune responses against invading pathogens. The role of these cells in respiratory viral infections still remains largely unknown. Human metapneumovirus (hMPV) belongs to the Paramyxoviridae family and is a leading respiratory pathogen causing severe symptoms such as bronchiolitis and pneumonia in young children, the elderly and immunocompromised patients. In this study, we investigate the role of neutrophils in hMPV infection using an experimental mouse model. Our results demonstrate that neutrophils predominate in the lungs of infected mice (>80%) during the acute phase of infection. In order to define the role of this robust response of neutrophils, we specifically depleted those cells using a monoclonal antibody. Our data indicate that neutrophil-depleted hMPV-infected mice exhibited significantly higher levels of inflammatory cytokines, pulmonary inflammation than competent mice. Moreover, depleted mice showed a more severe clinical disease (as indicated by an enhanced body weight loss and clinical illness) than competent animals. Interestingly, the recruitment of neutrophils to the site of infection was regulated by the expression of the hMPV G (attachment) protein. These novel findings indicate that neutrophils are critically important for the control of hMPV-induced disease and further our knowledge on the role of PMNs in respiratory viral infections.
TGF-β production associated with allergic asthma prevents influenza virus induced immunopathology (VIR5P.1160)
Authors: Furuya Yoichi , Sanfilippo Alan , Metzger Dennis
Published: January 2023

Asthma is believed to be a risk factor for influenza infection, however little remains known about how the asthmatic phenotype influences anti-viral immune responses. We therefore investigated the impact of asthma on susceptibility to influenza infection by using a mouse model of allergic airway inflammation. Unexpectedly, asthmatic mice were highly resistant to primary infection with the H1N1 2009 pandemic strain. Notably, the increased resistance was not attributed to enhanced viral clearance but instead was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, we show that asthmatic mice have significantly increased levels of TGF-β1 and that the heightened resistance of asthmatic mice is abrogated in the absence of TGF-β receptor II. Thus, we conclude that the transient increase in TGF-β expression following an acute asthma episode is responsible for temporarily protection via suppression of influenza-induced immunopathology.
FcµR expression by B cells regulates maximal induction of plasma cell differentiation in vivo and in vitro (VIR5P.1136)
Authors: Nguyen Trang , Baumgarth Nicole
Published: January 2023

Secreted (s)IgM functions in immune defense and regulation. It supports B cell development, selection and the size and quality of the humoral response. We aimed to identify the mechanisms underlying the reduced IgG responses in sIgM-/- mice after influenza virus infection. To distinguish effects of sIgM on B cell development from those on immune regulation, we generated sIgM-/- x wild-type (wt) F1 mice. All B cells develop normally in these mice but 50% lack the ability to secrete IgM. Wt and sIgM-/- B cells from F1 mice were transferred into sublethally irradiated wt controls, which were infected with influenza A/Puerto Rico/8/34. sIgM-/- B cells mounted reduced antiviral serum IgG responses compared to controls, demonstrating a non-redundant function for sIgM in B cell response induction. Studies in mice lacking the FcµR only on B cells suggested that sIgM-mediated regulation is due at least in part to sIgM-FcµR direct interaction. These mice mounted IgG and Hemagglutination Inhibition titers that were reduced by about half, and their sera had strongly decreased passive protective capacity compared to wt mice. ELISPOT and FACS-analysis revealed reduced plasma cell differentiation in draining mediastinal LNs at day 8, and long-lived plasma cells in bone marrows at day 70 after infection, as well as following stimulation of B cells in vitro. The data identify sIgM-FcµR direct interaction as a non-redundant B cell differentiation signal after B cell activation.
Murine cytomegalovirus specific response of non-classical CD8+ T cells (VIR6P.1175)
Authors: Anderson Courtney , Reilly Emma , Brossay Laurent
Published: January 2023

MHC class Ib-restricted CD8+ T cells have been shown to participate in the immune response towards intracellular bacterial pathogens. In contrast, the role of non-classical CD8+ T cells during viral infections is largely unknown. We sought to further investigate their functions using murine cytomegalovirus (MCMV), a well-established model for human CMV infections. To circumvent the demonstrated role of classical CD8+ T cells, we utilized KbDb-/- mice, in addition to KbDbCD1-/- mice that also lack natural killer T (NKT) cells. Following MCMV infection, we find that a subset of non-classical CD8+ T cells undergoes expansion and acquires an effector phenotype. We also demonstrate that this response is not due to a bystander reaction. These innate-like T cells exhibit a memory-like phenotype, but do not express the transcription factor PLZF. Interestingly, despite the absence of both classical CD8+ T cells and NKT cells, these mice are relatively resistant to infection. However, there is a prolonged inflammatory phenotype not present in wild-type animals, which clear MCMV. Taken together, these data demonstrate that MHC class 1b-restricted CD8+ T cells contribute towards the immune responses to viral infection.
Assessing the capability of the mycobacterial ribosome to elicit a CD4+ T cell response (APP5P.115)
Authors: Kennedy Steven , Johnson Alison , Seidel Ronald , Chan John , Jacobs William , Porcelli Steven
Published: January 2023

Mycobacterium tuberculosis (Mtb) infects one third of the world’s population, and an effective vaccine is desperately needed to curb the spread of disease. We have identified that the CD4+ T cell response is critical in combating Mtb. Using an attenuated version of Mycobacterium smegmatis, IKEPLUS, our lab previously identified the mycobacterial ribosome as a potential target to enhance CD4+ T cell responses to Mtb in mice. To directly assess the CD4+ T cell response mounted against the individual mycobacterial ribosomal proteins, we used an E. coli expression system to express and purify all fifty-seven proteins that make up the Mtb ribosome. This Mtb ribosomal library was then used to identify which proteins of the mycobacterial ribosome elicited a CD4+ T cell response in IKEPLUS infected animals by IFN-γ ELISPOT. When CD4+T cells of infected animals were isolated and restimulated with the Mtb ribosomal protein library, twenty-five of the fifty-seven total proteins elicited significant CD4+ Tcell responses. These ELISPOT results indicate that the Mtb ribosome is a highly immunogenic structure with the ability to stimulate strong T cell responses in mice. Further studies will focus on the use of the mycobacterial ribosome and isolated ribosomal proteins as potential components of vaccination regimens for inducing protective immunity against Mycobacterium tuberculosis.
Thymic development of insulin reactive T cells (BA11P.128)
Authors: Lee Thomas , Tully Natalie , Bettini Maria , Bettini Matthew
Published: January 2023

Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease that involves the destruction of insulin (Ins) producing β cells found within the pancreatic islets of Langerhans. While multiple studies have characterized the pathogenicity and diabetogenic potential of β-cell specific T cells, we have limited mechanistic insight into autoimmune T cell development and their escape from thymic selection. Utilizing the retrogenic mouse technology, we show that ectopic expression of InsB9-23 or insulin agonist mimetope (R22E) in the thymus influences the onset of diabetes. Our results demonstrate that increasing InsB9-23 expression in the thymus is insufficient to induce insulin reactive CD4+ T cell deletion, and upon escape from thymic selection, these insulin reactive T cells infiltrate the pancreas of NOD mice. Interestingly, these pancreatic-residing autoreactive CD4+ T cells are no longer able to induce spontaneous diabetes development. In contrast, when R22E mimetope is expressed, thymocyte development is halted at the double positive stage and insulin reactive T cells undergo negative selection. As a result, thymic expression of R22E completely prevents autoreactive thymocytes selection and diabetes development. Collectively, these results suggest that antigen dose or peptide:MHC complex stability of self-antigen presentation in the thymus can lead to multiple fates of insulin reactive CD4+T cell development and autoimmune outcome.
Self-lipid specific T cells, restricted by CD1b, contribute to the development of hyperlipidemia-associated skin inflammation (BA11P.133)
Authors: Bagchi Sreya , Zhang Hong , He Ying , Wang Chyung-Ru
Published: January 2023

Psoriasis, a chronic inflammatory skin disease, is associated with hyperlipidemia. While conventional T cells usually exacerbate psoriasis, the role of self-lipid reactive CD1-restricted T cells requires further study. CD1 molecules present lipid antigens to T cells and are divided in to two groups. Group 1 includes CD1a, CD1b, CD1c while CD1d belongs to group 2. Humans express both group 1 and group 2 CD1 molecules whereas mice only have CD1d. Thus, due to the lack of a suitable animal model, the role of autoreactive group 1 CD1-restricted T cells in hyperlipidemia-associated inflammatory diseases is unknown. To overcome this challenge, our lab generated mice that expressed human CD1b and a CD1b-autoreactive T cell receptor (hCD1Tg/HJ1Tg). hCD1Tg/HJ1Tg mice were crossed to the ApoE-/- background to examine the role of CD1b-restricted T cells in hyperlipidemia (hCD1Tg/HJ1Tg/ApoE-/-). Interestingly, hCD1Tg/HJ1Tg/ApoE-/- mice developed severe psoriasis-like skin inflammation characterized by T cell and neutrophil infiltration along with a Th17-biased cytokine milieu. They also showed significantly increased polar lipid accumulation in the skin as compared to hCD1Tg/HJ1Tg/ApoE+ mice. This suggested that the presence of excessive lipids in the skin resulted in activation of CD1b-autoreacitve T cells, leading to the onset of skin pathology. This work demonstrates that autoreactive group 1 CD1-reactive T cells might serve as a link between inflammatory processes and hyperlipidemia.
Aryl hydrocarbon receptor expressing Treg17 cells suppress type 1 diabetes in NOD mice (BA11P.140)
Authors: Singh Bhagirath , Nikoopour Enayat , Bellemore Stacey , Lee-Chan Edwin , Krougly Olga
Published: January 2023

The role of Th17 cells in type 1 diabetes (T1D) remains inconclusive. We found polarizing CD4+ T cells with TGF-beta+IL-6 or with IL-23+IL-6 cytokines induces different subsets of Th17 cells. Cells polarized with TGF-beta+IL-6 express high levels of transcription factor aryl hydrocarbon receptor (AhR) and IL-10 but minimal amounts of IL-22. Cells from T cell receptor transgenic BDC2.5 NOD mice polarized with IL-23+IL-6 produced large amounts of IL-22, expressed little AhR expression but induce T1D in young NOD mice. Cells derived with TGF-beta+IL-6 and termed as Treg17 cells were nonpathogenic and did not induce T1D. All adoptive transfer studies were done in young NOD mice and not NOD.SCID mice to prevent the conversion of Th17 cells into Th1 cells. We have also found IL-22-producing Th17 cells in the pancreas of diabetic NOD mice. The expression of IL-22 receptor in the pancreas of NOD mice increased with disease progression. Neutralization of IL-22 by antibody in vivo during adoptive transfer of pathogenic Th17 cells or splenocytes from diabetic mice did not significantly alter disease progression in the recipient mice. Therefore, Th17 derived IL-22 is not directly pathogenic to beta cells. We conclude regulatory Treg17 cells induced by TGF-beta+IL-6 that express high levels of AhR are protective while Th17 cells with a very low level of AhR induced by IL-23 + IL-6 are pathogenic. Both EN and SMB equally contributed to this work.
Loss of tolerance due to post-translational modification of beta cell peptides (BA11P.124)
Authors: McGinty John , Chow I-Ting , Greenbaum Carla , Odegard Jared , Kwok William , James Eddie
Published: January 2023

Post-translational modification (PTM) of self-proteins generates neo-antigens that are clinically relevant to autoimmune disease and are increasingly implicated in T1D. Here we demonstrate that enzymatic transglutamination and citrullination of beta cell antigens enhances presentation by the high risk HLA class II alleles DR4 and DQ8. We show CD4+ T cells specific for these epitopes are present at elevated frequencies ex vivo in the peripheral blood of patients with long standing T1D and are of Th1 phenotype. Such neo-antigens can induce distinct T cell specificities, while others cross-react with wild type-specific T cells. In each case, autoreactive T cells preferentially recognize modified epitopes resulting in enhanced proliferation and cytokine production. Mechanistically, modification at key binding pockets enhanced peptide binding to HLA class II, creating a stable pMHC that could bind autoreactive CD4+ T cells. Alternatively, modification of residues outside of binding pockets altered TCR interaction to permit T cell recognition. In one instance, dual modification of HLA and TCR contact points was required to induce a T cell response. Interrogation of autoantibody positive, healthy “non-progressors” revealed an intermediate T cell frequency, representing a break in tolerance without progression to overt diabetes. These findings implicate an important role for PTMs in broadening the antigenic repertoire and exacerbating the evolving autoimmune response in T1D.
IL4RA regulates lymphocytes trafficking in sclerodermatous graft-versus-host disease. (CAM4P.154)
Authors: Urso Katia , Alvarez David , Cremasco Viviana , Tsang Kelly , Lafyatis Robert , von Andrian Ulrich , Aliprantis Antonios
Published: January 2023

Scleroderma is an autoimmune disease characterized by inflammation and fibrosis in skin and internal organs. We utilized a murine Graft Versus Host Disease (GVHD) model of Scleroderma (SclGVHD), in which lymphocytes are transferred from B10.D2 mice (graft) into MHC-matched, lymphocyte deficient Balb/c Rag2-/- mice (host, sclGVHD mice). Over the course of 6 weeks, sclGVHD mice develop skin inflammation and fibrosis. Previously, we identified IL13 pathway activation in the skin of sclGVHD mice and scleroderma patients. Accordingly, host mice lacking IL4RA, a functional subunit for the IL13 receptor complex, are protected from sclGVHD. Here, we show that sclGVHD-IL4Ra-/- mice display a defect in lymphocyte trafficking, with more activated T cells in subcutaneous lymph nodes (sLNs) but fewer T cells in lymph, blood and skin compared to control mice. In addition, sphingosine 1-phosphate (S1P) kinase 1 was decreased in the sLNs and in sorted lymphatic endothelial cells (LEC) from sclGVHD-IL4Ra-/- mice. Since S1P is the major regulator of lymphocyte egress from sLNs, these data suggest that IL4Ra promotes sclGVHD by inducing the production of this mediator from LECs, which drives activated T cells into the periphery. These experiments highlight a potential novel role for IL4RA in inflammatory diseases, and indicate that strategies which impede lymphocyte trafficking may be effective therapeutically in the context of GVHD and scleroderma.
Tissue-resident specialized T cells promote the migration of local tissue dendritic cells: immune regulation at the local tissue level (CAM4P.155)
Authors: Gutierrez Miguel-Angel , Eriksson Anna , Randhawa Darshan , Kim Peter , Singh Ram
Published: January 2023

Dendritic cells (DC) that primarily reside in tissues carry antigens to local lymphoid organs to induce immunity or tolerance. We posit that a defect in the migration of tissue-DCs may predispose to autoimmunity in a tissue-specific manner; and an improved migration of tissue-DC may ameliorate disease in the respective organ. Here, we demonstrate that treatment of lupus dermatitis-prone MRL-lpr and MRL+/+ mice that exhibit a profound defect in the migration of skin-DC with a glycolipid αGalCer reduced the severity of dermatitis and enhanced the migration of skin-DCs, more so in Langerhans cells (LC; p=0.004) than in Lang+dDC (p=0.03). This effect of αGalCer was independent of its effect on iNKT cells, but required the presence of CD1d. Furthermore, αGalCer treatment enhanced the numbers of epidermis-resident γδ T cells in MRL-lpr mice that had reduced numbers of γδ T cells as compared to MHC-matched control mice. Finally, gd T cell-deficient mice had reduced skin-DC migration, and isolated skin-gd T cells directly promoted the migration of LC via CD40L-CD40 interaction. These data elucidate a new mechanism of regulation of skin-DC homeostasis whereby skin-gd T cells normally facilitate LC migration from skin to cutaneous lymph node. Such ‘local’ control of migratory behavior of tissue-DC can regulate immune response in a tissue-specific manner. This mechanism of skin-DC homeostasis is disrupted in lupus dermatitis, but can be repaired by treatment with a glycolipid.
Adipokine-induced activation responses in vascular endothelial cells: An inflammatory trigger in adipose microenvironment (CAM4P.160)
Authors: Singh Manindra , Benencia Fabian
Published: January 2023

Obesity is a global epidemic and leads to premature death due to its association with metabolic diseases. Evidence suggests that a low-grade, chronic inflammation of adipose tissue contributes to metabolic disorder of visceral adipose tissue. Adipose tissue-derived hormones, also called as adipokines, regulate variety of homeostatic functions, including metabolism and inflammation. We examined the function of adipokines: visfatin, vaspin and leptin, and tested the hypothesis that adipokines regulate immunoinfiltration of adipose tissue by inducing activation of endothelium in the adipose microenvironment. A large scale microarray was performed for the identification of soluble chemokines produced by adipokine-treated endothelial cells, and a decrease in the levels of chemokine ligand-17 was observed (n=5). Further, ELISA analysis confirmed significant upregulation for pro-inflammatory chemokines: Monocyte chemoattractant protein-1 (MCP-1) and granulocyte monocyte-colony stimulating factor (p<0.05, n=5). RNA was isolated from the adipokine-treated endothelial cells and real-time PCR analysis confirmed significant upregulation of pro-inflammatory genes interleukin-6 and MCP-1 in response to visfatin treatment (p<0.05, n=5). The surface expression of adhesion markers was measured by flow cytometry, and vascular cell adhesion protein-1 was found upregulated in response to visfatin. Overall, our results suggest that the studied adipokines are functional link for ECs and leukocyte crosstalk in the context of the adipose tissue.
Atheroprotective potential of CXCR4 on B1 cells (CAM4P.145)
Authors: Upadhye Aditi , Morris-Rosenfeld Samuel , McSkimming Chantel , Taylor Angela , McNamara Coleen
Published: January 2023

Atherosclerosis is a chronic inflammatory condition in which lipid deposition in arteries leads to the recruitment, activation, and accumulation of immune cells in the artery wall. Immune cells can have protective or detrimental effects on disease development. Existing evidence demonstrates that B1 cells hinder atherosclerotic lesion formation by secreting natural IgM antibodies that recognize modified self-epitopes present in diseased arteries. However, the mechanisms underlying B1 cell migration to arteries require further study. Our lab utilizes flow cytometry of circulating human immune cells in patients undergoing intravascular ultrasound of coronary arteries, in order to identify key surface markers that are associated with plaque burden. Expression of the chemokine receptor CXCR4 on human CD20+ CD3- CD27+ CD43+ B1 cells associated with reduced plaque burden in coronary arteries. No correlation was found between plaque volume and CXCR4 expression on naïve or memory B cell subsets. Prior evidence demonstrates that the helix-loop-helix transcription factor Id3 regulates B cell homing to the aorta and B cell-mediated atheroprotection. Loss of Id3 in the Apoe-/- mouse model of atherosclerosis led to systemic increase in surface expression of CXCR4 on B1 cells. Loss of Id3 additionally led to attenuated lesion formation, as measured by immunohistochemical lesion analysis of the aorta. These data suggest a potential atheroprotective function of CXCR4 on B1 cells.
The role of interleukin-17 in type I interferon mediated renal damage associated with lupus nephritis (CCR5P.202)
Authors: Ramani Kritika , Maers Kelly , Biswas Partha
Published: January 2023

Lupus nephritis (LuN) is a major clinical manifestation of systemic lupus erythematosus (SLE) resulting in significant morbidity and mortality. SLE is type I interferon (IFN-I) driven where IFN-I plays a critical role in irreversible damage in LuN. More recently, studies have implicated interleukin-17A (IL-17) in the pathogenesis of LuN. Little is known about the cross talk between IFN-I and IL-17 in mediating LuN. The objective of this study is to define the role of IL-17 in IFN-I mediated LuN. IFN-I driven LuN was induced with polyinosinic: polycytidylic acid (poly I:C) in lupus prone C57BL/6-Faslpr/lpr mice (B6.lpr). We show that renal damage is reduced in the absence of IL-17 receptor A (IL-17RA) signaling. Diminished renal pathology in B6.lprIL-17RA-/- mice correlated with diminished number of kidney infiltrating M2 macrophages implicated in the pathogenesis of poly I:C induced LuN. Kidneys of IL-17RA-/- mice also displayed reduced expression of osteopontin (Opn) produced by M2 macrophages and mediates Th17 regulation via IFN-I. Opn is involved in macrophage recruitment to sites of tissue damage. Finally, we showed that IFN-I and IL-17 could synergistically upregulate Opn gene expression in bone marrow derived macrophages. Therefore, these findings for the first time reveal a synergistic role for IL-17 and IFN-I in the pathogenesis of LuN. These studies may inspire development of novel therapeutic strategies for the treatment of LuN and other chronic kidney disorders.
Tregs expressing high Tl1a are proinflammatory and do not protect from T cell induced colitis. (CCR5P.204)
Authors: Sidhu Maninder , Shih David , Targan Stephan
Published: January 2023

Patients with Crohn’s disease have increased mucosal expression of TL1A. TL1A co-stimulates T cells and can promote expansion of activated and regulatory T cells. Mice generated with sustained and elevated T cell expression of Tl1a (L-Tg) have increased Foxp3+ Treg cells yet develop worsened ileitis, proximal migration of colitis and intestinal fibrosis. The aim of this study was to investigate the role of Tl1a in the differentiation and function of Tregs.In the L-Tg mice, the CD4+ T cell population was comprised of GFPhigh and GFPlowcells that correlated with the level of Tl1a expression.Foxp3 expression and suppression function was significantly lower in the Tregs expressing high Tl1a. Indeed, addition of high levels of exogenous rTl1a under iTreg differentiation conditions advances the expansion of IL-13 producing effector T cells. The percentage of GFPhigh Foxp3+ Tregs co-expressing GATA3 were significantly higher explaining the possible Th2 polarization of Tregs expressing high Tl1a. Additionally, in the T cell transfer model of colitis, RAG mice cotransferred with GFPlowFoxp3+ Tregs were better protected from inflammation and weight loss compared to mice cotransferred with GFPhighFoxp3+ T cells.These data reveal a novel role for TL1A in regulating T cell mediated immune responses such that the level of Tl1a (high v. low) can differentially influence the expansion of effector and Treg cells leading to either a pro-inflammatory or protective mucosal environment.
Dimeric IL-2 induces necrosis of Interleukin-2 receptor positive cells (CCR6P.217)
Authors: Ganesan Ramya , Arumugam Prakash , Clabaugh Suzanne , Kozak Ashot , Wrenshall Lucile
Published: January 2023

Mice deficient in T regulatory cells (T regs) die from systemic inflammation. Breeding these mice with IL-2KO mice clears inflammation in lungs and skin, suggesting that IL-2 has pro-inflammatory functions beyond promoting survival of T regs. We recently identified a dimeric form of IL2 mammalian tissues that is cytotoxic to cells expressing IL-2 receptor (IL-2R), which provides an explanation for the above. Exposure of IL-2R+ cells to dimeric IL-2 results in rapid release of LDH. We hypothesized that dimer-mediated cell death is consistent with the characteristics of necrosis. To test this hypothesis, we exposed Jurkat cells to dimeric IL-2 and processed them for electron microscopy at 5 minutes. EM revealed swollen nuclear membranes and mitochondria consistent with early necrosis. Exposure of vascular smooth muscle cells (VSMC) to dimeric IL-2 induced 50% loss of cellular ATP at 5 minutes and nearly complete loss at 20 minutes. Using the mitochondrial membrane potential indicator JC-1 in VSMC, nearly all mitochondria lost their MMP by 10 minutes. These data indicate that dimeric IL-2 kills cells by necrosis. Our finding that dimeric IL-2 is cytotoxic to cells expressing IL-2R, lymphoid or non-lymphoid, has implications beyond the immune system. These studies combined with prior studies showing presence of matrix-bound IL-2 in arteries, suggests dimeric IL-2 may contribute to vascular inflammation in processes such as atherosclerosis.
Real time visualization of the differential contribution of medullary thymic epithelial cells versus dendritic cells to central tolerance in the thymus (HEM7P.233)
Authors: Lancaster Jessica , Ehrlich Lauren
Published: January 2023

Tolerance for tissue-restricted antigens (TRAs) is established through interactions of thymocytes with medullary thymic epithelial cells (mTECs) and dendritic cells (DCs) in the thymic medulla, though their relative contributions remain unclear. Previous studies using genetic models to impair antigen presentation by mTECs or DCs showed that either subset could contribute to negative selection; however, because interactions between thymocytes and stromal cells are necessary for maintenance of a differentiated stromal compartment, results from these models may not reflect physiologic contributions of mTECs or DCs. Thus, we adopted a two-photon fluorescence microscopy approach to directly quantify interactions between thymocytes and mTECs or DCs at the outset of negative selection in an intact thymic environment. We find that both mTECs and DCs activate TCR signaling in CD8+ and CD4+ thymocytes in the context of secreted and membrane-bound forms of a negatively selecting TRA. mTECs account for more selection than DCs, regardless of the TRA form, and their contribution is greatest with a membrane-bound TRA. Strikingly, while interactions with mTECs and DCs account for all antigen-driven signaling of CD8+ thymocytes, our data suggest other cells also present TRAs to CD4+ thymocytes. Altogether, we find that mTECs make the most significant contribution to negative selection against Aire-driven TRAs, though DCs also contribute in the context of a physiologic thymic microenvironment.
An ultra-conserved element in Hoxa2 is required for the normal expression of Hoxa3 and normal thymus development (HEM7P.223)
Authors: Condie Brian , Buac Kristina , Mallo Moises
Published: January 2023

We analyzed thymus development in a Hoxa2 mutant mouse (Hoxa21) that carries a deletion of an ultraconserved element in the protein coding sequence of Hoxa2. Our phenotypic analysis revealed that the Hoxa21 mutants exhibit hypoplastic and ectopic thymii. Further analysis demonstrated delayed Foxn1 expression within the developing thymus of the mutants. These phenotypes resemble the alterations in thymus development found in Hoxa3 mutant embryos. Intriguingly, analysis of thymus development in mutants carrying a different Hoxa2 null allele that contains an intact ultraconserved element (Hoxa22) revealed no alteration in thymus development. This findings suggested that the abnormal thymus development seen in the Hoxa21 mutants may be due to alterations in Hoxa3 expression. Consistent with this hypothesis Hoxa21 mutants exhibited greatly reduced Hoxa3 expression in neural crest-derived mesenchyme and pharyngeal endoderm, embryonic cell types required for normal thymus development. In addition analysis of Hoxa21+/- Hoxa3 null/+ compound heterozygotes revealed thymus phenotypes that were very similar to Hoxa3-deficient mutants. These data suggest that Hoxa2, although expressed in thymic primordium, does not play a direct role in thymic organogenesis. Our results suggest that the ultraconserved enhancer element located the first exon of Hoxa2 is necessary for normal Hoxa3 expression in cell types necessary for normal thymus development.
Endothelial cells promote endogenous thymic regeneration via BMP4 signaling and activation of Foxn1 (HEM7P.224)
Authors: Dudakov Jarrod , Wertheimer Tobias , Velardi Enrico , Brede Christian , Young Lauren , Ginsberg Michael , Nolan Daniel , Beilhack Andreas , Rafii Shahin , Butler Jason , Van Den Brink Marcel
Published: January 2023

Although the thymus has a remarkable capacity for repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Here we reveal a critical role for endothelial cells (ECs) in aiding thymic recovery following acute injury. We found that ECs represent a highly damage-resistant niche in the thymus and, moreover, rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate tissue repair. In particular, we demonstrated that after damage ECs induce upregulation by cortical thymic epithelial cells (TECs) of Foxn1, a key transcription factor implicated in TEC development and thymic regeneration. Further investigation revealed that the mechanism by which ECs induced upregulation of Foxn1 was via a soluble factor that could be inhibited by Noggin, strongly suggesting the involvement of BMP signaling. Consistent with this, ECs were found to be potent producers of BMP4 after thymic damage and inhibition of BMP4 signals by ECs, achieved either pharmacologically or genetically, led to significantly worse thymic recovery after acute injury caused by TBI. These findings not only provide the first evidence of the role of ECs in endogenous thymic regeneration but, given the significant clinical need for strategies to promote immunity, also offer an innovative therapy to boost thymic function.
Transcriptional landscape of immune responses to naturally acquired influenza (HUM6P.248)
Authors: Allen E. , Ohlson Maikke , Finkelstein David , Rosenberger Carrie , Thomas Paul
Published: January 2023

Several human population studies have sought to determine what components of the immune system confer protection to influenza infection. The aim of our study was to detect transcriptional changes contributing to the illness outcome in patients with naturally acquired influenza infection. Patients with naturally acquired influenza infection were recruited into a five year prospective, longitudinal cohort. Whole blood was sampled at day of enrollment (Day 0) and Days 3, 7, 10, and 28. Additionally, clinical data was collected during the course of infection including symptoms (upper respiratory, lower respiratory, systemic, and gastrointestinal symptoms). RNA was extracted from PBMCs collected at Day 0 in a subset of influenza infected patients (N=45) and was analyzed by RNA-Seq. Genes were ranked using Median Absolute Deviation (MAD) and then hierarchical clustering analysis was conducted using Spotfire and WGCNA. We detected two distinct clusters of individuals with large differences in their transcriptional landscapes falling into four clusters of genes. Principal components analysis was unable to define the clusters using any clinical traits including age, race, gender, and illness severity. One of the four clusters was characterized as an interferon response, where the other clusters have more complex annotations. Ongoing analyses are exploring upstream mechanisms that might lead to the observed divergent transcriptional outcomes.