bluuebunny/crossref_metadata_2025 huggingface dataset:
A humanized mouse model for generation of tumor antigen specific T cells for preclinical research and anticancer therapy (75.4)
Authors: Hu Zheng , Wargo Jennifer , Yang Yong-Guang
Published: January 2023

Adoptive transfer of tumor-specific TCR-engineered T cells is a promising cancer therapy, but mispairing of endogenous TCRαβ chains with introduced TCR subunits remains a major limiting factor. T cells with mispaired TCRαβ chains, not only lose anti-tumor activity, but also convey potential risk of graft-vs.-host manifestations. In this study, we attempted to generate human T cells expressing only the engineered, tumor-specific TCR in humanized mice, which were made by transplantation of NSG mice with HLA-A2+ fetal human thymic tissue (under kidney capsule) and CD34+ fetal liver cells (i.v.) that were lentivirally transduced with HLA-A*0201-restricted Mart-1-specific TCR gene. MART-1:26-35(27L)/HLA-A*0201 tetramer+CD8 T cells became detectable in blood by 6 wks and reached to more than 70% by 14 wks. Following immunization with Mart-1 peptides, over 60% tetramer+CD8 T cells converted from a naïve phenotype to CD45RA-CCR7- effector/memory phenotype and acquired the ability to produce IFN-γ, while tetramer-CD8 T cells remained a naïve phenotype and incapable of IFN-γ production. Almost all tumor-infiltrating T cells were tetramer+CD45RA-CCR7-CD8+. Tetramer+ CD8 T cells mediated efficient specific killing of HLA-A2+Mart-1+ melanoma cells, and the cytolytic activity retained after substantial in vitro expansion (up to thousands of times) for about two months. Thus, this hu-mouse model may provide an efficient means of generating human T cells with potent anti-tumor activity.
T cell responses targeting subdominant epitopes of Mycobacterium tuberculosis antigen ESAT-6 resist acquisition of bacteria-driven functionality and enhance protection in vivo. (75.8)
Authors: Woodworth Joshua , Aagaard Claus , Agger Else Marie , Andersen Peter
Published: January 2023

Immune-mediated pathology drives M.tuberculosis (Mtb) disease and transmission. Hyperconservation of dominant T cell epitopes in Mtb’s genome suggests that the T cell response to infection is actually evolutionarily beneficial to Mtb. Vaccine-induced CD4 T cells recognizing novel subdominant epitopes of ESAT-6 that are not targeted during natural infection can improve protective. We investigate differential characteristics of dominant vs subdominant T cells in protective immunity to Mtb in vivo. Methods. CB6F1 Mice were vaccinated with either rESAT-6 or a truncated ESAT-6, which lacks the dominant epitope and induces subdom T cell responses. Before and after Mtb infection, epitope-specific T cell responses were evaluated by proliferation, cytokine secretion, flow cytometric assays. Results. Pre-infection, subdom CD4 T cells had enhanced proliferation, but delayed cytokine kinetics. Post-infection, vaccine-induced T cells had a markedly different cytokine profile relative to infection-driven T cells, including memory-like polyfunctionality and IL-17 production. However, over time, a larger proportion of dominant T cells developed an infection-driven cytokine and KLRG1/PD-1 profile. Conclusion. Subdom epitope CD4 T cell have enhanced maintenance of vaccination-induced cytokine profile following infection in vivo, associated with their ability to enhance protection, and therefore may be uniquely able to resist bacterially-driven modulation and immune-mediated disease.
Bystander chronic infection negatively impacts the development of CD8 T cell memory (76.6)
Authors: Stelekati Erietta , Shin Haina , Doering Travis , Dolfi Douglas , Ziegler Carly , Beiting Daniel , Liboon Jennifer , Wolski David , Katsikis Peter , Shen Hao , Roos David , Haining W. , Lauer Georg , Wherry E.
Published: January 2023

Accumulating epidemiological evidence suggests that chronic infections compromise immunity against antigenically unrelated infections, but the underlying mechanisms are unclear. We investigated whether antigenically unrelated “bystander” chronic infections impacted the development of memory CD8 T cells, the hallmarks of protective immunity against intracellular pathogens. We demonstrate that chronic bystander infection and inflammation substantially impair memory CD8 T cell responses in several mouse models with similar changes in non-human primates and in chronically HCV -infected humans. Type I IFN -induced molecular pathways were specifically dysregulated in memory CD8 T cells in inflammatory environments and induction of type I IFN by chronic poly(I:C) administration similarly impaired memory CD8 T cell development and function. Chronic inflammation increased the expression of the proapoptotic protein Bim in bystander CD8 T cells, while inhibiting their IL15-induced rescue from apoptosis ex vivo. Two transcriptional regulators of T cell development, Blimp-1 and T-bet, integrated the effects of inflammatory mediators in impairing optimal memory CD8 T cell development. Thus, exposure to chronic bystander infections deregulates the normal effector to memory CD8 T cell transition. These results have direct relevance concerning current therapeutic approaches and vaccination strategies for patients with persisting infections or chronic inflammatory conditions.
Evaluation of immune response in dorsal root ganglion of mice infected with Herpes Simplex Virus type 1 (HSV-1) (76.8)
Authors: Silva Jaqueline , Talbot Jhimmy , Lopes Alexandre , França Rafael , Fonseca Benedito , Cunha Thiago , Cunha Fernando
Published: January 2023

Herpes Zoster (HZ) is a disease caused by reactivation of latent herpesvirus Varicella Zoster in the sensory ganglion, characterized by dermal rash and severe pain. A model of mice infected with HSV-1 on the skin of the hind paw has been used to study the pathophysiology of HZ, since they develop HZ-like skin lesions and pain-related responses. There are no data available about the immune response in dorsal root ganglion (DRG) of these mice. Thus, the aim of this study was to evaluate cells and inflammatory mediators present in DRGs and its relationship with hypernociception during HSV-1 cutaneous infection. Mice developed hypernociception from day 3 to 7 post inoculation in the ipsilateral (Ips) paws, but not in the contralateral (Cl) paws. A higher viral load, measured by qPCR, was detected in DRGs L3, L4 and L5 of infected mice, when compared to control mice or naïve mice. By flow citometry analysis, we observed an inflammatory infiltrate composed by CD4+, CD8+, CD11b and Gr1+ cells in DRGs L3, L4 and L5, but not in spinal cord, that correlated with virus presence previously found. A higher expression of COX-2, IFN-γ, GFAP and IL-22 was detected in the same DRGs of Ips paws from infected mice, when compared to the same ganglion of Cl paws or ganglion from naïve mice. Our results show the presence of an inflammatory infiltrate in DRGs of infected mice and the expression of inflammatory mediators which might contribute for the development of hypernociception.
A potential role for HLA-DM in linking the antigen uptake and antigen presentation pathways in B cells. (P5007)
Authors: Macmillan Henriette , Strohman Michael , Ayyangar Sashi , Mellins Elizabeth
Published: January 2023

Antigen presenting cells (APC) are key regulators of immunity. They are involved in stimulating naïve and effector cells and are critical for orchestrating protective responses to infectious agents as well as aberrant responses to self-antigens. Among APC, B cells are uniquely capable of presenting small amounts of antigen, which they capture and take up through surface immunoglobulin (Ig). The uptake of antigen by B cells is well studied, and so is the loading of peptides onto MHC II, but how these two pathways intersect is less well elucidated. We propose that HLA-DM is a key factor in linking these pathways by interacting with Ig. Here we show that HLA-DM and Ig can be co-precipitated from B cell lines. We also utilized proximity ligation assays and confocal microscopy to show a close interaction between HLA-DM and Ig in fixed B cell lines. In addition, we show by western blot and FACS staining that mutant cell lines lacking HLA-DM have lower levels of Ig than their parent, DM-expressing cell lines. Our data suggest that HLA-DM is involved in linking the antigen uptake and antigen presentation pathways by interacting not only with MHC II but also with Ig.
Evolution of HLA class II through diversification in protein stability levels (P5009)
Authors: Miyadera Hiroko , Ohashi Jun , Tokunaga Katsushi
Published: January 2023

Allelic diversity in human leukocyte antigens (HLA) have been explained by balancing selection acting on polymorphic residues in the peptide binding groove of HLA that diversify binding spectrum for pathogenic epitopes. Here we show that HLA class II (HLA-DQ) allele products are extremely diverse in protein stability levels. We have profiled the relative stability of DQ proteins by a cell-based assay that measures cell surface MHC protein density (surface density index; SDI). The SDI varies over 100-fold among DQ allele products, indicating large stability differences. Through mutagenesis, we identified polymorphic residues that regulate the intrinsic stability of DQ protein, which includes variations on the exterior of the peptide-binding groove that mediate interdomain hydrogen bonds. One of the major stability regulator in DQA1 achieves high ratio of synonymous to non-sysnonymous substitutions per site (ds/dn), suggesting that variations at this site have been maintained through balancing selection. DQ genes of non-human primates and mammals also maintain these variatns. These data suggest that HLA and MHC have been evolving through diversifications in protein stability levels. Our study provides new functional insights into the mechanism of HLA evolution.
The development of thymic B cells and their role in T cell negative selection (P5039)
Authors: Perera Jason , Meng Liping , Huang Haochu
Published: January 2023

T cell negative selection is dependent on the presentation of self antigens by MHC expressing cells in the thymic medulla. In addition to dendritic cells and medullary thymic epithelial cells, thymic B cells also reside in the medulla and express high levels of MHC class II. The origin of these cells is unclear, with both intrathymic development and recirculation of mature B cells being reported. Using parabiosis studies and analysis of Rag-2-GFP BAC transgenic mice, we found that recirculation contributes minimally while development from thymic progenitors is the predominant pathway that maintains this population. Given their phenotype and high capacity to present cognate antigens, we hypothesized that thymic B cells play a role in T cell negative selection. Using the KRN TCR transgenic that recognizes the self antigen GPI, we tested how negative selection was affected when we altered B cell antigen presentation. When the KRN TCR was crossed to a GPI-specific BCR knockin that increased GPI presentation on B cells, negative selection of the KRN TCR was enhanced. When we eliminated thymic B cells by crossing the KRN TCR onto a μMT-/- background, negative selection was reduced, suggesting that the endogenous B cell repertoire contributes to negative selection of this physiologically relevant antigen. Given the high frequency of autoreactivity in immature B cells, we argue that B cell tolerance and T cell tolerance could be efficiently coupled in T cell negative selection.
Type I interferon induced by lymphotoxin-beta receptor signaling on dendritic cell is crucial for CD8 T cell infiltration into effector site by modulating the expression of tissue specific adhesion molecule VLA4 (P5045)
Authors: Ng Dennis , Ward Lesley , Rahbar Ramtin , Cummings Derek , Mossman Karen , Ohashi Pamela , Gommerman Jennifer
Published: January 2023

In non-infectious settings with minimal inflammatory stimulus, CD4 T cell help is needed for dendritic cells (DC) to cross-prime antigen in order to elicit a CD8 T cell response. Activated CD4 T cells express CD40-ligand (CD40L) and lymphotoxin-α1β2 (LTαβ), which interacts with their respective TNF receptors, CD40 and lymphotoxin-β receptor (LTβR) on DC, thereby significantly increases the DC immunogenic potential. Our early work showed that LTβR signaling induces Type I IFN expression in DC which facilitates CD8 T cell expansion against soluble protein antigen. In this study, we identified that LTβR signals through TNF receptor-associated factor (TRAF)-3 for interferon regulatory factor (IRF)-3 phosphorylation and Type I IFN production. Furthermore, using an inducible diabetes model through the transfer of activated DC, we found that LTβR signaling on DC is absolutely required for the proper priming of CD8 T cells and diabetes induction. We further showed that LTβR-induced Type I IFN affect the expression of the adhesion molecule VLA-4 on antigen specific CD8 T cells and it affects their ability to infiltrate into the pancreas. LTβR deficient DC fail to induce diabetes, and the provision of exogenous IFN-α was sufficient to rescue the diabetes status. Together, these results describe a novel role for LTβR in the induction of Type I IFN by DC, and demonstrate its relevance in an autoimmune setting.
Intracellular antibody activates innate immunity (P4135)
Authors: Tam Jerry , McEwan William , Watkinson Ruth , Bidgood Susanna , Mallery Donna , James Leo
Published: January 2023

Immune responses are initiated by infection, yet pathogens evolve faster than the host receptors tasked with their detection. Antibodies can match pathogen evolution but are extracellular whereas viruses replicate intracellularly. Recently however, we showed that viruses carry surface bound antibodies into cells when they infect. Cytosolic antibody-coated viruses are detected by the Fc-receptor and E3 ubiquitin ligase TRIM21, which targets them for proteasomal degradation in a process known as Antibody Dependent Intracellular Neutralization. Here we show that the presence of antibody in the cytosol acts as a highly potent DAMP, alerting the cell to infection and initiating an antiviral state. Upon recognition of an antibody-coated pathogen (eg a DNA or RNA non-enveloped virus or bacteria), TRIM21 synthesizes lysine-63 polyubiquitin chains and activates the NFκB, AP-1 and IRF pathways. Activation by TRIM21 is sufficient to drive production of proinflammatory cytokines, such as IL-6, TNF, CXCL10; as well as modulate surface expression of MHC Class 1-like stress ligands, such as RAE-1, H60 and HLA-E (Qa-1). In nonprofessional cells, such as primary human lung fibroblasts, TRIM21 and antibody are required for detection of adenovirus infection. In primary murine macrophages, TRIM21 mediated signalling promotes a type I immune response. The combination of intracellular antibody and ubiquitous cytosolic Fc-receptor provides a highly potent and rapidly adaptable pathogen sensor.
Involvement of natural immunoglobulin M secreted by epithelial cells in innate immune response (P4155)
Authors: Shao Wenwei , Hu Fanlei , Xiao Lin , Ma Junfan , Huang Jing , Qiu Xiaoyan
Published: January 2023

It is well known that B-1 B cells are the main cell type which is responsible for the production of natural immunoglobulin M (IgM) and can respond to infection by increasing IgM secretion. However, in this study, we reported that some epithelial cells also can secrete natural IgM that has natural IgM characteristics. 1. Epithelial cell-derived IgM revealed germline-encoded and restricted VDJ/VJ rearrangement patterns. 2. Epithelial cells can spontaneously secrete IgM, however, TLR9 agonists, CpG, can increase the secretion of IgM in epithelial cancer cells. 3. The CD79A and CD79B, two molecules that are physically linked to membranous IgM on the surface of B cells to form the B cell antigen receptor (BCR) complex, were also expressed on the cell surface of epithelial cancer cells and co-located with IgM to form BCR-like complex. 4. The epithelial cancer cell-derived IgM showed oligo-reactivity, which can recognize single-stranded DNA, double-stranded DNA, lipopolysaccharide, and some autoantigens. Our findings further expand the understanding for the significance of epithelial cells in the innate immune response, especially in the anti-bacterial immunity via secretion of IgM. In addition, this finding suggested that B-1 B cells may be not the main cell type that is responsible for the production of natural IgM.
Innate immune responses of exfoliated epithelial cells. (P4165)
Authors: Srinivasan Mythily , Swaminathan Venkatesh , Negrini Thais , Arthur Rodrigo
Published: January 2023

Cell exfoliation is a characteristic feature of epithelia covering the external surface and the internal cavities of the body. While the benefit of cytology in infections and cancer diagnosis is well recognized, the clinical use of epithelial cells obtained from body fluids is relatively unexplored. As the first cells to encounter microbes, the epithelial cells exhibit toll like receptors (TLR) that bind specific conserved microbial patterns and transduce signals to thwart deleterious host responses to commensals and pathogens. We evaluated the potential application of epithelial cells derived from human saliva in investigating host responses to microbes. The salivary epithelial cells (SEC) derived from chronic periodontitis saliva exhibited distinct TLR-2 and TLR-4 expression profile and responded differentially to protein peptide derivative and lipopolysaccharides, ligand for TLR-2 and TLR-4 respectively. Interestingly, the ability of SEC to respond was not restricted to stimulation with oral microbes. Co-culture of SEC with Staphylococcus aureus biofilm, a transient cutaneous pathogen upregulated TLR-2 expression and induced increased secretion of inflammatory cytokines IL-6, IL-8, IL-12 and IFN-γ as compared to SEC exposed to a Staphylococcus epidermidis, a skin commensal. Collectively, it is postulated that the SEC is an efficient model to investigate the epithelial:microbe interphase and may serve as personalized specimen for evaluating individual response to biofilm.
Haptoglobin inhibits the pro-inflammatory activity of HMGB1 (P4177)
Authors: Yang Huan , Ju Zhongliang , Lu Ben , Ochani Mahendar , Li Jianhua , Chavan Sangeeta , Tracey Kevin
Published: January 2023

HMGB1 is a cytokine mediator in the pathogenesis of inflammatory diseases including sepsis (Yang et al, BBA, 2009). Recently we demonstrated that haptoglobin (Hp), a naturally occurring serum protein, binds to HMGB1 and suppresses HMGB1-stimulated TNF and IL-8 release in cultured macrophages. Hp knockout mice subjected to cecal ligation and puncture (CLP)-induced sepsis had significantly higher serum HMGB1 levels and higher mortality rates compared to wild type animals (75% survival in wild type vs. 34% in Hp knockout mice; P<0.05). Wild type mice subjected to CLP and received injections of Hp were twice as likely to survive (63% survival in Hp-treated vs. 33% in vehicle control; P<0.05), suggesting the therapeutic potential of exogenous Hp in sepsis. Hp is composed of alpha and beta subunits in a polymeric form (Yueh, J. Chromat B Life Sci. 2007). Structure-functional analysis revealed that Hp beta subunit alone is sufficient to recapitulate effects of Hp to neutralize HMGB1 in vitro in macrophages. The survival advantage of Hp in CLP-induced sepsis was also fully reproduced by Hp beta. Surface plasmon resonance analysis showed that Hp beta binds HMGB1 with high affinity (Kd = 29 nM). Thus, our data reveal unexpected roles of Hp as an endogenous antagonist of HMGB1, preventing the harmful HMGB1-induced inflammation in sepsis. The essential domain of Hp maps to the beta subunit, making it a target in the design of therapeutics.
β-defensins activate human mast cells via Mas-related Gene-X2: cross-regulation by LPS (P4179)
Authors: Subramanian Hariharan , Gupta Kshitij , Lee Donguk , Bayir Arzu , Ahn Harry , Ali Hydar
Published: January 2023

Human β-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells and cause increased vascular permeability in wild-type but not mast cell-deficient rats. Here, we sought to determine if hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells in vitro and had no effect on vascular permeability in vivo. By contrast, they induced sustained Ca2+ mobilization and substantial degranulation in human mast cells with hBD3 being more potent. While human mast cells endogenously express G protein coupled receptor (GPCR); Mas-related gene X2 (MrgX2), murine mast cells and rat basophilic leukemia, RBL-2H3 cells do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD2/3-induced degranulation and ectopic expression of MrgX2 in RBL-2H3 cells restored these responses. Interestingly, lipopolysaccharide (LPS) inhibited hBD3 but not hBD2-induced Ca2+ mobilization and degranulation. Thus, hBDs activate human mast cells via MrgX2 and that the resistance of murine mast cells likely reflects the absence of this GPCR. Furthermore, the ability of LPS to inhibit hBD3/MrgX2-mediated mast cell activation provides a novel complexity of the role of mast cells in host defense and inflammation.
Maturational differences between neonatal and adult neutrophils (P4183)
Authors: Lawrence Shelley , Eckert Jeffrey , Pereira Anne
Published: January 2023

Our laboratory has shown that gene expression of multiple microbicidal RNAs is greatly up regulated in neonatal versus adult neutrophils, and that neonates have a higher percentage of circulating immature neutrophils than adults. The aims of this study were to determine if there was a difference in number of immature neutrophils or in the production of microbicidal proteins based on exposure to labor. Design: The ratio of immature to total neutrophils was determined via flow cytometry and complete blood counts. The maturational stage of the neutrophil was verified by neutrophil granule RNA production via Luminex QuantiGene Plex Assays and protein levels via ELISA. Results: Exposure to labor did not affect the number of circulating immature neutrophils in neonates (p=0.864), but those delivered without labor had more neutrophils at the earliest stages of development. There was no difference in α-defensin protein levels between samples, but lactoferrin was elevated in adult as compared to neonates without labor (p=0.0003), but not to those exposed to labor (p=0.087). Conclusion: Labor does not affect the number of immature neutrophils in neonates, but may “push” them to mature. While the gene expression of microbicidal proteins is greatly up regulated in neonatal neutrophils, the amount of protein produced is similar to or lower than adults. Studies are ongoing to determine if protein levels can be increased after stimulation by bacteria or other activators.
Phytoalexin protects dopaminergic neurons against neurotoxicity by modulating myeloperoxidase levels in glial cells (P4199)
Authors: Chang Chi Young , Yoon Hee Jung , Park Eun Jung
Published: January 2023

Myeloperoxidase functions as a key molecular component of the host defense system against diverse pathogens. We have previously reported that increased myeloperoxidase levels and activity is a distinguishing feature of rotenone-exposed glial cells, and that either overactivation or deficiency of myeloperoxidase leads to pathological conditions in the brain. Here, we provide that modulation of myeloperoxidase levels in glia by resveratrol confers protective effects on rotenone-induced neurotoxicity. We show that resveratrol significantly reduced myeloperoxidase levels but did not trigger abnormal nitric oxide production in microglia and astrocytes. Resveratrol-induced down-regulation of myeloperoxidase markedly attenuated rotenone-triggered inflammatory responses including phagocytic activity and reactive oxygen species production in primary microglia and astrocytes. In addition, impaired responses of primary mixed glia from MPO knockout mice to rotenone were relieved by treatment with resveratrol. We further show that rotenone-induced neuronal injury, particularly dopaminergic cell death, was attenuated by resveratrol in neuron-glia co-cultures, but not in neurons cultured alone. Collectively, our findings provide that resveratrol influences glial responses to rotenone by regulating both myeloperoxidase and nitric oxide, and thus protects against rotenone-induced neuronal injury.
Forward genetic analysis of type I interferon responses to cytosolic deoxynucleotides reveals polymorphisms in Tmem173 gene of wild derived MOLF/EiJ mice. (P4201)
Authors: Surpris Guy , Fitzgerald Katherine , Poltorak Alexander
Published: January 2023

We identified a novel phenotype in wild derived inbred mouse strain, MOLF/EiJ (MOLF). In response to cytosolic DNA or cyclic-diadenylate (c-di-AMP), MOLF macrophages exhibit high levels of IL-6 but very low levels of type I interferon, IFNβ, compared to classical inbred mouse strain C5BL/6J (B6). Furthermore, the IFNβ production is reduced in responses of MOLF macrophages to herpes simplex virus (HSV) and Listeria monocytogenes infection that release double stranded DNA and c-di-AMP into the cytosol, respectively. To identify loci that confer the trait, we used quantitative trait locus (QTL) mapping. We measured the IFNβ production of macrophages from a panel of 2nd filial generation (F2), B6/MOLF intercrossed mice, and genotyped the individual mice for inheritance of loci genome wide. The lack of IFNβ production mapped to a locus that contains Tmem173, a gene that encodes STING. STING is an innate immune cytosolic surveyor that mediates interferon response to cytosolic DNA and c-di-AMP. Sequencing the MOLF transcript of Tmem173 revealed multiple single nucleotide polymorphisms and an 18 base pair deletion in the MOLF allele of Tmem173. Almost all of these polymorphisms encode amino acid changes in the putative transmembrane domains of STING, while the cytosolic fraction is highly conserved. In vitro, expression of MOLF STING in Tmem173-/- murine embryonic fibroblasts (MEFs) shows greatly reduced activation of the IFNβ promoter, compared to C57BL/6.
Progranulin regulates endotoxin tolerance (P4143)
Authors: Tang Wei , Liu Lu , Zheng Lin , Lu Yi
Published: January 2023

The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, inflammation and host defense. The anti-inflammatory properties of PGRN have been reported in various pathologies and conditions, such as rheumatoid arthritis, but the mechanisms remain to be well resolved. Here we show that pretreatment of recombinant PGRN inhibits the immune response of mouse peritoneal macrophages to LPS, with decreased production of pro-inflammatory cytokines (TNFα, IL-12), and increased production of anti-inflammatory cytokines (IL-10), which displays an endotoxin tolerance-like phenotype. Most interestingly, PGRN deficient peritoneal macrophages show an increased sensitivity toward LPS and a deficient LPS-tolerance induction, and PGRN deficient mice exhibit more elevated lethality and lung injure compared with wild-type mice in response to a subsequent endotoxin. Collectively, these data demonstrate a critical and novel role of PGRN in endotoxin tolerance, and suggest the potential function of PGRN in endotoxin tolerance related diseases, such as sepsis or systemic inflammatory response syndrome (SIRS), hepatic ischaemia, acute coronary syndrome, cystic fibrosis and perhaps even cancer.
Cereblon is a novel E3 ubiquitin ligase regulator of CD28 signaling in T cells (P1007)
Authors: Epling-Burnette Pearlie , Han Ying , Rajadhyaksha Anjali , Mailloux Adam , Jessica McDaniel
Published: January 2023

E3 ubiquitin ligases (E3-UbL) regulate clonal anergy and immune self-tolerance. Of the known regulators, c-Cbl and Cbl-b regulate T-cell growth factor production and proliferation induced by IL-2 through the CD28 pathway. Investigations of immunomodulatory (IMiDsTM) drugs lenalidomide (LEN) and thalidomide in myelodysplastic syndrome and multiple myeloma show that these analogs are capable of activating T-cells in the absence of co-stimulation and reverse T-cell anergy. LEN increased IL-2 production (p<0.001), proliferation (p<0.001), and pCREB promoter binding in primary human T-cells after TCR stimulation alone without CD28-receptor ligation. Comparing LEN-induced response in sorted human CD28+CD8+ and CD28-CD8+ T-cells and in CD28 shRNA knock-down T-cells indicates that LEN acts to directly potentiate receptor function dependent on CD28 expression. A landmark study identified cereblon (CRBN), a RING-domain E3-UbL as the primary target of thalidomide. CRBN has also been shown to mediate LEN immunomodulatory activity in vitro. To examine the effect of CRBN on CD28 signaling, germ-line CRBN knock-out mice (crbn-/-) were studied and found to have normal numbers of mature T-cells, normal naïve and memory compartmentalization, and normal proliferative response, but increased contextual activation of IL-2 in the absence of CD28 ligation. These results reveal a novel E3-UbL regulator involved in CD28 signaling with importance in anti-tumor immunity.
Endosomal Toll-like receptors protect MyD88 from Complement Receptor 3-mediated degradation (P3001)
Authors: Reed Joanne , Buyon Jill , Gupta Vineet , Clancy Robert
Published: January 2023

Activation of Toll-like receptors (TLR) is a vital component of the inflammatory response in diseases characterized by nucleic acid-containing immune complexes. The identification of a biologic break to pathologic TLR signaling would be an important advance. Complement Receptor 3 (CR3) suppresses TLR-mediated inflammation in a murine sepsis model. The current study was initiated to evaluate whether a novel CR3 agonist, Leukadherin1 (LA1), downregulates TLR signaling in macrophages stimulated with hY3, a single stranded RNA that associates with the Ro autoantigen. Macrophages stimulated with either hY3, R848 (TLR7/8 agonist), lipopolysaccharide (TLR4 agonist), or interleukin (IL)-1β secreted TNFα. The addition of LA1 30 minutes prior to stimulation completely inhibited TNFα in all conditions and was accompanied by degradation of MyD88. When LA1 was administered 30 minutes after LPS and IL-1β, the CR3 agonist maintained its ability to inhibit TNFα. In contrast, the addition of LA1 after hY3 or R848 neither inhibited TNFα secretion nor degraded MyD88. These findings demonstrate that LA1 is a potent downregulator of signaling by surface “MyD88-dependent” receptors (TLR4, IL-1R). However, the anti-inflammatory properties of LA1 are negated if ligation of endosomal TLR7/8 has already occurred. In conclusion, CR3 agonists may be most effective in diseases mediated by surface TLR and limited to preventative therapy for autoimmune diseases triggered by RNA containing immune complexes.
The Immune Epitope Database (IEDB): A resource for immunological data related to infectious disease, autoimmunity and allergy. (P3024)
Authors: Vaughan Kerrie , Sette Alessandro
Published: January 2023

The Immune Epitope Database (IEDB) is a freely available repository of epitope data from infectious disease agents, allergy, autoimmunity and transplantation. The IEDB captures details of experiments associated with each epitope and thus provides an information rich resource and user-friendly platform for performing broad-based or more detailed queries. Recent analysis performed using the IEDB, including HCV, allergy, myasthenia gravis and diabetes; highlight the breadth of information accessible through the database, as well as tools and unique features provided therein. Each analysis serves to provide an inventory of all related data, thereby revealing interesting trends, experimental bias and/or knowledge gaps. Features like the Immunomebrowser and Disease Finder can be used synergistically to explore comparative immune reactivity across entire genomes or individual antigens for humans and animal models of disease, and tools like homology mapping can enable visualization of 3D discontinuous B cell epitopes. Moreover, these works provide outreach to the individual community of scientists, help elicit feedback to identify areas for improvement and can serve as ‘tutorials’ for those interested in using the database.
Bcl-XL overexpression prevents B cell receptor driven autophagy in IgM+ lymphoma (P3026)
Authors: Carey Gregory , Sesay Alphius , Roy Sanjit
Published: January 2023

We previously showed that B cell receptor (BCR) induced apoptosis in lymphoma required suppression of PI-3K [Carey GB, Scott DW (2001) J. Immunol, 166: 1618-]. PI-3K stimulates mTOR, a suppressor of autophagy and mitophagy. In addition, Bcl-XL blocks BCR-driven apoptosis in lymphoma, although the underlying mechanism is unclear. In contrast, a recent study demonstrated that Bcl2 overexpression blocked BCR-driven apoptosis, but not the induction of autophagy [Watanabe et al BBRC (2008) 19;374(2):274-]. Therefore the present study examined autophagic and mitophagic responses following BCR crosslinking on parental WEHI-231 lymphoma cells or on a derivative expressing high levels of Bcl-XL (WEHI-231/Bcl-XL). The results confirmed dramatic mitochondrial dysfunction followed by apoptosis in WEHI-231 compared to WEHI-231/Bcl-XL. BCR-crosslinking resulted in rapid and massive induction of the autophagic response in parental WEHI-231 compared to WEHI-231/Bcl-XL. Furthermore, treatment with STF-62247, a small molecule inducer of autophagy, resulted in apoptosis in WEHI-231 but not WEHI-231/Bcl-XL cells. These data suggest that Bcl-XL prevents BCR-driven apoptosis in lymphoma via blocking autophagic and mitophagic responses. Hence, these studies demonstrate specific roles of BclXL and bcl2 in BCR-driven death responses. Lastly, combinations of small molecule autophagy inducers and Bcl-XL antagonists may prove to be effective in addressing lymphoma.
Intracellular free Mg2+ is required to maintain NKG2D expression necessary for controlling EBV infection in XMEN disease (P3028)
Authors: Li Feng-Yen , Chaigne-Delalande Benjamin , O'Connor Geraldine , Lukacs Marshall , Zheng Lixin , Shatzer Amber , Matthews Helen , Su Helen , Cohen Jeffrey , Uzel Gulbu , Lenardo Michael
Published: January 2023

Epstein-Barr virus (EBV) is a B cell-tropic gammaherpesvirus present in latent form in approximately 90% of people worldwide. However, a subset of immunocompromised hosts develop malignancy from uncontrolled EBV infection. While the etiology of such immunodeficiency states is sometimes apparent, as in HIV and post-transplant immunosuppression, the underlying immune deficiency is other cases remains unknown. X-linked immunodeficiency with Mg2+ defect, EBV infection, and neoplasia (XMEN) disease is a novel primary immunodeficiency characterized by deleterious mutations in magnesium transporter 1 (MAGT1), chronic EBV infection and increased susceptibility for EBV-associated lymphomas. We had previously demonstrated that MAGT1 mediates a second messenger role for Mg2+ during T cell activation. Here, we show that loss of MAGT1 also decreases the basal level of intracellular free Mg2+ and abolishes the expression of NKG2D on natural killer cells and cytotoxic T lymphocytes, leading to impaired cytotoxicity against EBV-transformed lymphoblastoid cell lines. Interestingly, both these defects can be restored in vitro and in vivo by exogenous Mg2+ supplementation, leading to decreased EBV-infected cells in vivo. This not only revealed the first specific molecular function of basal free Mg2+ in eukaryotic cells but also identified a novel Mg2+-dependent immune arm of defense against EBV lymphomagenesis that has profound diagnostic and therapeutic implications for XMEN disease.
Possible involvement of transmembrane protein 173 in RANKL-induced osteoclastogenesis (P3012)
Authors: Kim Ju , Shin Yun Ji , Choe Chung Hyeon , Park In Sun , Yu Kang-Yeol , Jang Yong-Suk
Published: January 2023

Osteoclasts play a central role in bone development and remodeling via the resorption of bone and are thus important mediators of bone loss, which leads to osteoporosis. In this research, we investigated the effect of Tmem173 on RANKL-induced osteoclastogenesis via the study of osteoclast differentiation associated gene expression, TRAP activity and NO on Tmem173 overexpressed RAW264.7 cells. The mRNA levels of c-Fos, NFATc1, TRAP and TRAP activity were decreased in Tmem173 overexpressed RAW264.7 cells stimulated by RANKL. In addition, we observed down-regulation NO production and the mRNA levels of NO associated factors such as COX-2 and iNOS. Interestingly, we discovered up-regulation of the Tmem173 by osteoprotegerin (OPG) on Tmem173 overexpressed RAW264.7 cells. OPG prevents interaction of RANKL to RANK by competitive binding to RANKL. These results provide new additional information that Tmem173 played a regulatory role in RANKL-mediated osteoclast differentiation.
IL-4 upregulates Igα and Igβ protein, resulting in augmented IgM maturation and amplified BCR-triggered B cell activation in vitro and in vivo (P6230)
Authors: Guo Benchang , Rothstein Thomas
Published: January 2023

Interleukin 4 (IL-4) is critical for optimal B cell activation in T-dependent responses; however, the underlying mechanism remains elusive. In the present study, we demonstrate that IL-4-pretreatment markedly up-regulates STAT6-dependent Igα and Igβ protein expression in a translational manner in primary B cells. Elevated Igα and Igβ protein significantly promotes IgM maturation and IgM surface expression as evidenced by isolated overexpression of Igα and Igβ in a B cell line and this increased surface IgM is associated with augmented BCR-initiated ERK phosphorylation. In vivo, Tfh-derived IL-4 is critical for germinal center B cell expansion as shown by significant reduction of germinal center B cells in SRBC-immunized IL-4 receptor alpha deficient mice relative to wild type control mice. We find that IL-4 facilitates BCR-initiated pre-germinal center B cell activation by up-regulating Igα, Igβ, surface IgM expression and BCR-triggered pERK, all of which were reversed by administration of neutralizing anti-IL-4 antibody. In sum, this study elucidates a novel mechanism for crosstalk between the IL-4 receptor and B cell receptor.
IL-12 is required for mTOR regulation of memory CTLs during virus infection (P6232)
Authors: Garcia Karla , Xiao Zhengguo
Published: January 2023

Inflammatory cytokines are critical in the induction of memory CTLs, and inhibition of mTOR by rapamycin can enhance memory CTL generation. However, it is unknown if important inflammatory cytokines such as IL-12 are involved in the regulation of memory CTLs by mTOR during infection. We show here that inhibition of mTOR by rapamycin represses CTLs expansion and enhances memory CTL generation during vaccinia virus infection in mice. In addition, rapamycin promotes central memory phenotype in CTLs. However, the absence of IL-12 signal in CTLs diminishes rapamycin regulation on both CTL expansion and subsequent memory CTL formation, despite that rapamycin substantially increases memory CTLs when IL-12 signal is missing. Moreover, rapamycin-regulated memory CTLs in the absence of IL-12 are impaired in secondary expansion upon pathogen challenge, and subsequently form abolished secondary memory. In conclusion, IL-12 contributes to the strength of rapamycin regulation on primary memory CTLs, and lack of IL-12 signal leads to impaired secondary expansion of memory CTLs. Thus, the IL-12 signal is required for mTOR regulation on functional memory CTLs.
Novel CX3CR1+hi monocyte-derived dendritic cell subtype with inflammatory properties (P6244)
Authors: Beceiro Susana , Cortado Hanna , Partida-Sanchez Santiago
Published: January 2023

Blood monocyte-derived macrophages (MØ) and dendritic cells (DCs) are essential for effective control of infections. Exacerbated recruitment of these cells, in the other side, contributes to the pathogenesis of chronic inflammatory diseases. The main blood monocyte subsets differ in the expression of the differentiation marker Ly6C and the chemokine receptors CCR2 and CX3CR1. We recently found a subset of Ly6Clow/CCR2low/CX3CR1hi associated to inflammatory responses induced by urinary tract infection in the mouse. Therefore, here we analyzed monocyte-derived inflammatory cell subsets in a model of non-bacterial peritonitis using CCR2 and CX3CR1 KO mice. We found that CCR2 is not required for tissue recruitment of monocyte-derived MØ under homeostatic conditions. In contrast, CCR2 expression is required for the recruitment of two Ly6Chi/CX3CR1low and Ly6Clow/CX3CR1hi subpopulations of inflammatory cells. Furthermore, sorted Ly6Clow/CX3CR1hi cells produced high levels of IL-6, MIP-1 and TNF-α cytokines. These cells showed dendritic cell morphology, low levels of F4/80 but CD11c expression, as well as Class-II, CD80, and CD86 DC markers. In addition, Ly6Clow/CX3CR1hi cells were capable of picking-up antigen and trafficked to lymph nodes demonstrating specific DC functions. In summary, our studies indicate that blood monocytes expressing Ly6Clow/CX3CR1hi give rise to a novel inflammatory DC subset recruited to tissue during infectious or non-infectious inflammatory conditions.
CCR2 blockade of monocyte migration is less potent at higher CD11b integrin levels in HIV-1 infection (P6213)
Authors: D'Antoni Michelle , Jalbert Emilie , Shikuma Cecilia , Barbour Jason
Published: January 2023

Monocytes are a key contributor to HIV inflammation and HIV-associated neurocognitive disorders by migrating into the brain in response to chemokine gradients, such as MCP-1 (a CCR2 ligand). We sought to characterize the migration capacity of monocytes from HIV infected adults. Cryopreserved peripheral blood mononuclear cells from HIV-1 infected adults (n=6) were incubated with BMS-22 (blocks MCP-1 binding to CCR2) and transferred to a transwell migration system, and put under an MCP-1 gradient. Migrated cells were collected and stained for CD3, CD14, CD16, HLA-DR, CCR2, CCR5 and CD11b. Cells were acquired on a Fortessa flow cytometer and analyzed using FlowJo. MCP-1 caused a significant increase in migration compared to baseline (3.0 fold change, p = 0.01) in the absence of BMS-22. There was a significant inverse correlation between CD11b mean fluorescence intensity (MFI) and degree of inhibition of migration by BMS-22 (rho = -0.88, p = 0.03). A trend between higher CCR2 and CCR5 MFI and lower migratory rates was also observed. Higher CD11b rates associated with poorer inhibition of migration by BMS-22, a CCR2 but not CD11b antagonist. Blocking MCP-1 is being pursued as a novel treatment method in HIV. The incomplete inhibition of monocyte migration at higher CD11b expression levels by a CCR2 antagonist highlights compensatory mechanisms between CCR2 and integrins, and indicates CD11b levels could guide effective use of CCR2 antagonist based therapies in humans.
An in vivo induced membrane antigen of Lyme disease pathogen triggers murine joint inflammation (P6258)
Authors: Yang Xiuli , Pal Utpal
Published: January 2023

Borrelia burgdorferi bba57 is a plasmid-encoded gene-product of unknown function. Here we show that bba57 is induced in vivo and facilitates early murine infection. While BBA57 is not required for pathogen persistence during later phases of infection, unlike wild type isolate, the mutants failed to induce severe disease. We show that BBA57 is an outer membrane and surface-exposed antigen that is essential for the genesis of murine joint inflammation. Using a PCR array, we further identified that deficiency of BBA57 reduces the expression of selected “neutrophil-recruiting” chemokines and associated receptors in the joints that ultimately results in severe impairment of neutrophil infiltration. Similarly, using an established chemotaxis assay we demonstrated that BBA57 is directly involved in neutrophil migration in vitro. Taken together, our data suggest that BBA57 is a major trigger of murine Lyme arthritis, and that interfering with BBA57 may contribute to the development of new therapeutic strategy to prevent Lyme arthritis.
Hantaan virus induces high expression levels of CXCL10 via TLR3, RIG-I, and MDA5 pathways. (P6221)
Authors: Zhang Yusi , Liu Bei , Ma Ying , Zhang Chunmei , Yi Jing , Zhang Yun , Zhuang Ran , Jin Boquan
Published: January 2023

Hantaan virus is a major agent causing hemorrhagic fever with renal syndrome, a high-mortality-rate disease. However, the pathogenesis of HFRS remains to be elucidated. We found that CXCL10 expresses highly in the HFRS-patients’ sera and the increased CXCL10 is positively correlated with the severity of HFRS. To clarify the molecular mechanisms of HTNV infection inducing CXCL10 expression, the HTNV-infected human umbilical vein endothelial cells model has been used. The results indicated that the dsRNA, produced in the process of HTNV replication, could activate TLR3, RIG-I, and MDA5 pathways, which facilitated the translocation of IRF7 and NF-κB. Afterwards, the two transcription factors bound directly to the CXCL10 promoter region and promoted the transcription and expression of CXCL10. Furthermore, we found that the expression levels of CXCR3, the receptor of CXCL10, increased on the surface of monocytes at the early stage of HFRS. It suggested that CXCL10 may recruit monocytes, which may be involved in the cytokine storm and the pathogenesis of HFRS. Further studies are still needed to reveal the function of CXCL10 in the pathogenesis of HFRS. In conclusion, we reported that the increased CXCL10 level was associated with the state of the HFRS. The activation of innate immunity induced by HTNV replication may be of importance in the regulation of CXCL10 expression and the abundant CXCL10 may play an important role in the pathogenesis of HFRS.
Flowcytometric evaluation of T lymphocyte suppression by Euphorbia hirta (P6226)
Authors: Albakheet Saleh , Ahmad Sheikh
Published: January 2023

Euphorbia hirta is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, anti-inflammatory activities etc. The objective of the study was to investigate immunomodulatory effects of the ethanol extract of E. hirta on humoral antibody & delayed-type hypersensitivity (DTH) immune responses, graft rejection, T-lymphocytes (CD3+, CD4+ and CD8+ receptors), B- lymphocyte (CD19+ receptor) and Th1 cytokines (IL-2, TNF-α & IFN-γ) in dose dependent manner in mice. Assessment of T and B cell receptors and their intracellular content of (Th1) cytokines was carried out by flowcytometric analysis in Balb/c mice. E. hirta ethanol extract at 25-200 mg/kg doses was given orally for seven days from the day of immunization with sheep red blood cells (SRBC) as an antigen. Cyclosporine 5 mg/kg and levamisole 2.5 mg/kg was used as standard drug. E. hirta maximum inhibition at 100 and 200 mg/kg p.o was found to significantly block the production of the cell mediated immune response, (CD3+, CD4+, CD8+ and CD19+ receptors) and Th1 (IL-2, TNF-α & IFN-γ) and also prolongs graft rejection. E. hirta also showed a decrease of DTH response and dose related decrease in the primary antibody response. Based on the data, it can be suggested that E. hirta is a potent and non-toxic immunosuppressor, which can be further explored for the development of potent immunosuppressor.