bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of LPS Tolerance in Macrophages (48.2)
Authors: O'Carroll Christine , Fagan Ailis , Carmody Ruaidhri
Published: January 2023

Lipopolysaccharide (LPS) tolerance is an essential immune-homeostatic response which prevents unnecessary over-activation of the inflammatory response. LPS tolerance induces a state of reduced responsiveness to repeated challenges with LPS, resulting in decreased expression of pro-inflammatory modulators, up-regulation of antimicrobials and other mediators promoting the resolution of inflammation. LPS tolerance is a transient state and LPS tolerant macrophages (MΦT) return to an LPS responsive state within days of initial stimulation. Macrophages which have recovered from a tolerant state (MΦR) remain largely uncharacterised. Gene expression profiling reveals that LPS tolerance recovery triggers an extensive modulation of the gene expression profile of macrophages distinct from that of (MΦT) and LPS activated macrophages (MΦA). Recovery from LPS tolerance at 88 hours post treatment leads to expression of key pro-inflammatory genes at levels comparable to MΦA. MΦR show increased expression of key modulators of the immune response not previously observed during classical activation. We propose that MΦR represent a novel macrophage phenotype. Furthermore we propose that the recovery from LPS tolerance promotes a global modulation of gene expression with distinct expression profiles between genes promoting inflammation and genes key to the resolution of inflammation. In addition we present an expression analysis of NF-κB and its regulators in naïve, MΦT and MΦR cells.
P-selectin Glycoprotein Ligand-1 Expression on T cells is Required for Optimal Development of T Cell Memory in Response to Influenza Virus Infection (49.22)
Authors: Tinoco Roberto , Baaten Bas , Douglas Mia , Bradley Linda
Published: January 2023

Influenza viruses represent a major global health concern as they have the potential to cause worldwide pandemics characterized by increased morbidity and mortality. T cells are critical for eliminating virus-infected cells and their localization to the respiratory tract is crucial to resolving infection. The underlying mechanisms regulating effector and memory T cell responses after influenza virus infection remain unclear. Using a mouse model, we demonstrated that the adhesion molecule P-selectin is induced on lung endothelium after infection. Virus-specific T cells acquired the ability to bind P-selectin through expression and glycosylation of its ligand PSGL1. P-selectin binding antigen-specific T cells produced increased levels of IFN-γ and granzyme B in the lung when compared to non-binding T cells. Furthermore, P-selectin binding antigen-specific T cells persisted as memory cells, expressed IL-7 receptor, and localized primarily in the lung and its draining lymph node. PSGL1 knockout (KO) T cells, which do not bind P-selectin, expanded to a greater extent than wildtype T cells during primary infection. However, fewer PSGL1 KO T cells persisted following contraction and there was a profound defect in PSGL1 KO memory cell re-expansion. Our findings highlight a critical role for the P-selectin-PSGL-1 interaction in regulating local T cell responses. Modulating this interaction may provide a novel way to boost T cell memory and immunity to influenza viruses.
Nasopharyngeal colonization by Streptococcus pneumoniae elicits long-lasting immune responses that confer protection from invasive lung infections. (49.23)
Authors: Maung Nang , Goodwin Aaron , Munoz-Elias Ernesto , Clark Stacie , Camilli Andrew , Gerstein Rachel , Leong John
Published: January 2023

Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection, but more commonly resides harmlessly in the nasopharynx. We demonstrated that nasopharyngeal (n.p.) colonization by S. pneumoniae in mice generates an antibody response that provides at least 10-week protection against lethal lung challenge. B cells were required for this protection, and transfer of sera from n.p.-colonized mice or antibodies against phosphorylcholine, a common S. pneumoniae surface antigen, protected naïve mice. Whereas T-independent anti-capsular antibodies were previously shown to be sufficient for protection against bloodstream infection , we found that T cells were required to generate a fully protective response following n.p. colonization. Depletion of CD4+ T cells at the time of pulmonary challenge or colonization of MHC class II-deficient mice did not compromise protection, suggesting a limited role for CD4+ T cells in our model. Sub-lethal irradiation of mice, which inactivates memory B cells but not long-lived plasma cells, did not significantly diminish protection, and transfer of bone marrow cells enriched for long-lived plasma cells from n.p.-colonized mice to B cell-deficient recipients increased pneumococcal-specific antibody titers and afforded significant protection. Our results suggest that n.p. colonization is capable of eliciting extended protection against invasive pneumococcal disease by generating long-lived antibody-secreting cells.
Th17 cells mediate clade specific mucosal immunity (49.26)
Authors: Chen Kong , McAleer Jeremy , Weaver Casey , Kolls Jay , Paterson David
Published: January 2023

The IL-17 family of cytokines phylogenetically pre-dates the evolution of T-cells in jawed vertebrates, suggesting that the ontogeny of the Th17 lineage must have arisen to confer an evolutionary advantage to the host over innate sources of IL-17. Using a model of mucosal immunization with the encapsulated bacteria, Klebsiella pneumoniae, we show that intranasal immunization strongly induces IL-17 producing cells in the lung. Further analysis reveals that these cells are conventional Th17 cells and are antigen specific as they proliferate and produce IL-17 upon re-stimulation with heat-killed K. pneumoniae but not heat-killed Escherichia coli or Staphylococcus aureus. In vivo, immunization induced Th17 cells but not Th1 cells undergo activation/expansion and contraction overtime and the Th17 responses together with anit-K. pneumoniae antibody responses lead to the protection of immunized mice from live K. pneumoniae infection. However, B-cell/antibody mediated protection is limited only to the vaccine strain. In contrast memory Th17 cells proliferate in response to conserved outer membrane proteins and confer protection against subsequent bacterial challenge with several serotypes of K. pneumoniae, including the recently described multi-drug resistant New Delhi metallolactamase strain. These data suggest that Th17 adjuvants can improve vaccination strategies against drug resistant encapsulated bacteria.
Potent anti-inflammatory activity of a novel of class Hsp90 inhibitors for multiple sclerosis (51.1)
Authors: Zhou Dan , Liu Yuan , Ye Josephine , Ying Weiwen , Chimmanamada Dinesh , Shin Ogawa Luisa , Inoue Takayo , Rao Patricia , Wada Yumiko
Published: January 2023

Current therapies for multiple sclerosis (MS) generally reduce the frequency, severity and duration of relapses, and may slow disease progression, but are not curative. Clinical benefit also comes with side effects that range from mild to life-threatening. Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of many proteins that modulate cellular response to environmental stresses. Due to its central role in multiple immune-mediated mechanisms, Hsp90 has recently emerged as a potential target for MS therapy. A number of the inflammatory mediators underlying MS are known Hsp90 client proteins, and Hsp90 blockade represents an attractive multi-targeted therapeutic approach given its alternative mechanisms of neuroprotection and inhibition of inflammation. Here we describe a novel class of small molecule inhibitors that preferentially target Hsp90 associated with immune-mediated pathways and which demonstrate potent inhibitory activity, high oral bioavailability and prolonged CNS exposure. Further, the lead compound displays broad activity in a number of relevant in vitro and in vivo models; including inhibition of cytokine production by human PBMCs, decreased nitric oxide production by primary rat astrocytes, and efficacy in an adoptive transfer model of MS. Taken together, this class of Hsp90 inhibitors may potentially provide new avenues to counter immune overstimulation in MS patients.
Comparison of protein expression by different lentiviral vectors (51.12)
Authors: Wang Nan , Rajasekaran Narendiran , Hou Tieying , Mellins Elizabeth
Published: January 2023

HIV1-derived lentiviral vectors have been widely used as gene delivery tools due to their potent transduction capacity and stable expression after chromosomal integration in dividing and non-dividing mammalian cells. Lentiviral vectors were screened for expressing the murine class II chaperone, invariant chain (Ii), in hematopoietic stem and progenitor cells (HSPC). We compared various lentiviral vectors using GFP as a reporter gene in 293T cells and HSPC. We assessed a dual promoter vector (DP) with separate promoters for Ii and GFP, a fusion protein vector (FU) that expresses Ii and GFP together under a single promoter, and T2A vector (T2A) in which Ii and GFP are separated by a self-cleaving 2A peptide. Similar percentages of 293T cells were positive for Ii expression after being directly transfected with DP and T2A. The percentage was slightly higher in FU transfected cells. 293T cells transfected with DP have the highest GFP expression. We packaged each vector into lentivirus and found that both Ii and GFP expression levels dropped in 293T cells. Further reduction in expression was seen in viral transduced HSPC. The trend toward different expression levels of Ii and GFP persisted whether lentiviral transduction or vector transfection was used. Only DP and T2A constructs, but not FU constructs, resulted in functional Ii expression. Based on these results, we hypothesize that the poor expression of Ii may be due to deleterious effects of Ii on assembly of competent virus.
Effect of targeted drug delivery with G5-MTX dendrimers on induced T regulatory (iTreg) CD4+ T cells in an inflammatory bowel disease (IBD) colitis model (52.1)
Authors: Belyakov Igor , Zimmermann Ellen , Isakov Dmitry , Fisk Elizabeth , Orr Nicholas , Schmiedlin-Ren Lee , Choi Seok Ki , Baker James
Published: January 2023

Inflammatory bowel disease (IBD) are complex immune mediated diseases involving the interactions of many cell types including the intestinal mucosal epithelia, macrophages, T-lymphocytes (Foxp3+ Treg, Th17, Th2) and dendritic cell subsets. Induced Treg (iTreg) play an essential role in immune tolerance in the control of chronic inflammation. Current approaches to IBD therapy utilize immunosuppressive medications including methotrexate (MTX), which is hepatotoxic. Targeted drug delivery of MTX for IBD treatment may be achieved by conjugating MTX to G5 PAMAM dendrimer nanoparticles. We investigated the effect of G5-MTX12 on inflammatory responses in dextran sulfate sodium (DSS) induced IBD in BALB/c mice. We evaluated the frequency of iTreg (Foxp3+Helios- CD4+ TCRαβ+ T cells) in mesenteric lymph nodes (MLN). We found that treatment with G5-MTX induces protection of mice against DSS colitis. We found that the weight of mice treated with G5-MTX was not significantly different from naïve animals. There was a 4.7-fold increase in frequency of iTreg in mesenteric lymph nodes in G5-MTX treated mice compared to naïve animals. Thus, we achieved a therapeutic effect with targeted G5-MTX in the IBD model in mice. This targeted drug effect may be mediated by iTreg that developed under sub-immunogenic antigen presentation during intestinal inflammation. Our data may suggest that a targeted drug delivery strategy with dendrimer technology may be effective with potentially less toxicity
Selenoprotein K is a novel target of m-calpain, and cleavage is regulated by toll-like receptor-induced calpastatin in macrophages (54.22)
Authors: Rose Aaron , Zhi Huang , Hoffmann Fukun , Norton Robert , Hoffamann Peter
Published: January 2023

Calpains are calcium (Ca2+)-activated cysteine proteases that modulate cellular function through cleavage of a variety of intra-cellular targets. Two major isoforms of calpain, u- and m-calpain, are expressed in different tissues and cell-types. Selenoprotein K (SelK) is a selenocysteine containing transmembrane ER protein involved in Ca2+ flux, and western blot analyses revealed one SelK band in mouse lymphocytes (both B and T cells) and two bands for SelK in mouse myeloid cells (macrophages, dendritic cells, and neutrophils). This led us to investigate cleavage of SelK and we found that m-calpain cleaved SelK in myeloid cells. The calpain-mediated cleavage of SelK occured between Arg81 and Gly82 resulting in the removal of the selenocysteine-containing portion of the cytosolic region of the protein. We have further linked this event to the activation state of macrophages. Resting macrophages were found to predominantly contain cleaved SelK while macrophages activated through toll-like receptors contained full length SelK. This was due to increased levels of calpastatin, which is the endogenous inhibitor of calpains. The identification of SelK as a novel target for calpain cleavage activity reveals a novel mechanism by which macrophage activation is regulated and studies are on-going to further characterize the role of calpain/calpastatin in macrophages.
Decreased expression of B- and T-lymphocyte attenuator (BTLA) is associated with progression of disease in idiopathic pulmonary fibrosis (IPF) (54.7)
Authors: Takahashi Stephenie , Blaine Kelly , Williams Jesse , Fu Yang-Xin , Noth Imre , Sperling Anne
Published: January 2023

IPF is an interstitial lung disease that affects over 128,000 people in the U.S., has a yearly mortality of 48,000, and has no effective treatment. Current theories on the etiology of IPF have not focused on the role of the immune system due to the inefficacy of steroid therapy. However, we have found in microarray studies that peripheral blood T cell co-stimulatory markers CD28, ICOS and BTLA, are associated and significantly predictive of early mortality in IPF patients. A significant association was found between decreased BTLA mRNA levels and decreased predicted diffusing capacity of carbon monoxide (DLCO) levels, a clinical measure of disease severity. Analysis of all samples demonstrated a significant and dramatically decreased BTLA surface expression on both peripheral CD4 and CD8 T cells, but not B cells, in rapidly progressing patients compared to controls or stable patients. Similar, but less dramatic decrease in surface expression is found for CD28 and ICOS. In a bleomycin-induced pulmonary fibrosis model, BTLA-/- mice, and to a lesser extent ICOS-/- mice, show increased numbers of lung CD4+ T cells and macrophages, significantly decreased weight, and more severe lung fibrosis when compared to controls. These results support the role of altered co-stimulatory molecules in the progression of IPF implying that these molecules are involved in the pathogenesis of disease progression.
Asthma: A missense SNP in the FCER2 (CD23) gene favors increased IgE production by human B cells (57.2)
Authors: Chan Marcia , Gigliotti Nicole , Rosenwasser Lanny
Published: January 2023

Asthma now is the most common chronic condition in children. Many asthmatics are unresponsive to therapy and genetic predisposition likely contributes. Pharmacogenetic studies have highlighted novel sequence variants in several genes. One such gene is fcϵr2, encoding the low affinity IgE receptor CD23 (FcϵRII). CD23 is expressed on several cell types and has several roles in IgE-mediated immunity, including antigen focusing and presentation to TH2 cells; mediating production of pro-inflammatory cytokines by monocytes; transporting IgE and allergen-IgE complexes through gastrointestinal epithelium; and participating in both positive- and negative-feedback mechanisms in regulation of IgE synthesis by B cells. Single nucleotide polymorphisms (SNPs) in fcϵr2 are associated with increased risk of exacerbations in asthmatics treated with inhaled corticosteroids but functional relevance of these SNPs in fcϵr2 remains unknown. One SNP (rs2228137) encodes a nonsynonymous amino acid change (R62W). We established stable human B cell transfectants expressing either wild type CD23b-R62 or CD23b-R62W. Stimulation through CD23 causes differences in ERK1/2 activation between the transfectants. Transfectants expressing CD23-R62W exhibited increased activation of the transcription factor Egr-1 (found in the IL-4R promoter region), and increased expression of IL-4R. These data suggest that the R62W SNP may favor increased IgE production through increased responsiveness to IL-4.
Development of mRNA signatures tools to detect in vivo basophil phenotypes: IL-3 signatures (57.5)
Authors: MacGlashan Donald
Published: January 2023

There are several well-recognized phenotypes of peripheral blood basophils (e.g., non-releasers) but the underlying bases for the phenotypes are not explained. IL-3 has many effects on basophil maturation and function and is known to induce considerable changes in basophil phenotype. Purified human basophils were cultured with or without IL-3 for 24 hours and the microarray expression profile, relative to pre-culture, analyzed for transcripts that acted in a reciprocal fashion depending on the condition. A 51- gene transcript set was identified that was composed 34 transcripts that increased with IL-3 and decreased without IL-3 and a further 17 transcripts that did the reverse. The changes were stable in time, with 3-day cultures generating a concordant signature. The inverted nature of the response to ±IL-3 suggested that the normal circulating basophil is in a sensitive balanced state of IL-3 exposure. For example, it could detect the early generation of IL-3 that follows IgE-mediated stimulation of basophils. When this signature tool was applied to freshly isolated basophils, it was observed that the signature was stable in time for the same subjects’ basophils. But when comparing the non-releaser and releaser phenotypes, the signature was consistent with a low-level deficiency in exposure to IL-3 or an IL-3-like cytokine in the “non-releaser phenotype. These studies identified an IL-3 signature to probe changes in basophils occurring in vivo.
Methods for quantification of In vitro cell-substrate contacts. (58.1)
Authors: Cammer Michael , Vardhana Santosh , Novak Hila , Sacristan Rock Catarina , Liapis Anastasia , Depoil David , Dustin Michael
Published: January 2023

A common problem in in vitro immunology research is characterizing cell spreading on substrates by morphology and by protein or other molecule distributions. Here we describe automated and semi-automated methods for measuring protein distributions at the cell-substrate interface which are key indicators of biological function. These methods may be applied to images from live or fixed cells. Using macros in ImageJ, we have developed methods to quantify the symmetry of attaching or spreading cells and the radial distributions of proteins. These distributions may be correlated with morphometry and provide insight into cell physiology. Examples in T Cells are shown regarding the relative localizations protein kinases under various treatments and of ICAM and TCR in a myosin mutant. In addition, a simple method is described to measure the movement of proteins in live cells where automated computer detection is not feasible.
The susceptibility of CXCR3KO mice to EAE is associated with the increased numbers of Th17 infiltrating in CNS (60.4)
Authors: Chung Chen-Yen , Liao Fang
Published: January 2023

CXCR3 and its ligands are highly associated with neuronal inflammation. Given that Th17 cells play a critical role in EAE pathogenesis and that Th17 cells have been shown to express CXCR3, we intended to investigate the effect of CXCR3 on EAE, particularly focusing on Th17 cells. In this study, we demonstrated that CXCR3KO mice developed more severe EAE as compared with wild-type mice. Of interest, CXCR3KO mice exhibited higher numbers of Th17 cells infiltrating in CNS as compared with wild-type mice. In accordance with this result, using the passive EAE model, we showed that mice receiving Th17 cells derived from MOG-immunized CXCR3KO mice developed more severe EAE as compared with mice receiving Th17 cells derived from MOG-immunized wild-type mice. Furthermore, intracellular staining revealed that CXCR3KO mice exhibited the increased numbers of Th17 cells expressing GM-CSF, a recently identified encephalitogenic cytokine that contributes to the effector phase of EAE. Altogether, we provide evidence that CXCR3 plays a protective role rather than a immunopathgenic role in EAE via negative regulation of Th17 cells infiltrating in CNS. The mechanism underlying the increased numbers of Th17 cells infiltrating in CNS of CXCR3KO mice leading to the susceptibility to EAE is currently under investigation.
Contribution of CD11c+ GR1+ cells to the initiation of lymph node vascular growth after immunization via the secretion of IL-1β (61.8)
Authors: Benahmed Fairouz , Tian Sha , Lu Theresa
Published: January 2023

Immune stimulation results in draining lymph node swelling and vascular-stromal growth. Lymph node endothelial cell proliferation is upregulated at day 2 after immunization and this early event is dependent on CD11c+ cells and VEGF. Fibroblast-type reticular stromal cells (FRC) are the principal VEGF-expressing cells within lymph nodes. Here, we test the hypothesis that CD11cmed MHCIImed GR1+ cells that accumulate in the draining lymph node plays a role during the upregulation of endothelial cell proliferation at day 2. We show that depletion of GR1+ cells induces a reduction in endothelial cell proliferation in comparison with control mice. In vitro, CD11c+ cells isolated from immunized lymph nodes are able to stimulate VEGF secretion by FRCs. CD11c+Gr1+ cells express IL-1β, and anti-IL-1β treatment reduces lymph node endothelial cell proliferation. Our results suggest that the CD11c+GR1+ cells contribute to the initiation of lymph node vascular growth via the secretion of IL-1β, which then induces increased VEGF expression by FRCs.
Ceramide deficiency in the epidermis leads to development of psoriasis-like lesions associated with IL-23-dependent proliferation of γδ-17 cells (62.1)
Authors: Nakajima Kimiko , Kataoka Sayo , Goto-Inoue Naoko , Terao Mika , Setou Mitsutoshi , Katayama Ichiro , Takeda Junji , Sano Shigetoshi
Published: January 2023

It has been recognized that ceramide levels are decreased in the epidermis of patients with atopic dermatitis and psoriasis. However, the underlying mechanism by which ceramide deficiency leads to skin inflammation still remains unclear. Here, we generated Sptlc2 targeted mice under control of the keratin 5 promoter (referred to as K5-SPT-KO mice), by which their keratinocytes were devoid of serine palmitoyltransferase (SPT), the rate-limiting enzyme for de novo sphingolipid synthesis. K5-SPT-KO mice have decreased levels of ceramide in the epidermis and these mice demonstrate barrier dysfunction. From 2 weeks of age, they develop skin inflammation, showing psoriasis-like histopathologic changes and inflammatory cell infiltrates in the dermis. While expression of IFN-γ was not observed in the inflamed skin and draining lymph nodes, increased levels of IL-17 and IL-22 were detected. Strikingly, K5-SPT-KO mice showed increased numbers of γδ-T cells that produce IL-17 (γδ-17) in the skin lesion and lymph nodes and most of them also produce IL-22, similar to Th17 cells. However, much fewer γδ-17 cells are detected in the skin and lymph nodes of wild-type mice. In vivo administration of anti-IL-12/23p40 antibody ameliorated the skin lesions and reduced the number of γδ-17 cells in K5-SPT-KO mice. Therefore, we conclude that ceramide deficiency in the epidermis results in the development of psoriasis-like lesions, likely mediated by IL-23-dependent γδ-17 cells in mice.
Pre-clinical efficacy of dual PI3K δ/γ inhibitors in airway inflammation (64.2)
Authors: Nagarathnam Dhanapalan , Govindarajulu Babu , Routhu Kasiviswanath , Rajagopalan Prasanna , Veeraraghavan Sridhar , Viswanadha Srikant , Vakkalanka Swaroop
Published: January 2023

Dual δ/γ inhibition is strongly implicated as an intervention treatment in allergic and non-allergic inflammation of the airways and autoimmune diseases. The objective of this study was to explore the therapeutic potential of RP6047, a novel, small molecule PI3Kδ/γ inhibitor in airway disorders. Specificity of lead molecule, RP6047, towards PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and several cell based assays. Pre-clinical efficacy of RP6047 was confirmed in animal models of airway inflammation. RP6047 demonstrated significant potency against PI3Kδ (19.7 nM) and γ (31.2 nM) with several fold selectivity over α and β isoforms and subsequent pAKT inhibition. Additionally, the compound inhibited neutrophil functionality and mast-cell degranulation at nanomolar concentrations besides reversing CSE induced dexamethasone insensitivity by >90%. RP6047 displayed excellent efficacy in inhibiting LPS-induced TNFα (36% at 10 mg/kg/po) and neutrophilia (ED50 = 1.35 mg/kg/po) in a rat model. Administration of compound also resulted in a near complete reduction in inflammatory cell infiltration into the lungs in CSE treated mice over a 4-day period. Results demonstrate the therapeutic potential of RP6047 in asthma and related airway disorders acting via the PI3Kδ/γ pathway. Further in vivo studies are in progress to evaluate the efficacy of the compound in airway hyper-responsiveness prior to the initiation of clinical trials.
Neutrophils: key players in mast cell-mediated thermal pain in mice (67.4)
Authors: Chatterjea Devavani , Willenbring Robin , Balsells Evelyn , Paredes Luisa , Allen Juliann
Published: January 2023

Mast cells are involved in a range of physiologies and pathologies including allergic disease, defense against pathogens, and neutralization of venoms. Recently, these cells have been shown to play significant roles in inflammatory pain. Acute inflammation is a beneficial mechanism designed to prevent further tissue damage; however, it can also lead to a decreased quality of life and, as such, is a pervasive health concern. The involvement of mast cells in this process has not been fully characterized. We show, in a mast cell-dependent model of thermal pain that degranulation of peripheral mast cells in the plantar tissue of mice is accompanied by neutrophil influx as well as edema and upregulation of pro-inflammatory cytokines. When mast cells are stabilized with sodium cromoglycate, pain, edema and neutrophil influx are abrogated. Additionally, when neutrophil influx is blocked with pre-administration of the selectin blocker fucoidan, pain and edema are also abrogated. This suggests neutrophil influx is a key event in the development of mast cell-initiated inflammatory pain.
A novel autocrine-paracrine loop of TSLP in atopic dermatitis is modulated by p63. (71.4)
Authors: Kubo Terufumi , Ye Rui , Ara Shihoko , Matsumiya Hiroshi , Kojima Takashi , Kikuchi Tomoki , Sato Noriyuki , Ichimiya Shingo
Published: January 2023

Atopic dermatitis (AD) is a common disease affecting over 10% of children and adults, which severely impairs quality of life. Accumulating evidence has revealed that thymic stromal lymphopoietin (TSLP) of a pleiotropic cytokine play a critical role in the maintenance of AD. Currently, epidermal cells of AD are postulated as a major producer of TSLP, eventually leading to unfavorable local inflammation. However a property of the epidermal cells of AD is largely unknown. In the present study we assessed a functional significance of an epitheliotropic transcription factor p63 of a p53 homologue in the skin lesions of AD. Interestingly immunohistochemical analysis indicated constitutive downregulation of p63 of the epidermal cells in AD-affected skin areas. Further in vitro experiments using primary epidermal cells demonstrated that TSLP stimulation efficiently allowed cells to secrete TSLP as well as several inflammatory cytokines. Moreover it was found that the levels of expression of TSLP receptor (TSLPR) were under the control of p63. Collectively these indicate that the downregulation of p63 may cause chronic inflammation through providing a harmful TSLP-TSLPR axis to epidermal cells. Together with the evidence that innate ligands to TLRs like TLR3 could steadily suppress p63, our data would provide new insight into the pathogenesis of AD, especially with respect to the operation of innate immune responses by epidermal p63.
Ligation of CTLA-4 to the TCR inhibits T cell activation and directs differentiation into Foxp3+ regulatory T cells (72.2)
Authors: Zhu Yunxiang , Karman Jozsef , Jiang Ji-Lei , Gumlaw Nathan , Zhao Hongmei , Campos-Rivera Juanita , Sancho Jose , Zhang Jinhua , Jiang Canwen , Cheng Seng H.
Published: January 2023

ABSTRACT Crosslinking of ligand-engaged cytotoxic T lymphocyte antigen-4 (CTLA-4) to the T cell receptor (TCR) during the early phase of T cell activation attenuates TCR signaling, leading to T cell inhibition. To promote this event, a bispecific fusion protein comprising a mutant mouse CD80 (CD80w88a) and lymphocyte activation antigen-3 (LAG-3) was engineered to concurrently engage CTLA-4 and crosslink it to the TCR. Crosslinking is expected to attain via ligation of CTLA-4 first to MHC II and then indirectly to the TCR, generating a CTLA-4-MHC II-TCR tri-molecular complex that forms between T cells and antigen-presenting cells (APCs) during T cell activation. Treating T cells with this bispecific fusion protein inhibited T cell activation. In addition, it induced the production of IL-10 and TGF- β and attenuated AKT and mTOR signaling. Intriguingly, treatment with the bispecific fusion protein also directed early T cell differentiation into Foxp3+ regulatory T cells (Tregs). This process was dependent on the endogenous production of TGF- β. Thus, bispecific fusion proteins that engage CTLA-4 and co-ligate it to the TCR during the early phase of T cell activation can negatively regulate the T cell response. Bispecific biologics with such dual functions may therefore represent a novel class of therapeutics for immune modulation. The findings presented here also reveal a potential new role for CTLA-4 in Treg differentiation.
Control of IL-2/ regulatory T cell axis by Calcium/calmodulin-dependent protein kinase type IV plays a key role in lupus-prone mice (72.4)
Authors: Koga Tomohiro , Ichinose Kunihiro , Mizui Masayuki , Crispín José , Tsokos George
Published: January 2023

Objective. Dysfunction of regulatory CD4+CD25+FoxP3+ T (Treg) cells has been detected in autoimmune diseases. IL-2 is critical for the development, survival, and function of Treg cells. Increased levels of Calcium/calmodulin-dependent protein kinase type IV (CaMKIV) cause defective IL-2 production in T cells from patients with SLE. Methods. We crossed CaMKIV deficient (CaMK4-/-) mice with MRL/MpJ-lpr/lpr (MRL/lpr) mice and examined survival rate, autoantibody levels, immune cells subpopulations, pro-inflammatory cytokine production and T cell function. Results. The survival rate was significantly higher in MRL/lpr.CaMK4-/- mice than in wild type MRL/lpr mice at 32 weeks (89% versus 25%, p<0.05). MRL/lpr.CaMK4-/- mice displayed decreased titers of anti-DNA antibodies, reduced numbers of double negative T cell, CD4+ activated/memory cells in the spleen. The production of IL-2 in response to anti-CD3/28 antibody by CD4+ T cells was restored in MRL/lpr.CaMK4-/- mice, and this was associated with decreased levels of phosphorylated cAMP response element modulator (CREM) in the nucleus. Notably, the number of Treg cells was increased in MRL/lpr.CaMKiv-/- mice. We further confirmed silencing of CaMKIV resulted in overexpression of FoxP3 gene in human SLE T cells. Conclusion. Our results suggest that CaMKIV is important in the expression of the disease in lupus-prone mice, and we propose that CaMKIV represents a valuable target for treatment in autoimmune diseases.
The role of TRIM68 in Toll-like receptor and RIG-I-like receptor induced interferon production (72.5)
Authors: Wynne Claire , Higgs Rowan , Biron Christine , Jefferies Caroline
Published: January 2023

Innate immune receptors such as Toll-like receptors (TLR) and RIG-I-like receptors (RLR) play key roles in viral recognition and interferon (IFN) production. Systemic Lupus Erythematosus (SLE) patients commonly present with an over production of type 1 IFNs which contribute to disease pathogenesis so understanding how to dampen down this response is of huge importance. The focus of this project is on the E3 ubiquitin ligase TRIM68, a known autoantigen in SLE. Results show TRIM68 to be a novel negative regulator of type 1 IFN production in both TLR- and RLR-dependent pathways. Proteomic analysis of TRIM68 containing complexes has allowed us to identify TRK-fused gene (TFG) as a TRIM68 target. The physiological role of TFG is still unknown but it has been shown to interact with NEMO and TANK, two proteins which modulate pro-inflammatory cytokine production. Thus the effects of TRIM68 on IFN production is via this novel regulation of TFG. To confirm the critical role of TRIM68 in IFN production we have knocked down TRIM68 in mice via injection of PLGA microparticles encapsulating shRNA against TRIM68 and studied the effects of TRIM68 knock down on viral-induced IFN production. TRIM68 knockdown mice display an increase in serum and peritoneum IFN levels post LCMV infection, indicating that TRIM68 is a key negative regulator of IFN production. This work carves the way for the development of novel therapeutics targeting TRIM68 for the treatment of IFN-driven disease such as SLE.
Expression of the tumor-associated antigen Neu5Gc-Sialyl-Tn in human carcinomas (74.6)
Authors: Padler-Karavani Vered , Deng Liwen , Garcia Andrea , Karp Felix , Naidu Natasha , Choudhury Biswa , Varki Nissi , Varki Ajit
Published: January 2023

Altered glycosylation is prevalent on cancer cells, due to aberrant expression of glycosyltransferases, and frequently involving variations in sialylation patterns. In humans, this altered sialylation can also result from metabolic incorporation of dietary N-glycolylneuraminic acid (Neu5Gc) that differs from the human sialic acid N-acetylneuraminic acid (Neu5Ac) by one oxygen atom. Neu5Gc is immunogenic in humans and anti-Neu5Gc antibodies can either promote tumor growth at low doses or cause tumor regression when administered in excess. Antibodies against Neu5Gcα2−6GalNAcα1-O-Ser/Thr (anti-Neu5Gc-Sialyl-Tn IgG) appear to be novel carcinoma biomarkers, but direct evidence for expression of this epitope on human carcinomas is lacking. This neo-sialoglycan resembles the well-known carcinoma-associated biomarker Sialyl-Tn, except that Neu5Ac is replaced with dietary Neu5Gc. Here, we characterize unique antibodies against Neu5Gc and Sialyl-Tn by sialoglycan-microarrays and use them for immunohistochemistry of human breast, prostate, ovary, colon and lung carcinomas revealing co-staining in some mucinous carcinomas. We also generate MUC1-Fc that is heavily glycosylated with Neu5Gc-Sialyl-Tn for affinity-purification of human anti-Neu5Gc-Sialyl-Tn-MUC1 IgG for further immunohistochemistry analysis. Finally, we report a mass spectrometry approach to analyze O-linked glycans from tissues to ultimately prove biomarker expression in positively-stained tissues.
The mechanisms of photodynamic therapy enhancement of anti-tumor immunity (75.1)
Authors: Wang Minan , Gollnick Sandra
Published: January 2023

Photodynamic therapy (PDT) has long been shown to be capable of killing malignant cells and inducing host immune response. Recent investigations have also established that PDT augments anti-tumor immunity. Tumor cells treated by PDT release “danger signals” that have been shown to stimulate dendritic cells (DCs). In this study, we investigate the role of danger signal receptors, specifically the toll-like receptors (TLR) and the NOD-like receptor NLRP3, in PDT generated tumor cell lysates activation of DCs. Stimulation of bone marrow derived DCs (BMDCs) with PDT treated tumor cells leads to dramatic increase in pro-inflammatory cytokine production, including IL-1β, which plays a critical role in PDT efficacy. Production and activation of IL-1β require both TLR and NLR signaling. IL-1β production induced by PDT vaccine in BMDCs is MyD88 dependent. NF-κB is also activated upon treatment of BMDCs with PDT treated tumor cell lysates. Caspase-1, an important component of NLRP3 inflammasome, is known for its role in maturation of IL-1β. However, we found that IL-1β release from DCs is caspase-1 independent. This study aims to understand how PDT enhances tumor immunogenicity and provides insight to improve both in situ PDT and PDT vaccine efficacy in treating secondary disease.
Targeting the OX40 (CD134) co-stimulatory receptor enhances mucosal secondary memory CD8 T cells (P270) (75.7)
Authors: Goulding John , Croft Michael , Salek-Ardakani Shahram
Published: January 2023

The expansion and maintenance of poly-functional memory CD8 T cells (mCD8) that rapidly migrate to sites of viral replication is paramount for the development of effective mucosal vaccines. This concept is epitomized by the limited success of vaccines that utilize attenuated or replication defective virus strains incapable of promoting mCD8 T cells that persist at mucosal sites. Consequently, strategies that enhance both the number and functionality of mCD8 T cells have attracted increasing interest. Recent studies have shown that targeting of the OX40 co-stimulatory receptor can promote primary mucosal CD8 T cell memory following infection with virulent and attenuated pox-virus vaccine strains. However, the ability of OX40 co-stimulation to enhance the reactivation of secondary mCD8 T cells is unclear. To mimic a prime boost vaccination strategy we transferred virus specific mCD8 T cells into naïve mice that were later immunized via dermal scarification. We show that OX40 agonism 24 hours following vaccination enhances both the number of virus specific secondary effector mCD8 T cells, defined by their expression of KLRG, IL7-R, CD62L and CD27, located within the lung and their capacity to produce both TNFα and IFNγ. These data provide evidence that targeting OX40 at the same time as an antigen boost (secondary challenge) can expand the numbers of effector mCD8 T cells that are capable of residing within lung tissue and thus protect against mucosal viral infection.
Presentation of cryptic peptides by MHC I molecules is enhanced by inflammatory stimuli. (P5003)
Authors: Prasad Sharanya , Starck Shelley , Shastri Nilabh
Published: January 2023

Cytolytic T cells eliminate infected cells by recognizing intracellular peptides presented by MHC class I molecules. The antigenic peptides are derived primarily from newly synthesized proteins including those produced by cryptic translation. Previous studies have shown that in addition to the canonical AUG codon, translation can be initiated at non-AUG codons . Furthermore, translation initiation at non-AUG codons such as CUG is mechanistically distinct from canonical translation initiation as it is resistant to protein synthesis inhibitors that cause global translation shutdown. Here, we show that Toll-like receptor (TLR) signaling pathways involved in pathogen recognition enhance presentation of the cryptic peptides. Moreover, infection of bone-marrow derived macrophages with MCMV, influenza viruses or pro-inflammatory cytokines also enhances cryptic peptide presentation. Thus, translation and presentation of cryptic peptides may allow the immune system to detect intracellular pathogens that inhibit host translation and presentation of peptides from conventional sources.
IgE enhances B cell-derived exosomal induced T cell proliferation. (P5013)
Authors: Martin Rebecca , Brooks Keith , Henningson Frida , Heyman Brigitta , Conrad Daniel
Published: January 2023

The immunostimulatory potential of IgE immune complexes is known to be dependent on CD23, the low affinity IgE receptor. This was originally thought to be due to CD23 mediated uptake and subsequent stimulation of T cells by B cell processed antigen. Recent findings have indicated that dendritic cells (DC) are required in this process and B cells are primarily needed for antigen transportation. This has called into question the role of B cells in this interaction and leaves a gap in the knowledge of how the antigen is transferred to the DC. Given the resurgent interest in exosomes as having the capacity to mediate antigen presentation, we tested whether B cell derived exosomes might provide an explanation for this transfer. B cell derived exosomes express CD23 as well as the costimulatory molecules CD80 and CD86. Exosomes isolated from B cells that had been cultured with anti-TNP-IgE and TNP-OVA were shown to induce proliferation of ova specific DO11 T cells in a dose dependent manner. In the absence of either CD23 or anti-TNP-IgE, specific T cell stimulation was severely reduced. Thus, B cell derived exosomes alone have the capacity to induce antigen specific T cell activation and could represent the mechanism for antigen transfer to DCs. Ongoing testing involves whether injection of exosomes with processed antigen, either iv or intrasplenic will give an enhanced T cell and anti-OVA response in a manner similar to the classical IgE immune complex injection.
Regulation of gene expression by the immunoproteasome (P5049)
Authors: Rouette Alexandre , de Verteuil Danielle , Khanafer Hassan , Hardy Marie-Pierre , Perreault Claude
Published: January 2023

Background: Proteasomes play crucial roles in regulating fundamental cellular processes in eukaryotic cells. Catalytic subunits β1, β2 and β5 of the proteasome are replaced by LMP2, MECL-1 and LMP7 to form the immunoproteasome (IPr). We have shown that the IPr modulates mRNA levels of several clusters of genes in dendritic cells, but the precise mechanism by which it regulates gene expression is unknown. Results: By comparing transcriptome of Lmp7+/+/Mecl1+/+ (WT) and Lmp7-/-/Mecl1-/- (dKO) thymocytes by microarray, we found that the IPr affects the expression of 50 transcripts. The genes source of these transcripts are clustered in the genome, and are enriched in cell cycle-associated functions. Moreover, an overlap of 2% is seen by comparing with transcripts modulated by the IPr in dendritic cells. Chymotrypsin-like activity is higher in dKO than in WT cells, due to β5 overexpression. Furthermore, ubiquitinated proteins levels are similar between WT and dKO cells whereas free ubiquitin is lower in dKO cells. We next investigated levels of ubiquitin on histones, since it represent an important reservoir of ubiquitin and it can regulate transcriptional activity. Our results show that H2B, but not H2A, ubiquitination is increased in dKO cells. Conclusion: The effect of IPr on transcription is tissue-specific and could be due to increased ubiquitinated H2B levels. While proteasomes clearly regulate transcription, the impact of IPr on transcription has never been investigated.
Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate recognition by human Vγ2Vδ2 T cells (P5051)
Authors: Morita Craig , Henry Olivier , Distefano Mark , Mönkkönen Jukka , Tanaka Yoshimasa , Wang Hong
Published: January 2023

The majority of human γδ T cells express Vγ2Vδ2 TCRs and play important roles in microbial and tumor immunity. Vγ2Vδ2 T cells use their TCRs to recognize self and foreign prenyl pyrophosphate intermediates (prenyl-PP) in isoprenoid synthesis. However, little is known about the molecular basis for this recognition. We find that a monoclonal antibody specific for butyrophillin 3 (BTN3) Ig superfamily proteins mimics prenyl-PPs. The 20.1 mAb stimulated Vγ2Vδ2 T cell clones regardless of their functional phenotype or developmental origin, and selectively expanded blood Vγ2Vδ2 T cells. The γδ TCR mediates 20.1 stimulation since IL-2 is released by β− Jurkat cells transfected with Vγ2Vδ2 TCRs. 20.1 stimulation was not due isopentenyl pyrophosphate (IPP) accumulation because 20.1 treatment of APC did not increase IPP levels. Nor was stimulation inhibited by statin treatment which blocks IPP production. Importantly, siRNA knockdown of BTN3A1 abolished stimulation by IPP that could be restored by re-expressing BTN3A1. Rhesus monkey and baboon APC presented IPP and 20.1 to human Vγ2Vδ2 T cells despite amino acid differences localized to the outer BTN3A1 face suggesting that the top and/or the inner face interact with its co-receptor. Full length BTN3A1 did not preferentially bind a photoaffinity prenyl-PP analog. Our results confirm and extend recent studies and demonstrate that BTN3A1 is required for the recognition of prenyl-PP but does not appear to bind the intermediates directly.
Heat shock protein 90 inhibitor radicicol attenuates matrix metalloproteinase expression in human monocytic cells (P4153)
Authors: Hsiao George , Lee Tzong-Huei , Jan Jing-Shium , Hsiao Che-Jen , Cheng Yu-Wen
Published: January 2023

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting target for anti-tumor and immunoregulatory therapy. In this study, we investigated the inhibitory effects and mechanism of radicicol, as a HSP90 inhibitor, on human monocytic matrix metalloproteinases (MMPs) and pro-inflammatory cytokine expression. Radicicol exhibited concentration-dependent inhibition of MMP-9 enzymatic activation induced by either tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) in human monocytic THP-1 cells. Western blot and RT-PCR analyses showed that radicicol concentration-dependently suppressed LPS-induced monocytic MMP-9 protein and mRNA expression. It was also shown that radicicol enhanced HSP70 expression. Besides, we clearly found that radicicol restore the degradation of inhibitor-kappaB-alpha and abrogate NF-kappaB reporter gene expression in activated THP-1 cells. In addition, activation of Akt and its total protein expression were reduced by radicicol. Furthermore, radicicol presented an inhibitory effect on both TNF-alpha and interleukin-6 productions by ELISA. In conclusion, we demonstrate that radicicol might involve interference with the NF-kappaB or Akt signaling pathway to attenuate production of MMP-9 and pro-inflammatory cytokines. These results provide a new opportunity for the development of anti-leukemia and cachexia-relief strategies.
Deletion of Gab adaptor proteins leads to impaired macrophage development and chronic colitis (P4175)
Authors: Vaughan Tamisha , Imhoff Barry , Wang Zhengqi , Denning Tim , Bunting Kevin
Published: January 2023

Inflammatory Bowel Disease (IBD), commonly Crohn’s and Ulcerative Colitis, is characterized by chronic inflammation of the gastrointestinal tract. Although the cause of this disorder is not known, alterations in immune responses and uncontrolled intestinal microflora are thought to be involved. Our lab has uncovered an important role for the Grb2 associated binding (Gab) adaptor protein family in the development and pathogenesis of IBD. In mice, Gabs include three family members that play critical regulatory roles in modulating cytokine receptor signaling in T-cells and macrophage. Our studies demonstrate that knock-out (KO) of only two of these, Gab2/3, leads to intestinal inflammation associated with spontaneous rectal prolapse as early as 8 weeks of age that is not observed in single KO mice. Bone marrow derived macrophage (BMDM) cultured from mice lacking Gab2/3 expanded poorly compared to wild-type mice or mice lacking only Gab2 or Gab3 but expressed mature markers. RT-PCR analysis showed dysregulated expression of both pro- and anti-inflammatory cytokines. Importantly, pro-inflammatory cytokines IL-6, TNF-α, IL-12p40 were significantly induced in Gab2/3 KO BMDM compared to control. Upon TLR4 activation, Gab2/3 KO cells had prolonged activation of signaling molecules IκBα and NFκB. Together these data support our conclusion that Gabs play a critical role in the pathogenesis of IBD and provide insight toward new therapeutic approaches to control and treat IBD.