bluuebunny/crossref_metadata_2025 huggingface dataset:
The IRF5-TNPO3 association has two components in systemic lupus erythematosus, which are shared with other autoimmune disorders (P3315)
Authors: Kottyan Leah , Zoller Erin , Kaufman Kenneth , Harley John
Published: January 2023

Improved technical and bioinformatics genetic methods now allows us to capture more complete relevant genetic variation for candidate causal disease risk variant identification. Exploiting genotyping, DNA sequencing, imputation, and trans-ancestral mapping, we modeled the IRF5-TNPO3 genetic association on chromosome 7, now implicated in two immunotherapies and seven autoimmune diseases. Specifically, in systemic lupus erythematosus (SLE) we resolve separate associations in the IRF5 promoter (all ancestries) and an extended European haplotype. We capture 7,650 IRF5-TNPO3 genetic variants across five ethnicities in 8,395 SLE cases and 7,367 controls. The six plausible causative IRF5 promoter variants are confined to 5.7 kb (meta-analysis p-value=6x10^-49; Odds Ratio=1.38-1.97). The 22 potentially causal variants of an 85.5 kb haplotype of IRF5 and TNPO3 (haplotypic p-value in European Americans=2.4x10^-45, Odds Ratio=0.58) (individual p-value in European Americans=10^-27 - 10^-32, Odds Ratio=1.7-1.81). The possible models from this sample virtually eliminate the previously purported IRF5 functional variants as causal. Finally, this model also appears to operate in Sjögren’s syndrome and systemic sclerosis (both components) and primary biliary cirrhosis (haplotype only), demonstrating the nuances of similarity and difference in autoimmune disease risk mechanisms at IRF5-TNPO3.
Functional characterization of the MECP2/IRAK1 lupus risk haplotype in human T cells and a human MECP2 transgenic mouse (P3319)
Authors: Sawalha Amr , Koelsch Kristi , Webb Ryan , Jeffries Matlock , Dozmorov Mikhail , Frank Mark , Guthridge Joel , James Judith , Wren Jonathan
Published: January 2023

Genetic polymorphism in MECP2/IRAK1 is a confirmed susceptibility locus for lupus. DNA methylation changes in T cells play a central role in the pathogenesis of lupus, and MeCp-2 is a master regulator of gene expression and is known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals, we demonstrate that the lupus risk variant in MECP2/IRAK1 increases MECP2 mRNA expression in stimulated T cells. By assessing DNA methylation levels at ~ 485,000 methylation sites across the genome, we further show that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. We also show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms. Importantly, we demonstrate that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.
rhIL-15 and rhIL-2 together enhances growth and function of TCR transduced T cells for adoptive immunotherapy (P4358)
Authors: Scurti Gina , Moxley Kelly , Mehrotra Shikhar , Nishimura Michael
Published: January 2023

The use of TCR transduced T cells for the treatment of melanoma has shown promise in recent years. However, there is a need to improve the function and growth of transduced T cells to allow for better clinical outcomes. We wanted to evaluate the impact of rhIL-15 on TCR transduced T cells. T cells from multiple donors were transduced with a retrovirus encoding the TIL 1383I T cell receptor. The transduced T cells were cultured and expanded for approximately 21 days under several different cytokine conditions including 300IU/mL of rhIL-2, 100ng/mL of rhIL-15 or both rhIL-2 and rhIL-15. For each culture condition, data was collected throughout the process for growth, function, phenotype and TCR expression. We found that the T cells cultured in rhIL-15 had a greater TCR expression and produced more IFN-γ in response to tumor than T cells cultured only in rhIL-2. rhIL-15 alone did not increase the growth of TCR transduced T cells when compared to rhIL-2 whereas the use of both rhIL-2 and rhIL-15 not only enhanced the TCR expression and anti-tumor activity but also improved the growth of the T cells. We also noticed that T cells cultured with rhIL-15 increased resistance to apoptosis during oxidative stress. Based on these results, we have initiated our TCR gene transfer clinical trial using rhIL-15 and rhIL-2 to transduce and expand our T cells.
Mutant HSP70i prevents effector T cell responses in autoimmune vitiligo (P4352)
Authors: Mosenson Jeffrey , Zloza Andrew , Nieland John , Garrett-Mayer Elizabeth , Eby Jonathan , Huelsmann Erica , Kumar Previn , Denman Cecele , Lacek Andrew , Kohlhapp Frederick , Alamiri Ahmad , Hughes Tasha , Bines Steven , Kaufman Howard , Overbeck Andreas , Mehrotra Shikhar , Hernandez Claudia , Nishimura Michael , Guevara-Patino Jose , Le Poole I. Caroline
Published: January 2023

A single amino acid change (Q435A) within HSP70i renders an isoform with treatment potential for vitiligo, as demonstrated by ablated depigmentation in gene gun vaccinated, vitiligo-prone TCR transgenic mice. We hypothesized that HSP70iQ435A inhibits T cell mediated immune responses. We probed skin from gene gun vaccinated P-mel1 and h3TA2 mice for T cell infiltrates and analyzed splenocytes for T cell activation markers by FACS, comparing findings to those in tissues from vitiligo patients and among cells collected from human skin explants exposed to HSP70iQ435A. Decreased skin infiltration was observed for T cells responsible for melanocyte killing in both mouse strains. Eomes/T-bet ratios were significantly decreased by wild-type HSP70i driving an effector phenotype, whereas vaccination with HSP70iQ435A maintains a quiescent phenotype. This ratio was similarly decreased in circulating and skin-infiltrating T cells from vitiligo patients where we also observed an increase in CXCR3 expression, supportive of skin homing. In cells from skin explants exposed to wild-type or mutant HSP70i the Eomes/T-bet ratio is upregulated by HSP70iQ435A whereas upregulation of skin homing receptor CLA followed exposure to wildtype HSP70. In conclusion, the wild-type HSP70i-induced T cell effector phenotype accompanied by elevated CXCR3 and CLA expression observed in vitiligo is countered by HSP70iQ435A exposure, providing a mechanism for HSP70iQ435A induced inhibition of autoimmunity.
cAMP-based adjuvants (LT, mLT, dmLT) promote Th17 and germinal center responses through dendritic cells, while direct CD4 T-cell contact induces a T-suppressor phenotype. (P4512)
Authors: Norton Elizabeth , Clements John
Published: January 2023

Enterotoxin-based adjuvants with the ability to induce cAMP (LT, mLT and dmLT) elicit multifaceted antigen-specific response after vaccination, including Th1, Th2, Th17 and T-regulatory (Treg) responses unlike related non-cAMP inducing proteins (LT-E112K and B-subunit). While cAMP induction is linked to enterotoxin immunologic activity, many studies have now demonstrated a key regulatory role for cAMP in directing Treg responses. We investigated these paradoxical abilities to promote both suppressive and stimulatory T-cell subset using a panel of LT enterotoxin based proteins for cAMP induction, adjuvanticity, and in vitro effects on immune cells. We found that direct stimulation with cAMP-inducing adjuvants (LT, mLT or dmLT) induced a T-suppressive phenotype linked to cAMP activity but not Foxp3 or IL-10 expression. However, cAMP-inducing adjuvants promoted both proliferation and secretion of all classical Th-defining cytokines, especially IL-17, via indirect stimulation of T-cells through dendritic cells (DCs). Neutralization of IL-17 during vaccination reduced germinal center development and antibody responses. These results indicate cAMP-based adjuvants strongly promote Th17 cells via stimulated DCs, while direct contact mediates T-suppressive phenotype. In addition, DCs play a key role in directing these adjuvant signals away from suppression and towards activation.
Inflammaging decreases the human B cell response to the influenza vaccine (P4524)
Authors: Frasca Daniela , Diaz Alain , Romero Maria , Landin Ana Marie , Blomberg Bonnie
Published: January 2023

Deficits in immune responses in the elderly contribute to their mortality and morbidity. In order to develop predictive markers for a beneficial humoral immune response, we evaluated the in vivo and in vitro response to the seasonal influenza vaccine in young and elderly individuals. We measured serum antibody response and associated this with the in vitro B cell response to the vaccine, measured by AID (activation-induced cytidine deaminase). AID is a measure of immunoglobulin class switch recombination, the process that generates protective antibodies, and is a good measure of B cell function. Both responses decrease with age and are significantly correlated. We hypothesize that the increased pro-inflammatory status of the elderly, called inflammaging, directly impacts B cell function, thus impairing the capacity of the individual to make protective antibodies and to respond to vaccination. Our data indicate that aged B lymphocytes themselves make TNF-α before vaccine challenge and this correlates with their impaired function, including reduced AID expression after B cell stimulation. Our results reveal new molecular mechanisms which contribute to reduced antibody responses in aging and suggest that these will have an impact for crucial development of effective vaccines to protect the elderly from infectious and other debilitating diseases.
Induction of linear and conformational V2-specific antibodies using HIV-1 gp145 clade B envelope protein as the immunogen (P4505)
Authors: Rao Mangala , Peachman Kristina , Jobe Ousman , Kuaban Alice , Billings Erik , Guao Guofen , Wieczorek Lindsay , Polonis Victoria , Pinter Abraham , Kim Jerome , Michael Nelson , Alving Carl , Rao Venigalla
Published: January 2023

TThe potential importance of binding antibodies (Abs) in preventing HIV-1 acquisition was recently demonstrated by the case control analysis of the RV144 phase III HIV-1 vaccine trial, which showed that binding Abs to a gp70 scaffolded V1V2 loop of gp120 correlated inversely with infection. We examined if immunization with a HIV-1 env trimer in the absence of any priming vector could induce functional V2-specific Abs. The clade B HIV-1 gp145 env protein consisting of gp120, gp41 ectodomain, and MPER, trimerized with foldon was expressed in 293F cells and purified. The protein adjuvanted with liposomes containing lipid A was used to immunize New Zealand white rabbits. Strong Ab binding titers (50,000-400,000) were induced to env epitopes including V2 and V3 peptides (linear) and MuLV gp70-scaffolded V1V2 protein (conformational) as measured by ELISA and Biacore. The sera neutralized infectious molecular clones and pseudoviruses in PBMC, monocyte-derived macrophage, and TZM-bl assays. Purified IgG neutralized primary US-1 in a dose-dependent manner in the macrophage assay. Depletion of linear V2 or V3 epitope-specific Abs retained the conformation-specific gp70 V1V2 binding Abs and did not completely abolish neutralization. Further depletion of gp70 V1V2-specific Abs significantly reduced neutralization. These data indicate that gp145 trimers induce linear as well as conformational V2-specific antibodies and might be a good candidate for designing a preventative HIV-1 vaccine.
Nanoparticle vaccines comprising the respiratory syncytial virus G Protein CX3C chemokine motif induce robust immunity protecting from challenge and disease (P4513)
Authors: Tripp Ralph , Choi Youngjoo , Powell Thomas , Boyd James , Palath Naveen , Haynes Lia , Anderson Larry , Jorquera Patricia
Published: January 2023

BALB/c mice immunized with nanoparticle vaccines comprising RSV G protein CX3C polypeptides in absence of adjuvant had a neutralizing antibody response associated with reduced lung titers following RSV challenge. These mice also had an increased level of RSV G protein-specific IL-4 and IFNγ secreting cells in the lung, and an increased level of RSV M2-specific IL-4 and IFNγ CD8 T cells. There was a significant increase in M2-specific CD8 T cells that trafficked to the lung following RSV challenge despite the mice being vaccinated with a G protein epitope. Pulmonary cell analysis revealed no significant increase in neutrophils (Ly6Ghi Ly6Cint CD125loSiglecF-) or eosinophils (Ly6Gint Ly6Chi CD125hi SiglecF+) in vaccinated mice before or after RSV challenge. The results show that RSV G protein nanoparticle vaccination is safe and effective, induces a neutralizing protective antibody response, increased RSV G protein- and M2-specific T cell responses, and is not associated with pulmonary disease pathogenesis.
Assessment of immune response against HIV-1 P24 antigen using Bacillus thuringiensis serovar dakota (H15) activated delta-endotoxin as an adjuvant (P4491)
Authors: Abouelhag Hussien , Safhi Mohammed
Published: January 2023

Bacillus thuringiensis var. dakota activated δ-endotoxin (BtDEAP) had many biological activity. Our previous experience recorded that BtDEAP had anti-malignancy activity in-vitro and in-vivo. There were many thoughts about the mode of action and one of them was their capability of immune modulation against tumor cell Ag. This work assesses the immune response in BALB/c mice immunized with HIV-1 P24 Ag individually and/or in combination with BtDEAP, complete Freund's adjuvant (CFA). The evaluation of the immune response against immunogenic preparations was carried out through estimating the immunoregulatory cytokines (IL-1α, Il-1β, IL-4, IL-10 and TNF-α) in the supernatant of splenocytes from mice that were immunized with HIV-1 P24 Ag alone and/or in combination with BtDEAP, CFA in relation to splenocytes from control mice. Also, the total IG level in the serum of tested and control mice were appraised. The results obtained conducted that the total IG level was raised remarkably by using CFA in comparing with BtDEAP while HIV-1 P24 Ag alone record the minimal raise in IG levels. These results was accompanied with raised levels of IL-4 and IL-10 by using CFA combination with HIV-1 P24 while IL-1 and TNF-α raised by immunization using BtDEAP in combination with HIV-1 P24 Ag over that recorded by HIV-1 P24 Ag alone. Based on the described above, BtDEAP, seems to be playing a significant role in preferential induction of Th1-dependent immune response against HIV-1 P24 Ag.
Hypothemycin inhibits tumor necrosis factor-α production by post-transcriptional regulation in lipopolysaccharide-stimulated RAW 264.7 cells (P5046)
Authors: Kang Jong Soon , Park Ki Hwan , Han Sang-Bae , Kim Youngsoo , Oh Soo Jin , Yun Jieun , Lee Chang Woo
Published: January 2023

Tumor necrosis factor-α (TNF-α) is a major inflammatory cytokine produced by activated macrophages and lymphocytes and involved in the development of many inflammatory diseases. Preventing the production or action of TNF-α is considered as a promising therapeutic strategy for various inflammatory diseases. The resorcylic acid lactone hypothemycin has been shown to exert an antitumor activity. In this study, we studied the inhibitory effects of hypothemycin on TNF-α production and underlying mechamisms. Hypothemycin dose-dependently suppressed lipopolysaccharide (LPS)-induced TNF-α secretion without affecting LPS-induced nitric oxide (NO) production in RAW 264.7 cells. Quantitative real-time reverse transcription polymerase chain reaction analysis revealed that TNF-α mRNA expression was only slightly reduced by hypothemycin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that hypothemycin down-regulated the stability of TNF-α mRNA in LPS-stimulated RAW 264.7 cells. In addition, hypothemycin suppressed the phosphorylation of p38 MAPK and ERK in LPS-stimulated RAW 264.7 cells. Knockdown of tristetraprolin (TTP), a major component of TNF-α mRNA decay machinery and direct substrate of p38 MAPK, by small interfering RNA reversed the stability of TNF-α mRNA decreased by hypothemycin treatment. Collectively, these results suggest that hypothemycin inhibits TNF-α production, at least in part, by TTP-dependent reduction of mRNA stability in LPS-stimulated RAW 264.7 cells.
Predominant Th2 cytokine gene expression in lymph node cell cultures from Andes virus-infected deer mice (Peromyscus maniculatus) (P6316)
Authors: Schountz Tony , Haddock Elaine , Feldmann Heinz , Prescott Joseph
Published: January 2023

Deer mice (Peromyscus maniculatus) are the principal reservoir hosts of Sin Nombre virus (SNV), while long-tailed pygmy rice rats (Oligoryzomys longicaudatus) are the principal reservoir hosts of Andes virus (ANDV). These viruses cause the great majority of hantavirus cardiopulmonary syndrome cases in North and South America. The disease in humans is principally an inflammatory immunopathology that results in a pronounced capillary leak syndrome with a 36% fatality rate due to cardiogenic shock. In contrast, infection of rodent reservoirs with their hantaviruses results in life-long persistence, without signs of disease, that appears to be mediated by regulatory T cells. Deer mice are experimentally susceptible to ANDV; however, they clear infection within 56 days. We infected deer mice with SNV or ANDV for 14 days and examined the immune responses to each virus. Lymph node cells from infected deer mice were harvested and cultured with viral antigens to examine expression of 94 immune-related genes by real-time PCR. We found that most of the same genes were expressed in both SNV- and ANDV-infected deer mice; however, the magnitude of gene expression, particularly Th2 genes, were significantly greater in lymph node cells from ANDV-infected deer mice. Together, these results suggest a quantitative effect occurs in the host response to a reservoir hantavirus (SNV) compared to a nonreservoir hantavirus (ANDV) and is associated with persistence or clearance.
Circulating autoantibody and intrahepatic inflammation profiles in patients chronically infected with hepatitis C virus (P6328)
Authors: Yu Qigui , Ghabril Marwan , Meng Zhefeng , Hu Sishun , Amet Tohti , Byrd Daniel , Lan Jie , Hu Ningjie , Chalasani Naga
Published: January 2023

The liver injury in hepatitis C virus (HCV) infection is thought to be mainly due to immunological disorders and attacks of inflammatory factors rather than a direct cytopathic effect of the virus itself. To investigate whether intrahepatic inflammation and immunological disorders are correlated in HCV-infected patients, we used a real-time RT-PCR-based array and a slide-based autoantigen array to analyze intrahepatic inflammation gene profiles in liver biopsy specimens from HCV-infected (n=16) and uninfected (n=8) individuals and their circulating autoantibody profiles, respectively. Compared with uninfected individuals, HCV infection markedly altered expression of 59.5% of 84 inflammation-related genes tested. Among these genes affected, the CXCR3-associated chemokines (CXCL9, CXCL10, and CXCL11) were among the most up-regulated genes. Patients chronically infected with HCV have 2-fold or greater increase in circulating autoantibodies of IgG and IgM to 28 and 23 of 84 autoantigens tested, respectively, than that in uninfected individuals. The antinuclear antibody IgG titers correlated significantly with the levels of intrahepatic CXCR3-associated chemokines (p = 0.002). Given that CXCR3-associated chemokines are involved in the development of autoimmune diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), these chemokines may also play a key role in the pathogenesis of HCV-related liver disease through enhancement of autoimmune responses.
Involvement of type I IFN signaling in lymphocyte migration and lymphoid structure during viral infection (P6306)
Authors: Ng Cherie , Teijaro John , Schreiber Robert , Oldstone Michael
Published: January 2023

During many chronic viral infections, the stromal cells of secondary lymphoid organs (SLO), essential to lymphocyte migration, activation, and survival, are disrupted, suggesting abrogation of necessary immune cell interactions. Little is known why and how these changes occur. To study the changes in these scarce stromal populations, we immortalized fibroblastic reticular cells (FRCs), which are stromal cells of T cell zones that are targeted by multiple viruses, and tested the effect of type I and II interferon (IFN). In reaction to IFN-γ, FRCs expressed MHC II, along with co-stimulatory ligands, and activated naïve CD4+ and CD8+ T cells but limited expansion of effector T cells. To IFN-I, interestingly, FRCs responded differentially, upregulating PD-L1 and MHC I in reaction to IFN-β but not IFN-α. To assess the effect of IFN-I on stromal cells, including FRCs, we blocked IFN-I signaling during persistent lymphocytic choriomeningitis virus infection in mice. Surprisingly, IFN-I blockade protected from the SLO destruction that occurs with chronic infection. FRC structure and T cell (and B cell) zones remained intact despite higher viral load and greater numbers of anti-viral T cells. IFN blockade increased FRC expression of T cell/DC chemokines, resulting in significant differences in naïve T cell homing. These data suggest that IFN-I signaling alters lymphocyte migration via stromal cells and regulates pathways that are involved in maintaining SLO architecture.
Novel mouse models reveal how expression of NKG2D and its ligands on various cell subsets affects immune responses (P4351)
Authors: Morvan Maelig , Champsaur Marine , Johnson Natrina , Sexl Veronika , Lanier Lewis
Published: January 2023

NKG2D is an activating receptor expressed by all Natural Killer (NK) cells, a subset of T cells, and activated macrophages. NKG2D and its ligands, which are expressed on stressed or damaged cells, play important roles during viral infections, cancers, and autoimmune diseases, both in mouse and human. While NKG2D is well studied in NK cells, its function on other cells remains unclear, particularly in vivo. In this study, we examine the consequences of cell-specific ablation of NKG2D, or tissue-specific expression of its ligand Rae-1, on innate and adaptive immunity, using two novel mouse models. By crossing Cre expressing strains to our two newly generated mice bearing a floxed allele of NKG2D, or a Rosa26-LSL-Rae-1 transgene, we obtain conditional knockouts of NKG2D, or mice that express Rae-1 in a cell-specific manner. We are now able to dissect the contribution of each cell subset expressing NKG2D in models of viral infection, tumor rejection, and autoimmunity. In addition, we observed that NK cells chronically interact with myeloid cells in vivo, since expression of Rae-1 on these subsets, but not others, leads to down-modulation of NKG2D expression and impaired NKG2D-dependent functions. Data from these two new mouse models provide a better understanding of the role of NKG2D by defining its impact on different immune cell functions, and by mimicking physiopathological situations of patients in which a human counterpart of Rae-1 is expressed on various tissues.
NKG2C+CD57+ CD56dim NK cells are enriched following Epstein-Barr virus transformed lymphoblastoid cell line co-culture with peripheral blood mononuclear cells from cytomegalovirus+ donors (P4359)
Authors: Saghafian-Hedengren Shanie , Sohlberg Ebba , Theorell Jakob , Queiroz-Carvalho Claudia , Nagy Noémi , Nilsson Caroline , Bryceson Yenan , Sverremark-Ekström Eva
Published: January 2023

Cytomegalovirus (CMV) is a persistent herpesvirus that promotes NKG2C+ Natural Killer (NK) cell differentiation. Highly mature CD57+ NKG2C+ NK cells have been proposed as the equivalent of mouse memory-like NK cells, which have features as long-term persistence and enhanced recall responses. AIM: To examine if co-infection with another common herpesvirus, Epstein-Barr virus (EBV), affects the NKG2C+ CD56dim NK cell population. METHODS: PBMCs from 45 five-year old children with known EBV and CMV serostatus were phenotyped. Further, PBMCs from 6 CMV sero+ EBV sero- infants were used for co-culture with EBV+ lymphoblastoid cell-lines (LCLs) during 72 hours. EBV- LCLs (Ramos) served as controls. Anti CD3, CD14, CD19, CD56, CD57, NKG2C and ki-67 antibodies were used for flow cytometric analysis. RESULTS: EBV and CMV co-infection was related to higher proportions of NKG2C+ and NKG2C+CD57+ CD56dim NK cells compared to infection with CMV or EBV alone, or seronegativity. In contrast to co-culture with Ramos, PBMC co-culture with EBV+ LCLs generally led to enrichment of NKG2C+CD57+ NK cells (stimulation index median 1.23, range 0.87-1.30), which was not related with higher NK cell proliferation. CONCLUSIONS: EBV co-infection seems to promote the existence of higher proportions of memory-like NK cells. Our data herein indicate that this is not due to enhanced proliferation of these cells; rather it suggests that memory-like NK cells may be maintained by EBV co-infection.
Zosteriform spread of Herpes simplex virus 1 as a model to study the role of leukocyte subpopulations in the mortality induced during acute infection (P4373)
Authors: Salinas Nohemí , Becerril-García Miguel , Hisaki-Itaya Emiliano , Estrada-Parra Sergio , Pavón-Romero Lenin , Velasco-Velazquez Marco , Pérez-Tapia Sonia
Published: January 2023

Immune response against Herpes simplex virus 1 (HSV-1) has been studied by various routes of inoculation in models of herpes. Zoteriform spread of HSV-1 infection occurs after primary inoculation of the skin of mice. This inoculation route has been used for study the pathogenesis of infection at nervous system level but little is known about the mechanism of immune response in this model. The aim of this work is to study the infiltrated of leukocytes in the inguinal lymph nodes (ILN) and correlate changes in leukocyte subpopulations with mortality in BALB/c mice cutaneously infected with HSV-1. We observed that infected mice showed 70-100% mortality during the acute infection. We removed inguinal and mesenteric lymph nodes (MLN) from infected mice and analysed phenotype of CD3, CD4, CD8, and CD19 cells from 0 to 15 days post-infection. The percentage of CD19+ cells and CD3+ cells in the ILN from infected mice changed from 20% to 50% and from 70% to 40% respectively. In contrast, such changes were not observed in MLN, suggesting that during acute infection immune response is elicited only at the region of the infection. Kinetic analysis of CD4+ and CD8+ T cells showed that the HSV-1-infection induced the expression of CD11c in T cells. Our findings show that HSV-1 infected mice retain a CD11c+ leukocyte infiltrate that could be involved in mortality. The role of CD11c+ cells will be further studied using immune-modulators that modify the percentage of those populations.
Type I interferon inhibits phosphorylation of AKT to impair T cell proliferation induced by interleukin-7 (P6315)
Authors: Sieg Scott , Bazdar Douglas
Published: January 2023

Type I interferons (IFN-I) have anti-proliferative activity and are elevated in chronic HIV infection. We hypothesized IFN-I may adversely affect T cell homeostasis by limiting T cell proliferation in response to interleukin-7 (IL-7). Peripheral blood mononuclear cells (PBMC) or purified CD4+ T cells were CFSE-labeled and incubated with IL-7 (5ng/ml) plus or minus interferon alpha (30-500 U/ml) in X-vivo serum-free medium. T cell proliferation was assessed by CFSE dye dilution at 7 d. T cell function was examined by stimulating cells with SEB and measuring IFN-g, IL-2 and CD40L expression by intracellular flow cytometry. Intracellular expression of P-STAT5 and pAKT were measured at 1-3 d. CD4+ T cells incubated with IL-7 proliferated less efficiently in the presence of IFN-I (n=10; p<0.05). Moreover, these cells were less functional as indicated by reduced IL-2, CD40L and IFN-g production, with the greatest impairments observed for IL-2 production. Inhibition of either pAKT or P-STAT5 with chemical inhibitors also blocked T cell proliferation indicating that these pathways are both important in IL-7-induced proliferation of T cells. Analyses of IL-7-induced signaling pathways indicated that IFN-I impaired pAKT signaling (p<0.05) but had limited effect on P-STAT5 signaling. These results suggest that IFN-I has the potential to interfere with T cell homeostatic proliferation by inhibiting pAKT activation.
Study of the PBMC response to TGN1412 and visilizumab in the “lymph node like” cytokine release assay format (P6327)
Authors: Serna Daniel , Scesney Susanne , Ibraghimov Alexander
Published: January 2023

Recently, a new format for the cytokine release assay (CRA) used to predict the in vivo safety of therapeutic antibodies was developed. In this assay, normal donor PBMCs are pre-incubated at high density for two days to make the cells more “lymph node like” (LNL) and then are incubated with soluble antibody. The assay was developed in response to the failure of previous in vitro stimulation formats to predict the strong cytokine storm shown in humans by TGN1412, a CD28 superagonist human IgG4 antibody. Similar to TGN1412, previous formats failed to predict the strong cytokine response elicited in human patients by visilizumab, a human IgG2a anti-CD3 specific antibody. Here we compare and contrast the PBMC response in the LNL format to TGN1412 and visilizumab. Activation of PBMC subset populations, cytokine profile and cellular source of cytokines were analyzed. For the majority of donors tested, the LNL format of CRA detected PBMC cytokine responses to both TGN1412 and visilizumab, which were consistent with those cytokine responses previoulsly observed in vivo. Our study of two human IgG antibodies targeting different T cell activating receptors suggests that the LNL format of CRA may be a valuable predictor of in vivo cytokine release by T lymphocyte specific biologics.
Interleukin 37 employs the IL-1 family orphan receptor SIGIRR and the alpha chain of the IL-18 receptor to inhibit innate immunity (P6293)
Authors: Nold Claudia , Nold Marcel , Rudloff Ina , Lo Camden , Mangan Niamh , Rotter Bjoern , Zepp Jarod , Azam Tania , Li Suzhao , Mansell Ashley , Bufler Philip , Garlanda Cecilia , Mantovani Alberto , Dinarello Charles
Published: January 2023

IL-37 is a powerful anti-inflammatory IL-1 family member with activity against a broad spectrum of inflammatory assaults. One mechanism by which this beneficial effect is mediated is via intracellular association with Smad3; however, the cell surface receptor for IL-37 remains unknown. Here, we show that to block inflammation, IL-37 requires IL-18Rα and SIGIRR, a so-far orphaned member of the IL-1R family. In PBMC, which naturally express IL-37, knockdown of SIGIRR increased IL-1β and IL-6 threefold. Silencing of SIGIRR in THP-1 cells significantly attenuated the anti-inflammation of IL-37 (83% less IL-1β with IL-37 and SIGIRR, only 34% less without SIGIRR). Similar effects were observed when IL-18Rα was depleted. To study SIGIRR’s contribution to IL-37 function in vivo, we created a strain of SIGIRR-/- mice that express IL-37. The protective effect imparted by IL-37 was markedly reduced in these crossbred mice when they (and wild-type, IL-37-transgenic and SIGIRR-/- animals) were injected with LPS. Using proximity ligation assays and FRET in PBMC, thus exploring interactions of the naturally occurring pairs IL-37-SIGIRR, IL-37-IL-18Rα and SIGIRR-IL-18Rα, we showed sub-40nm colocalization of each complex. The association peaked 30min after LPS stimulation and decreased to background by 4h. Thus, IL-37 limits inflammation by employing the properties of SIGIRR and by associating with IL-18Rα, suggesting that SIGIRR-IL-18Rα functions as the cell surface receptor for IL-37.
Selenoprotein K is required for palmitoylation of CD36 in macrophages: implications in foam cell formation and atherogenesis (P1102)
Authors: Fredericks Gregory , Meiler Svenja , Baumer Yvonne , Huang Zhi , Hoffmann Fukun , Rose Aaron , Norton Robert , Boisvert William , Hoffmann Peter
Published: January 2023

Selenoprotein K (Selk) is an endoplasmic reticulum transmembrane protein important for calcium flux and macrophage activation, but its role in foam cell formation and atherosclerosis has not been evaluated. Bone marrow-derived macrophages (BMDM) from Selk-/- mice exhibited decreased uptake of modified low density lipoprotein and foam cell formation compared to wild-type controls, and the differences were eliminated with anti-CD36 blocking antibody. CD36 expression was decreased in TNFα-stimulated Selk-/- BMDM compared to wild-type controls. Fluorescence microscopy revealed TNFα-induced clustering of CD36 in wild-type BMDM indicative of lipid raft localization, which was absent in Selk-/- BMDM. Immunoprecipitation showed Selk-/- BMDM have decreased CD36 palmitoylation, which is crucial for both stabilizing CD36 expression and directing its localization to lipid rafts. To assess if this phenomenon had a role in atherogenesis, high-fat diet was fed to irradiated Ldlr-/- mice reconstituted with bone marrow from Selk-/- or wild-type mice. Selk was detected in aortic plaques of controls, particularly in macrophages. Selk deficiency in immune cells led to reduction in atherosclerotic lesion formation without affecting leukocyte migration into the arterial wall. These findings suggest that Selk is important for stable, localized expression of CD36 in macrophages during inflammation, thereby contributing to foam cell formation and atherogenesis.
Resveratrol ameliorates high fat diet-induced fatty liver with alteration of Kupffer cell subset in mice. (P1106)
Authors: Nishikawa Kahoko , Kinoshita Manabu , Iwaya Keiichi , Fujiwara Yoko , Sakamoto Toshihisa , Seki Shuji
Published: January 2023

Background: We gave considerable attention to improve a high-fat diet (HFD)-induced fatty liver by Resveratrol from a standpoint of lifestyle disease prevention. Subject: To determine which protein is strongly regulated by Resveratrol in HFD-induced fatty liver, we investigated the modulation of liver protein and Kupffer cell subset in the HFD mice. Material and Methods: Mice (♂C57BL/6, 7 weeks) were fed HFD, HFD supplemented 0.2% Resveratrol or control diet for 8 weeks. Protein extraction from the liver was analyzed by LCMS-based proteomics. Immunostaining was performed using F4/80, CD68, CD11b and adipophilin (ADFP) Abs, and the Kupffer cell subset was analyzed by flow cytometry. TNF production by Kupffer cells was also measured. Results: Resveratrol decreased liver weights and hepatic triglyceride (TG) levels in the HFD mice without changing food intake, and improved fatty liver. Lipid metabolism was strongly down-regulated by Resveratrol. The most down-regulated protein was the ADFP that plays a role of TG storage in lipid droplets and macrophage. Resveratrol reduced lipid droplets and ADFP in the liver of HFD mice. Concurrently, Resveratrol suppressed increased CD68-CD11b+ Kupffer cell subset with suppressing their TNF production in the HFD mice. Conclusion: Resveratrol altered lipid metabolism, especially ADFP, in HFD mice, and suppressed TNF-producing CD11b+ Kupffer cells, thereby ameliorating fatty liver.
Investigating how bacteriophages influence the human immune response to Propionibacterium acnes (P3148)
Authors: Marinelli Laura , Modlin Robert
Published: January 2023

Numerous lines of evidence implicate the Gram-positive skin commensal, Propionibacterium acnes, in the pathogenesis of acne vulgaris, in part by eliciting a host inflammatory response. The emergence of antibiotic resistance in up to 60% of P. acnes isolates, along with the adverse side effects of current treatments has highlighted the need for the development of safer and more effective anti-acne therapeutics. We have investigated the bacteriophages that infect and kill P. acnes and found that they have limited genetic diversity and broad host ranges against clinical isolates, pointing to the potential utility of a phage-based acne treatment. Here, we explored whether these phages, by lysing P. acnes, influence the human immune response to the bacteria. Human monocytes were cultured in the presence of phage-killed or live bacteria, and cytokine responses were measured. We found that monocytes cultured with phage-killed bacteria secrete lower levels of pro-inflammatory cytokines, such as TNFα, IL-1β and IL-12p40, as compared to those infected with live bacteria; phage alone did not stimulate a response. This pattern was also observed when phage and bacteria were added concurrently, that is, during active phage infection, and in all cases, this was consistent across multiple donors. These data suggest that bacteriophage-mediated killing of P. acnes bacteria could help to ameliorate the inflammation associated with acne as a therapeutic modality.
Modulation of mucosal immune responses to Clostridium difficile by probiotic bacteria (P3073)
Authors: Viladomiu Pujol Monica , Hontecillas Raquel , Pedragosa Mireia , Philipson Cassandra , Bassaganya-Riera Josep
Published: January 2023

Commensal bacteria play a critical role in maintaining intestinal epithelium and mucosal immune homeostasis. Alterations in intestinal microflora predispose mammalian hosts to infection with pathogens such as Clostridium difficile, which has recently re-emerged as a pathogen causing nosocomial diarrhea, colitis and death. We investigated the mechanisms by which the probiotic VSL#3 ameliorates C. difficile-associated disease (CDAD) in mice. Oral VSL#3 administration reduced disease activity, body weight loss, and colonic inflammatory lesions after infection. Probiotic treatment also decreased Th17 and IL-17-producing TCRγδ cell populations at the colonic lamina propria as well as production of IL-17 and IL-1β in the colonic mucosa. Increased numbers of regulatory T cells (Treg) were seen in spleens of VSL#3-treated mice, which correlated to increased peroxisome proliferator-activated receptor γ (PPARγ) activity. PPARγ is a transcription factor and nuclear receptor that modulates immune responses and whose activation is essential for the maintenance of innate antimicrobial immunity in the colon. Mice treated with VSL#3 had an upregulation of calprotectin antimicrobial peptides S100A8 and S100A9 in the colon correlating to decreased fecal clostrial burden at day 4 post-infection. VSL#3 probiotic bacteria regulate colitis by increasing PPARγ activity and decreasing IL-17 responses and increase clostridial clearance by upregulating antimicrobial peptides in mice with CDAD.
CD73-generated extracellular adenosine regulates Toxoplasma gondii infection via NKT cell cytokine production. (P3057)
Authors: Toussaint Leon , Mahamed Deeqa , Bynoe Margaret
Published: January 2023

Toxoplasma gondii is an apicomplexa, obligate intracellular pathogen that evades the host immune system-mediated clearance by undergoing stage differentiation to persist indefinitely in susceptible hosts. We have previously shown that mice deficient in the ecto-enzyme CD73, which generates extracellular adenosine, were significantly resistant to acute and chronic toxoplasmosis after oral infection with the T. gondii type II ME49 strain. Resistance in CD73KO mice was due to a defect in parasite differentiation in the central nervous system (CNS). In contrast to oral infection, intraperitoneal infected CD73KO mice were highly susceptible to immune-mediated pathology, with significantly increased infiltration of neutrophils and T cells into the peritoneal cavity. The adenosine receptor agonist NECA offered protection against immunopathology in infected CD73-/- mice, suggesting the absence of CD73-generated adenosine led to increased susceptibility in these mice. Peritoneal exudate cells from infected CD73KO mice generated higher levels of the inflammatory mediators nitric oxide, TNFα, IL1β and IFNγ. Interestingly, despite the high IFNγ in CD73-/- mice, they produced little to no IL-12. We propose that the IFNγ production independent of IL-12 in these mice during T. gondii infection may be produced by NKT cells (proficient producers of IFNγ). Thus extracellular adenosine is a key molecule that regulates the immune response to an intracellular pathogen and promotes host survival.
Vav1 cooperate with Themis to modulate regulatory T cell suppressive functions (P1217)
Authors: Pedros Christophe , Chabod Marianne , Dejean Anne S , Bernard Isabelle , Lagrange Dominique , Andreoletti Olivier , Fournié Gilbert J , Saoudi Abdelhadi
Published: January 2023

Natural Foxp3+ regulatory T cells (Treg) are of paramount importance for establishing self-tolerance, controlling inflammatory responses and maintaining immune homeostasis. The identification of new molecules controlling Treg functions is nowadays an important challenge. We previously reported the implication of Themis, a new actor of TCR signaling, in Treg suppressive capacities. Indeed, Treg from Themis deficient BN rats were defective, leading to the development of an inflammatory bowel disease. In the present study, we show that Themis deficiency does not impair Treg suppressive functions and does not induce any pathological manifestations in LEW rats. These findings suggest that an interaction with other(s) gene(s) may contribute to the Treg defect and disease development in Themis deficient BN rats. To test this hypothesis and identify the involved gene(s), we performed a genetic dissection using different congenic lines. We show that the impact of Themis deficiency on Treg suppressive functions requires the expression of a constitutively active Vav1 variant. Together, these studies highlight the importance of Vav1 and Themis signaling hub in regulating the suppressive functions of Treg. Understanding the underlying molecular mechanisms may lead to advances in Treg biology and favor the identification of new pharmacogenetic markers and therapeutic targets in immune mediated diseases.
T cell-derived inducible nitric oxide synthase switches off Th17 cell differentiation (P1225)
Authors: Zhang Ruihua , Yang Jianjun , Xiong Huabao
Published: January 2023

Although it is clear that RORgt is necessary for the generation of Th17 cells, the molecular mechanisms for the regulation of Th17 cell. here, we show that activation of CD4+ T cells results in the expression of inducible nitric oxide synthase (iNOS), but not endothelial NOS (eNOS) or neuronal NOS (nNOS). iNOS deficient mice displayed enhanced Th17 cell differentiation but without major effects on either Th1 or Th2 cell lineages, while eNOS or nNOS mutant mice showed comparable Th17 cell differentiation compared to wild type control mice. Addition of dihydrochloride (L-NIL), the iNOS inhibitor, significantly enhanced Th17 cell differentiation and S-nitroso-N-acetylpenicillamine (SNAP), the NO donor, dose-dependently reduced the percentage of IL-17-producing CD4+ T cells. NO mediates nitration of tyrosine residues in RORgt leading to the suppression of RORgt-induced IL-17 promoter activation, indicating that NO regulates IL-17 expression at the transcriptional level. Finally, studies of an experimental model of colitis showed that iNOS deficiency results in more severe inflammation with an enhanced Th17 phenotype. These results suggest that NO derived from iNOS by activated T cells plays a negative role in the regulation of Th17 cell differentiation and highlight the important of intrinsic programs for the control of Th17 immune responses.
Impact of age and body fat on increased toxicity following systemic immunotherapy (P1235)
Authors: Mirsoian Annie , Bouchlaka Myriam , Sckisel Gail , Monjazeb Arta , Murphy William
Published: January 2023

Both aging and obesity are associated with increased distribution of visceral fat and the presence of a chronic pro-inflammatory state. Recent studies have shown that strong systemic immune stimulation with combined aCD40 and IL-2 immunotherapy (IT) of aged (>15 mos) mice results in rapid lethality within two days of treatment as opposed to young mice whom are able to tolerate the entire regimen. Aged mice exhibited lethal toxicities via increased pathological levels of serum proinflammatory cytokines (TNFa, IL-6), and increased multi-organ damage to the liver, lung and gut. Liver damage was further evidenced by heightened serum ALT. Given that aging is characterized by an increase in lipid deposition, we assessed the impact of fat content upon IT induced cytokine storm and tissue damage by comparing young obese (ob/ob) mice with aged caloric-restricted (CR) and age-matched control (WT) mice. Young obese mice showed greater serum cytokine levels of TNFa and IL-6 after immune stimulation, similar to aged mice, in comparison to age-matched controls but did not incur multi-organ pathology as seen in the aged cohort. These data demonstrate the intricate relationship between age and body fat suggesting that fat may be responsible for the exacerbated cytokine storm associated with aging but that aging itself may play a unique and distinct role in cytokine induced acute multi-organ toxicities, possibly through differential cytokine receptor expression in comparison to young.
Soluble IP4 limits NK cell effector functions by controlling PI3K signaling (P1198)
Authors: Sauer Karsten , Park Eugene , Siegemund Sabine , French Anthony , Wahle Joseph , Sternberg Luise , Rigaud Stephanie , Jonsson A. , Yokoyama Wayne , Huang Yina
Published: January 2023

NK cells have important functions in cancer immunosurveillance, bone marrow allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize pathogen-infected or tumor cells through invariant NK cell receptors (NKR), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble inositol(1,3,4,5)tetrakisphosphate (IP4). We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product phosphatidylinositol(3,4,5) trisphosphate (PIP3) and control PIP3-mediated signaling protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.