bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphisms in FcγRII (CD32) mediate abnormal antibody half-life in NOD SCID mice and reduce antibody-mediated tumour cell deletion.
Authors: Oldham Robert J , Cox Kerry L , Cragg Mark S
Published: January 2023

NOD SCID (NS) mice are commonly used for the assessment of monoclonal antibody (mAb) therapeutics and form the genetic background of NSG mice widely used in human patient derived xenograft models. However, relatively little is known regarding how the NS background effects monoclonal antibody (mAb) pharmacokinetics and how this might influence therapeutic response. To address this we examined mAb half-life and clearance of hCD20 transgenic EμTCL-1 tumours in NS mice following administration of anti-hCD20 hIgG1 mAb and showed mAb half-life to be significantly shorter in NS versus SCID mice, correlating with reduced therapy.

Reduced half-life was also seen with mIgG2a but not mIgG1 or hIgG2 isotypes. Additionally, NS mice lacking CD32 or the FcR γ-chain demonstrated this rapid loss was dependent on the expression of CD32 and not activatory FcγRs. Sequencing confirmed the NOD strains carry the rare ly17.1 variant of CD32. Recombinant ly17.1 displayed increased affinity for hIgG1 compared to the more common ly17.2 variant, providing a potential mechanism for its more rapid removal from serum.

Importantly, the reduced half-life was dependent on the presence of both SCID and NOD mutations. SCID mice lack endogenous IgG and so we reasoned this might accentuate the effects of the NOD CD32 polymorphic variant. Accordingly, reconstitution of NS mice with physiological levels of mIgG restored normal half-life. These findings highlight the importance of examining mAb pharmacokinetics in complex mouse strains and demonstrate that therapeutic studies in mice on the NS background should be performed in the presence of exogenous IgG.
Cell-intrinsic glycogen metabolism supports early glycolytic reprogramming associated with dendritic cell immune responses
Authors: Thwe Phyu M. , Beauchamp Saritha , Reisz Julie A , D’Alessandro Angelo , Amiel Eyal
Published: January 2023

Stimulation of dendritic cells (DCs) through toll-like receptors (TLRs) is accompanied by an increased metabolic demand, which provides metabolites and energy required for DC activation. This metabolic requirement is fulfilled by TLR-driven rapid glycolytic burst, and inhibition of glycolysis impairs the survival and effector function of DCs. Previous studies have shown an important role for extracellular glucose uptake via glucose transporter 1 (Glut1) in supporting TLR-driven glycolysis; however, the contributions of intracellular glucose stores to these processes have not been well characterized. While non-immune cells store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen in DCs has not been identified. We propose here that cell-intrinsic glycogen metabolism in DCs supports the early glycolytic reprogramming essential for TLR-driven activation. Our data indicate that glycogen metabolism supports the activation of DCs, particularly before the upregulation of Glut1. Here, we show that DCs express the rate-limiting enzymes for glycogen metabolism and possess intracellular glycogen stores. Inhibition of glycogen breakdown severely impairs the expression of key maturation-associated molecules as well as the ability of DCs to stimulate T cells. Additionally, our metabolomics data indicate that the glycogen metabolism in DCs generates both glycolytic and TCA cycle intermediates and that glycogen-derived carbons preferentially engage in metabolic pathways distinct from free glucose catabolism. Our work reveals a novel metabolic regulatory pathway by which DCs differentially utilize glycogen and glucose metabolism to support their activation.
Canonical and cross-reactive binding of NK cell inhibitory receptors to HLA-C allotypes is dictated by peptides bound to HLA-C
Authors: Sim Malcolm John Wyness , Malaker Stacy A , Khan Ayesha , Stowell Janet M , Shabanowitz Jeffrey , Peterson Mary E , Rajagopalan Sumati , Hunt Donald F , Altmann Daniel M , Long Eric O , Boyton Rosemary J
Published: January 2023

Human NK cell activity is regulated by killer-cell Ig-like receptors (KIR) that bind HLA class I. Combinations of KIR and HLA genotypes are associated with disease, including susceptibility to viral infection and disorders of pregnancy. KIR2DL1 binds group C2 HLA-C (Lys80), while KIR2DL2/3 bind group C1 HLA-C (Asn80). The goal of this study was to determine how the endogenous HLA-C peptide repertoire influences specific binding of inhibitory KIR to HLA-C allotypes.

HLA-C*05:01 (C2) and HLA-C*08:02 (C1) have identical sequences apart from the key KIR specificity determining residues at 77 and 80. Endogenous peptides were eluted from HLA-C*05:01 and used to test KIR2DL1 and KIR2DL2/3 binding to HLA-C*05:01 and HLA-C*08:02 and subsequent impact on NK cell function. Canonical binding of KIR2DL1 to C2 occurred with the majority of peptides tested, while KIR2DL2/3 binding to C1 occurred with a subset of peptides. Cross-reactive binding of KIR2DL2/3 with C2 was restricted to even fewer peptides. Unexpectedly, two peptides promoted binding of the C2 specific KIR2DL1 to the C1 allotype.

KIR2DL2/3 binding to C1 is more peptide selective than KIR2DL1 binding to C2, providing an explanation for why KIR2DL3–C1 interactions appear weaker than KIR2DL1–C2. Cross-reactive KIR binding is characterized by even higher peptide selectivity. These differences in peptide selectivity may aid the interpretation of human disease associations with KIR and HLA, while peptide sequence-driven KIR binding provides a potential mechanism for pathogen or self-peptide to modulate KIR binding and NK cell activation.
Corticotropin releasing factor receptor 2 exerts global suppression of mast cell degranulation and associated pathophysiology.
Authors: Mackey Emily , D’Costa Susan , Ayyadurai Saravannan , Gibson Amelia , Poopal Ashwini , Moeser Adam J
Published: January 2023

Psychological stress triggers mast cell (MC) activation and is a major risk factor in the onset and exacerbation of MC-associated disorders including allergy/anaphylaxis, irritable bowel syndrome, and autoimmune diseases. The mechanisms by which stress modulates MC function and disease susceptibility is poorly understood. Our previous studies showed that corticotropin releasing factor receptor 1 (CRF1) signaling potentiated stress-induced MC degranulation. Here we utilized CRF2 deficient (CRF2 −/−) mice and MCs to define the in vivo and in vitro biological importance of CRF receptor subtype 2, CRF2. Compared with WT mice, CRF2 −/− mice exhibited greater serum histamine levels following restraint stress (RS) (by 143%) and IgE-mediated passive systemic anaphylaxis (PSA) (by 415%). The heightened response was greater in CRF2 −/− females compared with males. CRF2 −/− mice exhibited greater RS-induced and PSA-induced elevations in intestinal permeability compared to WT mice. Bone marrow derived MCs (BMMCs) derived from CRF2 −/− mice exhibited greater release of β-hexosaminidase and histamine and heightened Ca2+ mobilization from intracellular stores in response to diverse MC stimuli including IgE/DNP, c48/80, and A23187. MC-deficient mice engrafted with CRF2 −/− BMMCs exhibited greater RS-induced intestinal permeability and PSA-induced hypothermia compared with WT BMMC-engrafted mice. Together, these results demonstrate that MC-CRF2 receptor signaling exerts a global suppression of MC degranulation and pathophysiology. Elucidation of the mechanisms by which CRF2 dampens MC degranulation could reveal novel therapeutic targets for hyperactive MC disorders.
The interaction of the RNA binding protein with microRNA regulates cytokine production in Th17 cells
Authors: Chen Jing , Yu Shiguang
Published: January 2023

GM-CSF produced by Th17 cells play an essential and non-redundant role in chronic inflammatory and autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of GM-CSF gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3′ untranslated region of many inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, the regulation of GM-CSF expression by HuR in Th17 cells has not been fully characterized. CD4+ Th17 cells from HuR knockout mice had decreased GM-CSF steady-state mRNA compared with wild-type Th17 cells. Moreover, we demonstrated that HuR directly binds to GM-CSF mRNA 3′ untranslated region (3′UTR) by using RNA immunoprecipitation and biotin pulldown assays. In addition, knockout of HuR increases certain levels of microRNAs in Th17 cells, among them, miR-466i has multiple potential binding sites in the 3′ UTRs of GM-CSF and IL-17 mRNAs. In vitro luciferase assay showed that overexpression of miR-466i reduces activity of luciferase reporter vector containing GM-CSF or IL-17 mRNA 3 UTR, suggesting that miR-466i has the potential to mediate GM-CSF and IL-17 mRNA decay. Taken together, these findings indicate that interaction of HuR with miR-466i orchestrates GM-CSF expression by Th17 cells.
Alterations in p53 expression in PMBCs of PTSD patients leads to dysregulation in miRNA biogenesis which promotes an inflammatory Th17 immune cell phenotype.
Authors: Busbee Philip Brandon , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Post-traumatic stress disorder (PTSD) is a neurological disorder brought on by a traumatic or life-threatening event, causing symptoms such as reoccurring memories of trauma, mental numbing, and avoidance of cues that make them recall the trauma in question. In addition to insufficient treatment options, PTSD patients often express other comorbid health issues, particularly inflammatory disorders. Our lab has previously published a report showing peripheral blood mononuclear cells (PBMCs) from PTSD patients exhibit an increased inflammatory CD4+ T helper (Th) cell phenotype that correlated with PTSD severity. However, key regulators that promote this CD4+ inflammatory phenotype in PTSD patients have not yet been identified. In the current study we performed microRNA (miRNA) array analysis on PMBCs collected from healthy control and PTSD patients. Profiles from miRNA indicated that PTSD patients had a widespread downregulation of miRNA when compared to controls. Further analysis revealed that many of the miRNA were associated with transcription factor p53. Our examination of PBMCs from PTSD samples showed that p53 is in fact downregulated in these patients when compared to healthy controls. Downregulated miRNA associated with p53 were shown to negatively regulate the promotion and function of Th17 T cells, particularly miRNA let-7a, which targets IL-17 and Th-17 promoting IL-6. We therefore transfected PMBCs with let-7a and were able to show that let-7a modulation alone was able to greatly influence Th17 development. Collectively, these data reveal important mechanisms that promote an inflammatory phenotype in PTSD patients and bring attention to the novel involvement of p53 dysregulation in this patient population.
Bcl11b, a novel GATA3-interacting protein, regulates T-helper-2-cell differentiation and function
Authors: Fang Difeng , Gurram Rama Krishna , Cui Kairong , Zhong Chao , Hu Gangqing , Oler Andrew , Sharma Suveena , Liu Pentao , Sun Bing , Zhao Keji , Zhu Jinfang (Jeff)
Published: January 2023

GATA-binding protein 3 (GATA3) acts as the master transcription factor for type 2 T helper (Th2) cell differentiation and function. Fine-tuning GATA3 transcription activity is critical for effectively expelling extracellular parasites and preventing allergic disorders. However, it is still elusive how GATA3 function is regulated in Th2 cells. Here, we report that the transcription factor B-cell Lymphoma 11b (Bcl11b) is a novel component of the GATA3 transcriptional complex executing GATA3-mediated gene expression. Bcl11b binds to GATA3 through protein-protein interaction; ChIP-Seq analysis showed co-localization of Bcl11b and GATA3 at many important cis-regulatory elements in Th2 cells. The expression of type 2 cytokines, including IL-4, IL-5 and IL-13, is up-regulated in Bcl11b-deficient Th2 cells both in vitro and in vivo; such up-regulation is completely GATA3 dependent indicating that Bcl11b inhibits GATA3-mediated cytokines production. Genome-wide analysis of Bcl11b- and GATA3-mediated gene regulation (RNA-Seq) and co-binding pattern (ChIP-Seq) suggests that GATA3/Bcl11b complex are involved in limiting Th2 gene expression as well as inhibiting non-Th2 gene expression. Thus, Bcl11b, a previously unknown GATA3 binding protein, fine-tunes GATA3-mediated gene expression in Th2 cells.
MicroRNA-132, miR-146a, miR-155 as potential biomarkers of Methotrexate response in patients with Rheumatoid arthritis.
Authors: Singh Ankita , Patro Pradeepta Sekhar , Aggarwal Amita
Published: January 2023

Introduction

MiRNAs regulate signaling pathways of inflammation and thus can serve as biomarkers. Patients with Rheumatoid arthritis (RA) have high expression of miR-155, miR-146a and miR-132 in their peripheral blood cells. As one third of patients do not respond to methotrexate(MTX), the first line therapy in RA, we studied if levels of these miRNAs can predict response to MTX.

Methods

Patients with RA (ACR 2010 criteria), DMARD naïve with active disease (DAS 28 >3.2) were enrolled. Blood sample was collected. All patients were treated with MTX by gradually increasing dose to a maximum of 25mg/week or the maximal tolerated dose. No corticosteroids were given. At 4 months disease activity was measured by DAS 28 and EULAR response was assessed. miR-132, miR-146a and miR-155 were measured using real time qPCR in the whole blood RNA. Let7a was used as a housekeeping gene.

Results

80 patients (83.8% females; mean age 41.05+12.19 years); mean duration of disease 2.23+1.95years; mean DAS28 4.56+ 0.71) were enrolled. Among 80 patients 59 were classified as good responders while 14 were moderate- responders and 7 were non-responders at 4 months. The baseline miRNA levels did not correlate with the baseline DAS28 score or any other clinical parameters of RA patients. All the three miRNA’s were lower in responders as compared to non-responders but it was significant only for miR-155 (p=0.016) with mean value −7.531±0.833 for responders (n=73) and −6.476±1.069 for non-responders (n=7). ROC curve analysis showed that miR-155 with cutoff value of −7.3(dtCt) is predictor of non-response to MTX treatment with a sensitivity of 67.1% and a specificity of 71.4% (AUC:0.777, p=0.016).

Conclusion

MicroRNA-155 may serve as a good biomarker of response to MTX.
Oral idebenone attenuates murine lupus
Authors: Blanco Luz P , Lightfoot Yaima , Seto Nickie , Carmona-Rivera Carmelo , Liu Yudong , Hoffmann Victoria , Kaplan Mariana J
Published: January 2023

A role for mitochondrial dysfunction has been proposed in the immune dysregulation and organ damage characteristic of systemic lupus erythematous (SLE) disease pathogenesis. We have now examined the role of drugs that may improve mitochondrial function in SLE symptomatology in murine models of lupus. Idebenone is a synthetic quinone analog compound of coenzyme Q10 that has been safely used in humans to treat diverse diseases where mitochondria function is deficient. Idebenone is considered a strong antioxidant able to protect cells against enhanced reactive oxygen species’ toxicity. Idebenone also improves electron transfer chain function in overloaded and/or damaged mitochondria. Idebenone was administered 250 mg/day/kg orally to two different mouse models of SLE starting in preclinical stages (10 weeks of age) for 6 weeks (MRL/lpr mice) or 4 months (NZM2328 mice). Disease attenuation was observed in both models at euthanasia. Specifically, proteinuria, intestinal inflammation and splenomegaly were decreased in both models upon exposure to idebenone, while levels of autoantibodies were not affected. In the MRL/lpr mouse model, skin lesions characteristic of this model were significantly decreased. In addition, levels of proinflammatory cytokines, type I IFNs and inflammasome-related genes were significantly decreased. No obvious toxicity was observed following exposure to idebenone. In conclusion, idebenone modulates lupus disease activity and organ damage severity and future studies should further address mechanism of action and potential role in human disease.
Farnesylthiosalicylic acid reduces disease severity in the collagen type-II induced arthritis mouse model by inhibiting Ras Signaling in pathogenic T cells
Authors: Zayoud Morad , Vax Einva , Sfadia Galit Elad , Kloog Yoel , Goldstein Itamar
Published: January 2023

Background

Ras proteins are vital for normal T cell activation, and downstream effectors of Ras include the MEK/ERK, PI3-kinase/AKT, and NF-kB pathways. T cells from Rheumatoid Arthritis patients exhibit abnormal activation of the Ras/MEK/ERK pathway. The small molecule Farnesylthiosalicylic acid (FTS) blocks the interaction between Ras proteins and their prenyl binding chaperones, attenuating plasma membrane localization and signaling.

Objectives

To investigate the immunomodulatory effect of FTS alone or combined with methotrexate (MTX) in the DBA/1 mouse collagen type-II induced arthritis (CIA) model.

Methods

Arthritis was induced in 8–10 week old male DBA/1 mice by immunization with collagen type-II (CII) and complete Freund’s adjuvant (CFA). Animals were treated semi-prophylactically with once daily oral FTS (100 mg/kg); weekly i.p injection of MTX (0.5 mg/kg), FTS combined with MTX, or daily oral vehicle solution (control). Arthritis severity was scored daily from disease onset until study termination and multiple immunological biomarkers of inflammation were analyzed.

Results

Our data from the mouse CIA model show that the therapeutic efficacy of FTS was similar to MTX, and both drugs significantly reduced arthritis severity compared to CMC controls. Importantly, FTS significantly inhibited the production of pathogenic anti-CII autoantibodies and upregulation of serum IL-6 and IL-17A compared to control arthritic mice. The in depth, multiplex, analysis of the effect of FTS on the T cell cytokine response to CII, revealed strong suppression of IL-22, IL-17, IL-9, GM-CSF and TNF production. Importantly, FTS therapy positively correlated with reduced p-ERK1/2 and p-AKT levels in splenic lymphocytes.
A comparison of the immunogenicity of a live attenuated herpes zoster vaccine (ZV) and the recombinant gE/AS01B candidate vaccine in older adults
Authors: Weinberg Adriana , Johnson Michael J , Kroehl Miranda , Lang Nancy , Reinhold Dominik , Levin Myron J
Published: January 2023

Herpes zoster (HZ) has high morbidity in older adults. ZV, which is recommended for individuals ≥60 y, is 51% effective against HZ in the 1st 3 y after vaccination; efficacy decreases with the age of the vaccinee; and wanes to ~40% at 5 y. gE/ASO1B is a recombinant adjuvanted vaccine with 97% efficacy against HZ that does not vary with age of the vaccinee and wanes to 87% at 4y. We compared the cell-mediated immunity (CMI) of ZV and gE/ASO1B at 30 d after the last dose of vaccine and at 1 y.

Subjects were 50–70+ yoa who never received ZV (1ary) and adults ≥70 yoa who received ZV ≥5 y previously (boosted). Participants received ZV at entry or gE/ASO1B at 0 and 60 d. Blood from days 0, 30 (ZV peak response), 90 (peak gE/ASO1B) and 365 was used to measure CMI to varicella zoster virus (VZV) and to gE by IFNg and IL2 fluorospot [spot forming cells/106 PBMC (SFC)] and by flow cytometry assessing VZV- and gE-specific proliferation and effector (Teff), effector memory (Tem), and central memory (Tcm) outcomes.

The higher CMI responses that best distinguished gE/ASO1B from ZV recipients after adjusting for age, gender, prior ZV administration and baseline CMI were: 1) VZV-specific IL2 and gE-specific IL2 and IFNg SFC at peak response (p≤0.004); 2) higher VZV-specific CD8+ and gE-specific CD4+ and CD8+ responses measured by proliferation at peak (p<0.05); 3) higher CD4+ Tcm% and Tem% and lower Teff% at peak response (p≤0.005); 4) higher VZV-specific and gE-specific IL2 and IFNg SFC at 1 y in all gE/ASO1B groups, but only in the boosted ZV group. CMI did not differ among gE/ASO1B recipients by age or prior ZV administration.

The higher memory CD4+ and CD8+ responses detected in gE/AS01B recipients might contribute to its superior efficacy and persistence of protection against HZ.
Modulation of DNA/ALVAC/gp120 vaccine immune-response by vaccination with Insulin-Like Growth Factor 1 (IGF-1)
Authors: Bissa Massimiliano , Galli Veronica , Schifanella Luca , Gorini Giacomo , De Castro Isabela Silva , Brown Dallas , Doster Melvin N , Liyanage Namal , Rosati Margherita , Felber Barbara K. , Pavlakis George N , Phogat Sanjay , Franchini Genoveffa
Published: January 2023

ALVAC-SIV/gp120 vaccine reduced the risk of infection in SIVmac251 model. Protection was associated with antibodies (Abs) to V2 region of gp120 protein, mucosal NKp44+ cells, and the expression of 11 genes in the Ras pathway. IGF-1 hormone is a Ras activator and its plasma level diminishes with aging. We hypothesized that IGF-1 might affect the efficacy of the ALVAC-SIV/gp120 platform by modulating the immune responses, particularly when administered in old macaques. In this study we enrolled 25 young (Group 1, av. 3 years) and 34 old macaques (Group 2, av. 9 years) having significantly different plasma levels of IGF-1, and we immunized them with a DNA-SIV/ALVAC-SIV/gp120 vaccine. During immunizations, half of the animals of each group were also administered IGF-1, as recombinant protein (Increlex, Ipsen) and as DNA vaccine. All the animals, including controls, were challenged intra-vaginally with repeated low-doses of SIVmac251. The estimated vaccine efficacy in young macaques of Group 1 was 69%. IGF-1 administration did not increase the efficacy; although, it determined a significant increase of serum Abs titers against V2 and frequency of mucosal NKp44+ cells. Furthermore, IGF-1 treated macaques showed higher frequencies of circulating classical (CCR2+CD14+CD16−) and intermediate (CCR2+CD14+CD16+) monocytes and lower frequencies of mucosal CD4+ T cells expressing α4β7 or CCR5 compared to the untreated animals. These data suggest that IGF-1 administration might improve protective immune responses and decrease possible SIV target cells in the mucosa. Therefore, IGF-1 administration during vaccination might particularly benefit older animals. Challenge exposure of older animals in Group 2 is ongoing.
Sustained delivery of a HIV subunit vaccine using silk microneedle skin patches
Authors: Boopathy Archana Vidya , Mandal Anasuya , Kulp Dan W , Kumari Sudha , Wang Wade , He Yanpu , Tokatlian Talar , Schief William R , Hammond Paula T , Irvine Darrell J
Published: January 2023

Many broadly neutralizing antibodies (bNAbs) against HIV exhibit high degrees of somatic hypermutation, suggesting that immunization approaches promoting germinal center (GC) stimulation and affinity maturation may be critical for an HIV vaccine. To this end, we recently demonstrated that sustained exposure of draining lymph nodes (LNs) to env immunogens through repeated injections over 2 weeks enhanced env capture on follicular dendritic cell networks in LNs, with Tfh expansion and GC induction. To obtain similar sustained vaccine kinetics for mass immunization, we developed a submicron-scale dissolving silk microneedle array (MN) for dermal vaccine delivery. We utilized silk protein to encapsulate an HIV envelope trimer (BG505 SOSIP variants) with adjuvants supported by a poly acrylic acid (PAA) base. Upon dermal application, rapid PAA dissolution is followed by silk beta sheet-dependent sustained release of encapsulated vaccine with minimal local inflammation. Antigenicity of SOSIP was maintained in silk as determined by binding to structure-sensitive bNAbs- PGT151, PGT145, PGT121 and VRCO1 without binding to non-bNAb 14e. Following immunization in mice, skin and draining LNs were subjected to lipid clearance using organic solvents and imaged by confocal microscopy. We observed SOSIP and silk co-retention at the skin site of microneedle application after one week. MN delivery resulted in higher SOSIP retention and increased SOSIP-specific T follicular helper cells in the GCs of vaccine draining LNs on day 14 post prime, and increased anti-SOSIP IgG titers on day 28 post prime compared to intradermal injections. Taken together, these results suggest the potential of silk MNs in modulating release of HIV subunit vaccine.
Protection and durable humoral and cellular immune responses in cynomolgus macaques following administration of a Zaire Ebola virus (EBOV) GP DNA vaccine delivered by intramuscular or intradermal electroporation
Authors: Patel Ami , Reuschel Emma L. , Kraynyak Kimberly Ann , Park Daniel H , Racine Trina , Yan Jian , Khan Amir , Morrow Matthew P , Muthumani Kar , Shedlock Devon J , Broderick Kate E. , Audet Jonathan , Jones Shane , Bello Alexander , Soule Geoff , Tran Kaylie N , Tierney Kevin , Qiu Xiangguo , Kobinger Gary P , Humeau Laurent , Sardesai Niranjan , Weiner David
Published: January 2023

The 2013–2016 Ebola virus disease (EVD) was the first time that the spread of the virus reached epidemic status. There are no licensed vaccines; however the most advanced candidate rVSV-ZEBOV vaccine, is a live-attenuated vesicular stomatitis virus encoding the Ebola virus glycoprotein (GP), and has demonstrated efficacy in a ring-vaccination trial. However, several EVD viral vector vaccine trials have reported adverse events that could limit administration to certain populations. The establishment of robust anamnestic responses has yet to be fully understood with these candidates and may be limited by potential anti-vector immunity. We designed EVD DNA vaccines as an alternative platform with a remarkable safety profile that is serology independent, allowing for possible repeat vector administration. We designed 3 novel synthetic Zaire Ebola virus (EBOV) GP DNA sequences which were tested alone or as multivalent formulations delivered by in vivo intramuscular (IM) or intradermal (ID) electroporation (EP). The EBOV-GP DNA vaccines were highly protective against lethal EBOV-Makona challenge in cynomolgus macaques, with 100% protection in NHPs receiving vaccine by ID-EP delivery and 75% protection in NHPs receiving 2 doses IM-EP. Vaccinated NHPs had no detectable viremia following challenge. Animals (n=4–5/group) injected with different IM-EP or ID-EP DNA regimens were followed to monitor long-term immunogenicity. NHPs have durable total IgG antibody titers and T cells responses to EBOV GP antigen, including polyfunctional CD4 and CD8 T cells and responses in memory subset populations. Together, the data strong support EBOV-GP DNA vaccine delivery for protection and the generation of robust memory immune responses.
The genetics and expression of canine antigen receptors analysed with a novel RNA-seq strategy.
Authors: Martin Jolyon N E , Ponstingl Hannes , Sargan David , Bradley Allan
Published: January 2023

INTRODUCTION

Dogs are an excellent model for human disease, e.g. the treatment of canine lymphoma is often predictive of the human response to that treatment. However, an incomplete picture of their antigen-receptor (AR) gene loci and a limited set of available experimental tools has restricted their use. This work advances the annotation of the canine AR loci, develops methods to interrogate their expression patterns, and looks into breed-specific features of the loci.

METHODS

A bioinformatic approach alongside unbiased RNA-seq was used to complete the annotation of the canine AR genes, and these sequences were used to query 107 whole genome sequences from 19 breeds. A 5’RACE strategy was developed to selectively amplify ARs for next generation sequencing, and this was applied to RNA isolated from canine peripheral blood samples.

RESULTS

>5,500 novel alleles were identified across the ~550 gene segments (of which 326 were newly annotated) of the AR loci, yielding insight into AR evolution as well as confirming the greater conservation between dog and human than mouse with either. The 5’RACE strategy was validated with a pilot study that cast light onto the baseline expression and mutation patterns of healthy dogs, finding they closely mirrored their human counterparts.

CONCLUSION

This work brings our understanding of the genetics and expression of ARs in dogs to the same high standard as mice and men. The large number of genome sequences serves as a reference for future studies, and has allowed statistically powerful conclusions to be drawn on the pressures that have shaped these loci. Finally, it confirms the dog as a superior immunological model, particularly in the context of the high conservation seen in both sequence and expression features.
Mycoplasma gallisepticum requires ATP to activate the canonical inflammasome in vitro.
Authors: McDaniel Dylan , Karetnyi Anastasia , Caswell Clayton , Allen Irving C
Published: January 2023

Mycoplasma gallisepticum (MG) is a bacteria which lacks a cells wall and colonizes mucosal epithelial cells. This colonization commonly occurs in the respiratory tracts of chickens and turkeys, leading to chronic respiratory disease. MG is an economically significant pathogen in poultry and can cause large economic losses on poultry farms because of decreased egg laying. Although the importance of MG as an economically relevant pathogen is understood, there is a paucity of knowledge in regards to its pathogenesis. One possible mechanism is via the activation of the inflammasome, a multi-protein complex leading to the maturation of IL-1β and IL-18 as well as the inflammatory form of cell death called pyroptosis. Interestingly, Mycoplasma hyorhinis (MH) was shown to activate the NLRP3 inflammasome in THP-1 and mouse bone marrow derived dendritic cells (BMDCs). In addition, HD-11 cells co-cultured with tracheal epithelial cells that were exposed to MG, show increased levels of IL-1β. In this study, we aimed to determine if MG activated the inflammasome in murine and chicken macrophages. We found that MG activates the canonical inflammasome in primary murine macrophages via the induction of IL-1b release. However, we determined that this MG-mediated activation of the canonical inflammasome requires the addition of ATP in vitro. In addition, we also found that MG alone is not sufficient to activate pyroptosis in vitro. Interestingly, MG also initiated the production of IL-6 in these cells, suggesting potential activation of the NF-κB pathway. We are currently working with the HD-11 chicken cell line to determine if MG activates the inflammasome in these cells as well.
Number and location of AID ‘hotspot sequences’ in germline variable genes of diverse species
Authors: Levings Randall
Published: January 2023

Generation of antibody (Ab) diversity is accomplished through five mechanisms: Heavy/Light chain combination, Variable-(Diversity)-Joining gene combination, junctional base addition/deletion, gene conversion, and somatic hypermutation (SHM). Different species have been shown to use the mechanisms to varying degrees. Species with relatively fewer variable (V) genes might be expected to have V genes containing more ‘hotspots’ for activation induced cytidine deaminase (AID), so as to ‘preposition’ the genes for SHM. Germline V sequences identified in the Im Muno Gene Tics (IMGT) database were analyzed for AID target sequences (RGYW, WRCY) relative to framework and complementarity determining regions as designated in the IMGT amino acid numbering system. Examples of functional germline sequences from six classes of vertebrates (only Agnatha was excluded) - including multiple orders of mammals - were analyzed. No clear pattern of AID hotspot number or location was observed to be associated with phylogeny or diversification strategy (when known). However, ‘outlier species/genes’ were noted. The results indicate that caution should be exercised in generalizing or extrapolating between species regarding Ab gene sequences. They also reinforce the importance of conducting gene studies including germline, fetal B lymphocytes, and mature B-cells across many species. Information on the origin and mechanisms of Ab diversity has relevance to the potential breadth of the immune response to infection and vaccination, and possibly to engineering effective Ab for immunotherapy.
Dairy cattle naturally infected with bovine leukemia virus exhibit abnormal adaptive immunity after primary and secondary challenges with keyhole limpet hemocyanin.
Authors: Frie Meredith , Sporer Kelly R. B. , Coussens Paul M.
Published: January 2023

Bovine leukemia virus (BLV) is a retrovirus that is widely prevalent in the US: over 83% of dairy herds and approximately 40% of all dairy cows are infected. While less than 5% of infected cows will develop leukemia or lymphoma as a result of BLV, new research demonstrated that BLV+ cows produce less milk and have shorter life spans than their BLV− herdmates. BLV+ cows likely have impaired immunity, putting them at risk for contracting other infectious diseases that negatively impact production and welfare. To investigate whether BLV+ cows develop abnormal immune responses to a primary infection, BLV+ and BLV− cows were challenged with the protein keyhole limpet hemocyanin (KLH) to elicit a primary immune response and T and B cell responses to KLH were characterized. 56 days after the primary challenge, BLV+ and BLV− cattle were re-inoculated with KLH to elicit a secondary memory immune response and in vivo T and B cell responses to KLH were characterized. BLV+ cows produced less IgM in response to KLH and exhibited abnormal γδ T and B cell populations. T and B cell responses to in vitro stimulation were also characterized. CD8+ and γδ T cells from BLV+ cows produced more IFNγ, and CD8+ T cells from BLV+ cows exhibited atypical IL4 production. In comparison to B cells from BLV− cows, BLV-expressing and BLV-non-expressing B cells from BLV+ cows exhibited altered MHCII expression. Activated B cells also exhibited a much higher proportion of BLV expression in comparison to unactivated B cells. Together, these data show that BLV+ cows exhibit a reduced and abnormal immune response even upon a primary immune challenge.
Epithelial expression of IL-17 mediates a gut-associated inflammatory response in the purple sea urchin larva.
Authors: Buckley Katherine M , Rast Jonathan P
Published: January 2023

Immune response to microbial disturbance in the gut involves coordinated activity of gut epithelial cells, motile immune cells and other cells throughout the organism. Many of these functions are ancient among bilaterians, although it is not yet clear how they relate among animal phyla. As morphologically simple organisms that share an important genetic heritage with vertebrates, sea urchin larvae offer a novel, experimentally tractable system in which to characterize the gut-associated immune response. We have developed a model to characterize gene regulatory control of this response using the marine bacterium Vibrio diazotrophicus. When exposed to Vibrio in the surrounding seawater, bacteria accumulate in the gut and later invade the body cavity. Bacteria that enter the blastocoel are rapidly cleared by a coordinated response of phagocytic and granular immune cells. Peripheral immune cells migrate through the body cavity and accumulate at the gut. RNA-Seq data indicate that the most acutely upregulated gene in the early phase of response are two types of IL17 genes. Whole mount in situ hybridization and BAC-based fluorescent protein reporters indicate that these genes are expressed within gut epithelial cells. Notably, perturbation of IL17 receptor signaling results in reduced levels of tnfaip3 (a negative feedback inhibitor of IL17), nfkbiz (an IL17 target gene in vertebrates), the transcription factors cebpα and cebpγ and soul1 (SOUL domains are evolutionarily widespread and involved in immune responses). These results indicate that the highly regulated IL17 expression in the gut epithelium and signaling through IL17R1 form a central axis of larval gut-associated immunity.
High expression of genes encoding signaling molecules (LAX1, BLK) and transcriptional factors (ICOS, GATA3) characterizes bovine neonatal immunity.
Authors: Kaushik Azad Kumar , Pasman Yfke , Merico Daniele
Published: January 2023

We analyzed 18 RNA-seq libraries from 3 bovine calves at day 0, 7, 14 and 28, together with their dams (day 0 and 7) for cell signaling and transcription factors gene expression. LAX1, lymphocyte transmembrane adaptor 1 is consistently expressed at higher levels post-birth. Similarly, BLK, a cell signaling tyrosine kinase required for from pro- to pre-B cell transition is expressed at high levels 4 weeks post-birth. A trend for increased expression of Igα (CD79A) and Igβ (CD79B) in neonatal B cells is noted day 0 to day 28 (LogFC 0.8 and P-value< 0.05 FDR <0.15). The CD40 expression is also significantly increased day 0 versus day 14 (LogFC 1.05, FDR .00039) to achieve steady levels. A lack of increase in T-cell signaling molecules CD3G and CD3D expression suggests neonatal T cell quiescence, but is higher than the dams (FDR<0.05). However, the ICOS (CD278) expression is increased from day 0 to day 7 post-birth (LogFC 1.11, FDR 0.00027). Similarly, an increased expression of FOXP3, relevant to the development of regulatory T cells, occurs in bovine neonates day 0 vs day 7 (LogFc 1.23, FDR 0.00063). The expression of GATA3 transcription factor, regulator of innate and adaptive immunity associated with TH2 differentiation, is high in neonates as compared to the adults (FDR<0.001). As would be expected, various transcriptional factors and cell signaling molecules contribute towards T and B cell proliferation and expansion, but a lack of increased expression of CD3G and CD3D genes suggests T cell quiescence early during ontogeny. [Supported by NSERC Canada Discovery Grant]
The Actin Cytoskeleton Controls FcgRIIB-Mediated Inhibition of B-Cell Signaling
Authors: Shu Zhou , Lazzaro Michelle , Liu Chaohong , Seeley-Fallen Margaret , Csiszer Ivan , Upadhyaya Arpita , Zhao Xiaodong , Song Wenxia
Published: January 2023

FcgRIIB is a major inhibitory coreceptor of B cell receptor (BCR), and its deficiency leads to B cell autoimmunity. Upon colligated with the BCR by immune complexes (ICs), this coreceptor activates SH2-containing inositol-5 phosphatase (SHIP) and inhibits BCR clustering in lipid rafts. However, the molecular mechanisms underlying FcgRIIB-mediated inhibition of BCR activation remain elusive. This study reveals a critical role of the actin cytoskeleton in FcgRIIB-mediated inhibition. Upon interacting with ICs tethered to lipid bilayers, an actomyosin ring is quickly formed surrounding the B cell membrane region interacting with the lipid bilayer (contact zone). Perturbing the actomyosin ring, by myosin inhibitors or B cell-specific gene knockout of non-muscle myosin IIA (NMIIA) or neuronal Wiskott-Aldrich syndrome protein (N-WASP), releases B cell spreading, BCR clustering and signaling from the inhibition by FcgRIIB. Using conditional gene knockout mice, we further show that NMIIA is recruited to the B cell contact zone in an SHIP-dependent manner, while N-WASP-mediated actin remodeling organizes NMIIA and actin into a ring. Surprisingly, WASP-mediated actin dynamics is required for the FcgRIIB-induced activation of SHIP but not the formation of the actomyosin ring. Our results suggest that FcgRIIB-triggered signaling induces unique actin remodeling involving NMII, WASP, and N-WASP, which limits B cell morphological changes and BCR lateral mobility and enhances the activation of inhibitory signaling. These data potentially provide a mechanistic explanation for the elevated autoantibody levels caused by WASP or N-WASP deficiency.
ITK is a magnesium sensor during T cell activation
Authors: George Alex B , Kanellopoulou Chrysi , Masutani Evan , Chaigne-Delalande Benjamin , Lenardo Michael J
Published: January 2023

Interleukin-2-Inductible T-cell Kinase (ITK), a member of the TEC family of kinases, serves a vital role in the activation and differentiation of T-cells. Following stimulation of the T cell receptor (TCR) and phosphorylation by LCK, ITK phosphorylates PLCy1 and initiates a transduction pathway that results in the release of intracellular Ca2+ stores and the transcription of IL-2. Although ITK’s role in this signal transduction pathway is known, there is still much to be characterized about the regulation of ITK activity. Previous studies have indicated that Mg2+ serves a key role as a second messenger in T lymphocyte activation and that attenuated intracellular Mg2+ levels inhibit the phosphorylation of PLCy1 and impair Ca flux. We propose that ITK serves as a sensor for the Mg2+ flux required for optimal T cell activation. Kinase assays show that ITK activity is enhanced by Mg2+ and suggest a model where two Mg2+ ions occupy the catalytic site. Molecular modeling also suggests that ITK possesses two Mg2+ binding sites in the catalytic domain. We are currently studying the effect of Mg2+binding on ITK kinase activity via site directed mutagenesis and are in the process of generating crystal structures of ITK complexed with Mg2+. Finally, we propose that this ‘second site’ Mg2+ is a general feature of kinases and may highlight the importance of divalent cations in kinase regulation.
ITK signaling via the Ras/IRF4 pathway regulates the development and function of type 1 regulatory T cells
Authors: Huang Weishan , Solouki Sabrina , Koylass Nicholas , Zheng Song-Guo , August Avery
Published: January 2023

Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging T cell receptor (TCR), express high levels of the immunosuppressive cytokine IL-10 but not Foxp3, and can suppress inflammation and promote immune tolerance. Here, using IL-10GFP/Foxp3RFP dual reporter transgenic mouse system, we show that ITK, a key modulator of TCR signaling, is required for the development of Tr1 cells in various organs following TCR activation, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell fate programming and regulates the balance between IL-10 and IFN-γ production during Tr1 cell differentiation. The requirement for ITK function during Tr1 cell development and suppressive function can be restored by the expression of the transcription factor IRF4. Downstream of ITK, Ras, and MAP kinases ERK1/2, p38 and JNK, are required for Tr1 cell differentiation, but not Th17 cell differentiation. We have previously shown that Th17 differentiation is also dependent on ITK signaling. However, in the absence of ITK, expression of the constitutively active HRasG12V rescued IRF4 expression and Tr1 cell differentiation, but failed to restore Th17 cell differentiation. We conclude that the TCR/ITK signaling through the Ras/MAPK/IRF4 pathway is specifically required for functional development of Tr1 cells, and that targeting these signaling components may be of therapeutic benefit.
Effect of genetics on the development of Hypersensitivity Pneumonitis (HP) using the BXD recombinant inbred mouse reference population
Authors: Fitzpatrick Elizabeth , Yoon Tae-Won , Williams Robert , Yi Ae-Kyung
Published: January 2023

HP is an interstitial lung disease that develops following repeated exposure to a variety of inhaled environmental antigens. The disease is characterized by alveolitis, granulomas and in some patients, develops into a chronic form associated with fibrosis. Not all individuals exposed to HP causative antigens develop disease suggesting that genetic factors play a role in susceptibility; however, the host co-factor(s) that play a role in susceptibility are unknown. We are using the innovative systems genetics tool, the BXD panel of recombinant inbred mice with the Saccharopolyspora rectivirgula (SR) model of HP to identify genetic loci and candidate genes associated with disease development. We exposed the parental strains, C57Bl/6 and DBA/2, and sixteen BXD strains to SR for 3 weeks and analyzed the lungs 18h after the last exposure. Bronchoalveolar lavage (BAL) was performed and cells recovered from the BAL fluid were analyzed by flow cytometry to determine the frequency of infiltrating immune cells. The results reveal variability in the cellular composition of the BAL; some BXD strains exhibited a neutrophilic alveolitis whereas other BXD strains exhibited a more lymphocytic alveolitis. BAL recovered from mice of the BXD45 strain was 68% neutrophils and 7% T lymphocytes, whereas BAL from the BXD73 strain was 92% neutrophils and 2% T lymphocytes. In addition, there were significant differences in the level of activated CD4+ and CD8+ T cells across the strains. H&E staining of lung tissue revealed significant differences in the extent of granuloma formation across strains. These data support the contention that genetic differences critically influence SR responsiveness given that environmental factors were held constant.
Resveratrol improves a murine model of asthma through alterations in the gut microbiome
Authors: Alharris Esraah Sattar , Alghetaa Hasan F , Busbee Philip Brandon , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Asthma is a condition characterized by bronchial spasms, inflammation, and mucous hypersecretion which leads to difficulties in respiration. Asthmatic patients are usually presented with recurrent attacks of coughing, wheezing, and shortness of breath which could be life-threatening. More than three million cases of asthma in the United States are diagnosed annually. Resveratrol, a polyphenolic stilbene, is known to be useful in controlling asthmatic attacks via different molecular mechanisms within the lung epithelium and infiltrating immune cells. However, few studies mentioned the effect of resveratrol on the microbiome in ovalbumin-induced asthma mouse model. In this study, we induce asthma in BALB/c mice by injecting ovalbumin (OVA) with aluminum hydroxide intraperitoneally followed by 7 days treatment with resveratrol (100mg/kg) by oral gavage. Intranasal ovalbumin was given on day 8 to induce asthma followed by 7 more days of treatment with resveratrol. We performed 16S rRNA microbial analysis on cecal flushes between disease and treatment groups, noting changes in microbial diversity among disease and treated groups. Specifically, we identified alterations in gut microbial composition, which included significantly higher levels of Prevotella in the disease groups, which was reduced upon treatment with resveratrol. In addition, Bacteroides acidifaciens species were significantly lower in the asthma-induced mice and significantly increased in resveratrol treated.

Supported in part by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641, MoHESR and R01AI129788
Atypical protein kinase PKCλ/ι on pathogenesis of asthma
Authors: Yang Jun-Qi , Yang Yingying , Dong Panpan , Guo Fukun , Zheng Yi
Published: January 2023

Asthma is a chronic airway inflammation in which Th2 and Th17 cells play critical roles in its pathogenesis. We have reported that atypical protein kinase PKCλ/ι is a new regulator for Th2 differentiation and function. Our recent findings indicate that PKCλ/ι may regulate Th17 cells. The secretion of Th17 effector cytokines, including IL-17, IL-17F, IL21 and IL-22, were inhibited from PKCλ/ι-deficient T cells under non-polarized or Th17-polarized culture conditions. Further studies indicate that impaired Th17 differentiation and function is associated with the down-regulation of Stat3, NFATc1 transcription factors in PKCλ/ι-deficient CD4+ T cells in culture. We developed a model of Th17 and neutrophil-involved allergic airway inflammation by intratracheal inoculation of allergenic extracts of house dust mite (HDM). PKCλ/ι-deficiency significantly inhibited airway inflammations. The infiltrating cells in the lungs and bronchoalveolar lavage (BAL) fluid were significantly reduced in conditional PKCλ/ι-deficient mice. Th17 effector cytokines were reduced in the BAL and lungs at protein and mRNA levels. Our findings suggest that PKCλ/ι might regulate allergic airway inflammation through the control of Th17 cells. The underlying molecular mechanisms are currently under investigation.