bluuebunny/crossref_metadata_2025 huggingface dataset:
RIG-ging the host innate immune response for vaccine adjuvant and antiviral therapy
Authors: Loo Yueh-Ming , Wilkins Courtney R. , Pattabhi Sowmya , Knoll Megan L. , Kaiser Shari , Mire Chad E. , Grigg John , Hemann Emily A , Probst Peter , Posakony Jeffrey , Ireton Renee C. , Geisbert Thomas W. , Bedard Kristin M. , Iadonato Shawn P. , Gale Michael
Published: January 2023

Retinoic acid inducible gene I (RIG-I) is essential for directing and priming the host immune response against many RNA viruses. A diverse small molecule library was used in a cell-based screening approach to identify drug-like compounds that could target the RIG-I signaling pathway and induce downstream IRF3 to drive innate antiviral immunity. We identified small molecule compounds that activate IRF3 to induce discrete subsets of innate immune and antiviral genes. We identified a class of hydroxyquinoline compounds that induce IRF3 activation and innate immunity of cultured cells to decrease viral RNA load and infectious virus production, thereby controlling infection by viruses of the families Flaviviridae, Filoviridae, Orthomyxoviridae, Arenaviridae and Paramyxoviridae. In addition, we identified a second class of benzo-bisthiazole compounds that directly engage and activate RIG-I to promote downstream IRF3 signaling and the expression of innate immune genes, cytokines and chemokines. Importantly, these compounds serve potent immune adjuvant functions to promote the maturation of dendritic cells to enhance antigen presentation and T cell activation. When administered to mice in combination with influenza A virus or West Nile virus vaccine, this compound conferred adjuvant activity to facilitate vaccine-mediated protection of mice from lethal virus challenge under conditions of otherwise suboptimal vaccine dose. Thus, we have identified novel small molecule RIG-I agonists that activate the innate immune response. Our studies reveal the strong potential of developing small molecule RIG-I agonists as broad-spectrum antiviral agents to control RNA virus infection and as adjuvants to potentiate vaccine immunity.
Human and rat epithelial cells inactivate extracellular influenza A virus via H2O2-dependent hypothiocyanite
Authors: Gingerich Aaron , Hanson Jarod , Dlugolenski Daniel , Pang Lan , Tripp Ralph A , Rada Balazs
Published: January 2023

Influenza A virus (IAV) causes ongoing epidemics characterized by high morbidity and variable mortality in several species including humans and swine. Current intervention strategies incorporate either vaccines with several strains of IAV or antiviral drugs against which the virus is developing resistance. Identifying novel anti-influenza mechanisms of the respiratory innate immune system is a potential source of influenza therapies. The first line of defense against respiratory pathogens is the respiratory epithelium. One of the innate defense mechanisms respiratory epithelial cells use to prevent infection with pathogens such as IAV is an extracellular oxidative burst at the airway surface mediated by lactoperoxidase (LPO), thiocyanate anion (SCN−), and hydrogen peroxide (H2O2) to produce antiviral hypothiocyanite ions. Cellular H2O2 is produced by two isoforms of the NADPH oxidase dual oxidase (DUOX) enzymes, DUOX1 and DUOX2. Our data has shown that differentiated, primary human or rat tracheal epithelial cells supplemented with LPO and SCN− are capable of inactivating IAV in vitro. This system has shown a 4–5 log reduction in viral titers that was eliminated by the addition of catalase (an H2O2 scavenger) demonstrating its H2O2 dependence. Additionally, we have shown that this system is capable of inactivating human-origin, swine-origin, and recombinant IAV strains. These results suggest that by modulating the DUOX system IAV infection could be prevented or at least modulated to improve viral killing thereby reducing virus shedding which could alter IAV infection dynamics within a population.
Carbohydrate specific T cell stimulation by HIV envelope glycoprotein
Authors: Sun Lina , Ozdilek Ahmet , Middleton Dustin R , Avci Fikri Y
Published: January 2023

HIV has been a major threat to human health and a protective AIDS vaccine has not as yet been developed. The design of the current generation of vaccines does not make use of specific scientific knowledge to maximize stimulation of critical immune cells (i.e., helper T cells) involved in producing protective antibodies. HIV-1 surface is decorated with a heavily glycosylated envelope protein called gp120, whose interaction with the CD4 molecule is the key step for the virus’s entry into CD4+ T cells. Gp120 glycans have been shown to play critical roles in Env structure, viral infectivity, antigenicity, receptor binding and HIV-1 transmission. Our goal is to identify CD4+ T cell repertoires recognizing the glycan shield of HIV-1 envelope glycoprotein, gp120. We have gained significant evidence for glycan/glycopeptide presentation on the antigen presenting cell (APC) surfaces serving as potential epitopes to induce carbohydrate-specific T cells (Tcarb) response. Additionally, we demonstrate that certain populations of CD4+ T cells differentiate between glycosylated and non-glycosylated gp120 epitopes, suggesting carbohydrate recognition by CD4+ T cells. Using the discovery of such Tcarbs and their glycan epitopes, we can design and develop knowledge-based, new-generation HIV vaccines that will elicit strong and long lasting immune response to protect from HIV. We believe our study will yield a new platform to develop a new-generation of protective HIV vaccines.
Profiling system-wide immune signaling in chronic viral infection and its response to viral clearance
Authors: Angel Cesar Joel Lopez , Furman David , Pham Edward , Fram Benjamin , Nguyen Thai , Ahmed Aijaz , Grant Philip , Maecker Holden T , Glenn Jeffrey , Davis Mark M
Published: January 2023

There are many billions of chronic viral infections in humans, as each of the ~7.4 billion individuals alive today each host ~10 chronic infections. The clinical significance of these infections vary widely, yet all individuals with chronic viral infections rely on persistent antiviral immunity to keep these infections in check. Over time, chronically infected individuals exhibit impaired immune function as evidenced by their poor response to vaccination and progressive T cell dysfunction – a process known as exhaustion. The impact of chronic viral infection on other immune cell types is understudied, and whether the immune landscape can be restored upon viral eradication is unknown. To address these questions, we have prospectively recruited a cohort of HCV-infected patients who underwent standard of care treatment leading to viral clearance in 12 weeks, a cohort of virologically suppressed HIV-infected patients, and an aged-matched cohort of healthy individuals. With the use of single-cell mass cytometry (CyTOF) and systems bioinformatics analyses, we comprehensively characterized the molecular pathways and functional signature underlying immune exhaustion. Here we report an immune signaling signature that differentiates individuals with chronic viral infections from uninfected individuals. While this signature is largely unaltered following viral clearance, we identified a set of functional parameters that are restorable upon successful viral eradication which represent a previously unobserved level of plasticity of the human immune system.
A role for CXCL17 in Protective Vaginal CD8+ T cell Immunity Against Genital Herpes Infection and Disease
Authors: Benmohamed Lbachir , Srivastava Ruchi , Hernández Marcela , Khan Arif A , Zlotnik Albert
Published: January 2023

Conventional memory CD8+ T cells include effector memory (TEM) and central memory (TCM) CD8+ T cell subsets that circulate through the blood to access lymphoid and non-lymphoid tissues in order to control peripheral infections. A distinct non-recirculating tissue-resident memory CD8+ T cell subset (CD8+ TRM) develops and is retained within peripheral non-lymphoid mucosal tissues. Chemokines play a crucial role as chemoattractants for conventional memory CD8+ T cells. However, the identity of the chemokines involved in the homing of tissue-specific mucosal resident CD8+ TRM cells remain to be fully elucidated. We screened several prominent mucosal chemokines to identify those relevant to viral infections in the vagina. This screen indicated that the mucosal chemokine CXCL17 is strongly expressed in the vagina. We used herpes simplex virus type 1 (HSV-1) to determine the role of CXCL17 in immunity in the vaginal mucosa. Following HSV-1 infection, CXCL17 is strongly induced in the vaginal mucosa, and its expression parallels an increase in the number of tissue-resident CXCR8+CD8+ TRM cells. These changes are associated with a decrease in genital herpes infection and disease. Furthermore, a CXCL17−/− mouse failed to contain genital herpes when infected with HSV-1. These results strongly suggest a pivotal role for CXCL17 in protective vaginal CD8+ T cell immunity.
Elevated CD11c and CD80 levels on peripheral B cells are associated with reduced viral set points in SIV infected infant macaques
Authors: Wood Matthew P , Lippy Adriana , Fisher Bridget , Fuller Deborah , Sodora Donald
Published: January 2023

With 150,000 new infections in 2015, HIV continues to infect infants at an unacceptably high rate, partly due to a lack of an effective HIV vaccine. To better understand infant immune factors contributing to oral HIV transmission and disease in infants, we monitored several markers of peripheral immune cell activation in newborn infant Rhesus macaques. Infants in this study were orally challenged weekly until infected with SIVmac251. From 4–9 weeks post infection, macaques diverged into two distinct groups designated as High Viremic (HV) (7.2×107 – 3.9×108vRNA copies/mL plasma, n=4) and Low Viremic (LV) (3.8×105 – 6.2×106vRNA copies/mL plasma,, n=4) based on viral set points. Markers of immune activation on B cells, monocytes, T cells, and dendritic cells in blood were assessed by flow cytometry through 10 weeks post-infection. At 10 weeks post infection we observed significantly higher (p=0.015) inflammatory (CD14+, CD16+) monocytes in HV macaques, which has been observed in the literature in adult macaques. Unexpectedly, we also observed striking differences between these two groups of infants regarding the activation markers CD11c+ and CD80+ on B cells. LV infant macaques had significantly higher CD11c+ (p=0.036) and CD80+ (p=0.009) B cells by 5–6 weeks post infection compared to HVs (26 fold LV vs 13.2 fold HV at 10 weeks). These findings suggest that activation of B cells may contribute to reduced viral loads in these infants, or alternatively that high levels of SIV can suppress activation of B cells. Experiments are currently underway to explore B cell/antibody mediated viral control in LV infants as these findings have implications regarding development of SIV/HIV vaccines in infants.
The role of Id3 in regulating NKT cells of the adipose tissue
Authors: Buechel Heather Marie , Piccirillo Ann , D’Cruz Louise M
Published: January 2023

Disorders involving obesity, such as diabetes, heart disease, and metabolic syndrome have become a predominant health issues in the United States and around the world. According to the CDC 36% of adults in the US qualify as obese, leading to a variety of negative health outcomes and billions of dollars in medical costs.

One of the hallmarks of obesity is inflammation of the adipose tissue mediated by a combination of molecules such as hormones and cytokines produced by immune cells. Natural killer T (NKT) cells are a heterogeneous population of innate lymphocytes, defined in part, by their ability to quickly release cytokines after activation. NKT cells have been implicated in the maintenance of healthy adipose tissue, and loss of NKT cells in obesity has been correlated with the increased inflammation, glucose and insulin intolerance.

As a heterogeneous population, the exact manner by which adipose resident NKT cells modulate the adipose tissue is contentious. We hope to further elucidate NKT cells’ role in adipose tissue, after activation. We also hope to define the molecules responsible for mediating NKT cells’ activity in a healthy adipose environment, with the goal of modulating these cells to improve health outcomes in the disease state.

Our first molecule of interest is the inhibitor of DNA binding protein, ID3, which has been previously implicated in NKT cell development in the thymus. Here we use both a GFP reporter and a conditionally deficient line to determine the role that Id3 plays in NKT cell activation in healthy adipose tissue.
AP-1 transcription factor reprograms T cell epigenome during activation
Authors: Barski Artem
Published: January 2023

Upon encountering an antigen, naïve T helper (Th) cells are activated, differentiate into several lineages and contribute to immune response. Epigenetic changes at several cytokine and other effector genes during activation has been described previously, but the mechanism behind these changes is not understood. Here, we have profiled open chromatin during human T cell activation using ATAC-Seq approach and identified the regions where epigenetic changes take place. T cell activation requires antigen signaling via T cell receptor and co-stimulation via CD28 which in turn result in nuclear translocation of NFAT and AP-1 transcription factors. For this reason, we profiled genome-wide distribution of these proteins during activation by ChIP-Seq. Our results show that 73% of activation-induced open chromatin loci contain binding sites for AP-1, whereas 39% of them bind NFAT. Over 90 % of the binding sites for NFAT were shared with AP-1. We and others have previously shown that the lack of co-stimulation results in reduction or delay of AP-1 translocation into the nucleus. In order to confirm the role of AP-1 in establishment of the open chromatin structure during activation we mapped open chromatin in the cells that were activated without co-stimulation. Our data showed that ATAC signal was reduced at the open chromatin sites bound by AP-1 in the absence of co-stimulation, suggesting that CD28 signaling and AP-1 transcription factor are involved in open chromatin formation during T cell activation.
Macrophage Persistence in Adipose Tissue of Rhesus Macaques
Authors: Fahlberg Marissa , Alvarez Xavier , Midkiff Cecily , Didier Elizabeth , Kuroda Marcelo J
Published: January 2023

BACKGROUND

HIV/SIV establishes lifelong infection by integrating proviral DNA into the genome of susceptible long-lived immune cells. In order to eliminate the virus, cellular reservoirs must be removed. Long-lived macrophages are permissible to SIV infection and replication. Given recent publications describing adipose as a tissue reservoir for HIV in HAART-treated patients, we chose to investigate the abundance of macrophages in adipose tissue and the proportion that are long-lived to determine their potential as viral reservoirs.

METHODS

Mesenteric adipose tissue from three RM infected with SIVmac239 intravenously and three uninfected RM were analyzed by confocal microscopy to determine frequency of CD163+ macrophages among nucleated cells. An additional two macaques (one SIVmac239-infected, one uninfected) were injected with dextran intravenously, an inert molecule which can be phagocytosed by macrophages and detected at later time points. Tissues were analyzed by IHC and flow cytometry.

RESULTS

CD163+ macrophages constituted 10.5% ± 2.0 of all nucleated cells in the adipose of three uninfected macaques, which differed significantly from infected macaques where macrophages made up 22.0% ± 3.1 of all nucleated cells. We detected dextran in cells within adipose tissue from both injected rhesus macaques at the time of death. The time points at which they died were 43 days (uninfected animal) and 50 days (infected animal) post-injection. Macrophages constitute a significant proportion of nucleated cells in adipose tissue of both uninfected and infected macaques, and their incorporation of dextran is indicative of a long-lived tissue-resident macrophage subtype with potential to be a reservoir for HIV/SIV.
Do T cells from HIV-infected individuals have normal metabolic function?
Authors: Clutton Genevieve Tyndale , Council Olivia , Xu Yinyan , Warren Joanna , Lee De’Ashia , Fernandez Maria Abad , Archin Nancie , Kuruc JoAnn , Eron Joseph , Gay Cindy , Margolis David , Goonetilleke Nilu
Published: January 2023

Untreated HIV infection is characterized by generalized immune activation, T cell dysfunction, and ultimately the exhaustion of HIV-specific CD8 T cell responses. Durable virus suppression resulting from anti-retroviral therapy (ART) is associated with greatly improved immune function, but some phenotypic and functional abnormalities persist even after years of suppressive ART. We have observed that, relative to HIV-seronegative individuals, CD8 T cells from HIV+ participants on ART are skewed toward a more effector-like (T-bethigh Eomeslow) phenotype and have significantly reduced proliferative capacity in response to antigenic stimulation.

Metabolic pathways play a key role in shaping T cell maturation, phenotype, and function, and the ability to produce ATP via both glycolytic and oxidative phosphorylation (mitochondrial) pathways is critical to T cell function and longevity. However, whether underlying metabolic abnormalities contribute to residual CD8 T cell dysfunction in HIV+ individuals on ART has not been fully explored. Here we compare the metabolic phenotype of T cells from HIV+ participants on ART with those from HIV-seronegative participants. We are using MitoTracker® dyes to assess mitochondrial mass and polarization in T cell memory subsets, ex vivo and in response to antigen, and measuring expression of the glucose transporter GLUT1 and the mitochondrial biogenesis master regulator PGC-1α in different CD8 T cell effector subsets. Our findings will inform whether phenotypic and functional abnormalities in CD8 T cells from HIV+ individuals on ART reflect underlying metabolic dysfunction and if metabolic interventions could improve T cell function.
CD8+ T cells from healthy blood bank donors secrete antiviral levels of interferon-alpha
Authors: Killian M. Scott , Wegehaupt Abby , Roufs Ellen
Published: January 2023

Background

CD8+ T cells play important roles in the antiviral immune response to HIV-1 infection. Variation in the antiviral activities of CD8+ T cells among HIV-1-infected persons has been associated with disease state and clinical prognosis. However, the anti-HIV activities of CD8+ T cells from healthy uninfected blood donors have not been fully established.

Methods

In this study we assessed the ability of peripheral blood CD8+ T cells from healthy blood bank donors to inhibit HIV-1-replication. Co-culture assays were performed with CD8+ T cells and acutely HIV-1-infected primary CD4+ T cells. Also, CD8+ T cells were cultured and the derivative supernatants were evaluated in virus inhibition assays, ELISAs, and multiplex cytokine arrays.

Results

Variation was observed among CD8+ T cells from different blood donors in the ability to suppress HIV-1 replication. In the absence of stimulation, the CD8+ T cells, or their derivative fluids, lacked any appreciable anti-HIV activity. After PHA or anti-CD3/CD28 stimulation, the majority of CD8+ T cells and derivative fluids exhibited anti-HIV activity. Low levels of interferon-alpha were present in the CD8+ T cell fluids having increased anti-HIV activity, whereas CD8+ T cell fluids lacking anti-HIV activity contained undetectable levels of interferon-alpha. Other soluble factors present at increased levels in CD8+ T cell fluids having anti-HIV activity included TNF-beta, IL-8, IP-10, and TRAIL.

Conclusions

Our findings indicate that the secretion of antiviral levels of interferon-alpha is a normal function of CD8+ T cells in response to activation and stimulation. Variation in this immune response may influence host susceptibility to infection and disease.
Determining the effects of CCL21 nanomedicine therapeutic modalities in cancer
Authors: Poelaert Brittany J , Romanova Svetlana , Smits Kaitlin , Sliker Bailee , Moffitt Alexandra , Smith Lynette , Bronich Tatiana K , Solheim Joyce
Published: January 2023

Solid tumors demonstrate varying levels of immunogenic potential as well as the proficiency to undergo immune evasion, thus there is a need for novel therapies capable of augmenting the patient’s anti-tumor immune response in a concentrated and durable manner. We hypothesized that therapeutic modalities utilizing the chemoattractant C-C motif chemokine ligand 21 (CCL21) in a novel alginate nanoformulation will provide prolonged release of CCL21 and subsequent steady infiltration of immune cells into the tumor, which will abrogate disease progression and delay tumor growth significantly better than CCL21 alone. Nanoformulations of CCL21 were first analyzed for rates of release in vitro. We found that intratumoral administration of nanoformulation CCL21 significantly prolonged the survival of A/J mice with subcutaneous neuroblastoma tumors, compared to controls. Furthermore, we also determined that the tumor growth rate for nanoformulation CCL21-treated mice was significantly lower in comparison to the tumor growth rates of mice treated with buffer control, empty nanoparticles, or CCL21 alone. Notably, four out of nine nanoformulation CCL21-treated mice demonstrated complete tumor regression. When rechallenged, these four mice failed to produce tumors within a two-week period, therefore suggesting that a protective memory immune response had been induced by the nanoformulation CCL21 treatment. These results indicate that nanoformulation CCL21 therapy may be clinically relevant for the treatment of children and adolescents diagnosed with neuroblastoma. In addition, this novel nanoformulation may also have potential for future development as a means for slow-release delivery of other immunotherapies besides CCL21.
Leukemic cell expressed CTLA-4 suppresses T cells via down-modulation of CD80 by trans-endocytosis
Authors: Do Priscilla , Beckwith Kyle A , Beaver Larry , Griffin Brittany G , Mo Xiaokui , Liu Yang , Jones Jeffrey A , Hertlein Erin , Muthusamy Natarajan , Byrd John C
Published: January 2023

Despite clinical utility in targeting the immune checkpoint, Cytotoxic T Lymphocyte Antigen 4 (CTLA-4), on T cells, its function on non-T cells remains unaddressed, especially in the context of therapy. We define an immunosuppressive role for tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). Most primary CLL samples were intracellularly CTLA-4+ (12/14) but surface CTLA-4− (14/14). Co-culture with activated T cells induced surface expression of CTLA-4 on CLL B cells. To mechanistically study CLL B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL cell lines to inducibly express CTLA-4. These cell lines express the ligands for CTLA-4, CD80 and CD86. Expression of CTLA-4 on Mec1 and OSU-CLL resulted in decreased CD80 expression on CD80+ cells with rescue upon CTLA-4 blockade (N=3). Co-culture of CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with CD80-GFP+ Hek293 cells revealed transfer of CD80-GFP into primary CLL cells (N=2) and the Mec1 cell line. This finding was consistent with the ability of CTLA-4+ T cells to trans-endocytose CD80. Co-culture of T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production (N=3, p=0.0172). The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. We generated an in vivo model to study tumor CTLA-4 using the TCL1 murine model of CLL. Murine CTLA-4+ (mCTLA-4) TCL1 tumor cells were engrafted into human CTLA-4+/+mFcγR−/− immune competent mice. MCTLA-4 on the tumor cells but not human CTLA-4 on the host cells was blocked with an anti-mCTLA-4 antibody. Effect on disease progression and overall survival are currently being assessed. We present a paradigm shift whereby CTLA-4 is an immunosuppressive protein irrespective of expression on T cells.
Anti-viral immune responses within the tumor microenvironment decrease tumor progression
Authors: Chesson Charles Brent , Newman Jenna H , Dobias Natalie S , Sadimin Evita , Schenkel Jason M , Zloza Andrew
Published: January 2023

We recently reported that acute concomitant infection in a tissue distant from the tumor results in the trafficking of tumor-specific CD8+ T cells from the tumor microenvironment to the infection site and to accelerated tumor growth and host death. However, we hypothesized that anti-viral responses within the tumor (rather than distant from it), possibly even without productive viral replication, can reprogram the local tumor microenvironment and lead to long-term tumor regression. Indeed, we discovered in a mouse model of melanoma (B16-F10), that injection of live influenza (A/H1N1/PR8), heat-inactivated influenza, or live vesicular stomatis virus (VSV) results in no observable mordibity or mortality, but significantly inhibits tumor growth (with 60% of the mice surviving more than 50 days). We observed similar results in a 4T1 breast cancer model where, likewise, such virus-associated treatments significantly decreased the tumor area compared to vehicle-treated controls. Interestingly, prior influenza exposure completely abrogated the anti-tumor response after local re-exposure, suggesting that primary anti-viral responses, but not secondary immune responses, at the tumor site may be a novel type of immunotherapy. This finding may explain why infections in cancer patients have been reported to both decrease and increase cancer-specific mortality. Future studies will seek to determine if combination immunotherapies utilizing anti-viral immune responses may augment tumor regression and whether specific viruses are favored based on the cancer type. The overall goal of our work is to translate these findings into clinical trials and cancer patient treatment regimens that improve and save cancer patient lives.
Human gingiva-derived mesenchymal stem cells modulate the fate of macrophages and alleviate atherosclerosis
Authors: Wang Julie , Zhang Ximei , Zheng Song Guo
Published: January 2023

Atherosclerosis is the major cause of cardiovascular diseases. Current evidence indicates that macrophages and inflammation are involved in the pathogenesis of atherosclerosis. Human gingiva-derived mesenchymal stem cells(GMSCs) have displayed anti-inflammatory and immunomodulatory effects on autoimmune and inflammatory diseases. However, whether GMSCs can modulate macrophages and eventually prevent and treat atherosclerosis is far from clear. Here, we show that GMSCs markedly inhibited the foam cell formation and the inflammatory macrophages activation, converting inflammatory macrophages to anti-inflammatory macrophages in vitro. Additionally, infusion of GMSC to AopE−/− mice significantly reduced the frequency of inflammatory monocytes and decreased the size of plaque and lipid deposition. Thus, our study has revealed an unexpected role of GMSCs in modulating the function of macrophages and alleviating atherosclerosis. Transplantation of GMSCs to atherosclerotic patients might be a new therapeutic strategy to improve atherosclerosis.
Using transgenic MHC class II/self-peptide reactive cytotoxic T cells as a method of ameliorating established EAE
Authors: Lees Jason R , Nurmukhambetova Saule , Kim Yong Chan , Zhang Ai-Hong , Blanchfield Jennifer Lori , Evavold Brian D , Scott David W.
Published: January 2023

In multiple sclerosis (MS) CD4+ T cells are thought to coordinate initiation and maintenance of neuroinflammation following recognition of central nervous system (CNS) antigens in the context of MHC class II molecules. Re-activation of these CNS antigen specific CD4+ T cells within the CNS is thought to be crucial to recruitment of secondary immune effector cells and resultant tissue inflammation. MHC class II is thought to be expressed on professional antigen presenting cells (APC) but not on oligodendrocytes and neurons, two cellular targets for MS pathology. Here we examine the use of defined MHC class II/peptide restricted transgenic T cell receptor expression in mature CD8 T cells to generate cytotoxic cells specific for CNS antigen presenting APC. We suggest that auto-antigen/MHC class II specific APC elimination can ameliorate disease while sparing MHC II negative neurons and oligodendrocytes. This study examines a novel potential therapeutic modality for its efficacy in neuroinflammation.
Erdr1 is a potential therapeutic molecule in inflammatory skin disease, psoriasis
Authors: Houh Youn Kyung , Kim Kyung Eun , Cho Daeho
Published: January 2023

Psoriasis is a common skin disease accompanied by chronic inflammation. Pro-inflammatory cytokines are increased in psoriatic lesional skin and have important roles in psoriasis pathogenesis. Especially, as a representative pro-inflammatory cytokine, IL-18 has been known as a biomarker for psoriasis activity. In previous studies, erythroid differentiation regulator 1 (Erdr1) was shown to have a negative correlation with IL-18 in several inflammatory skin diseases such as melanoma and rosacea, suggesting the opposite effects with IL-18. The present study investigated whether Erdr1 in negatively regulated by IL-18 in human keratinocytes in vitro and the level of cutaneous expression of Erdr1 was compared in skin tissues from psoriasis patients and healthy donors. Erdr1 expression was significantly reduced in psoriatic lesional skin whereas enhanced IL-18 expression. Based on the negative correlation, It was hypothesized that Erdr1 may exert anti-inflammatory effects in contrast to the pro-inflammatory effect of IL-18. To investigate the anti-inflammatory effects of Erdr1 in psoriasis, recombinant Erdr1 was injected intraperitoneally into a psoriasis mouse model. Recombinant Erdr1 (rErdr1) significantly alleviated the symptoms of psoriasis-like skin inflammation and reduced the mRNA of various psoriasis-related markers. Additionally, rErdr1 inhibits Th17 cell distribution in psoriatic lesional skin through regulation of CCL20 expression. Taken together, this results provides the first clinical evidence that Erdr1 is downregulated in lesional skin and acts as a novel regulator for psoriasis pathogenesis which can be a potential therapeutic molecule.
The impact of using web conferencing system on student learning, engagement and performance in Immunology Course
Authors: Hussein Mohamed , Reda Betlhem , Bizuayehu-Mekonnen Menorwork , Fazal Nadeem
Published: January 2023

The increasing complexity of pharmacy curricula has been a continuing challenge for both students and faculty. The technology learning platforms currently used in Chicago State University College of Pharmacy are Moodle, Livetext and ExamSoft. The aim of this study was to develop and investigate certain tools instituted by Moodle. We wanted to study the impact of using podcasts on student learning, engagement and performance in Immunology. Our focus was on assessing the full functionality of Moodle features and study its effects in improving learning outcomes, classroom performance and student involvement. Our objective was to explore Moodle-based web conferencing system called BigBlueButton (BBB) in learning immunology. The study was an observational, cohort study with 86 students who were enrolled in Microbiology/Immunology offered to P-1 students at Chicago State University College of Pharmacy. BBB is a web conferencing system, which allows for distant lecture delivery and stores audio/visual content into Moodle. Delivery time was set up according to lecture time. Recordings were automatically uploaded into the course in Moodle within 30 minutes. BBB module was available at no charge on the Moodle service to supplement lecture information. Students have the ability to review recorded lectures at any time, anywhere and on any device with satisfactory picture and sound quality. Recorded podcasts can remain on the Moodle course for a year, and are easily transferable to an upcoming lecture course. Our data shows a positive correlation between the BBB videos watched by the students and their performance. Further studies may need to be done to assess the duration of time spent by students outside of the classroom in reviewing the podcasts.
Fc-fusion drugs have C1q/FcγR binding and signaling properties that can affect their immunogenicity.
Authors: Lagasse H.A. Daniel , Levin Ditza , Hengel Hartmut , Golding Basil , Sauna Zuben E.
Published: January 2023

Fusing the IgG1 constant region (Fc-domain) to therapeutic proteins increases their plasma half-life via neonatal Fc receptor (FcRn) binding. However, potential interactions with other Fc-binding molecules including complement C1q and Fc gamma receptors (FcγR) can have immunological consequences. Our previous studies in a mouse model suggest that engagement of the Fc-Factor IX (FIX) to FcγR on APC and NKT cells could alter the immunogenicity of the FIX moiety. Here, we conducted a comparative study of FDA-approved Fc-fusion proteins to assess their potential for C1q binding as well as FcγR binding and signaling. Binding of soluble drug to C1q was measured by ELISA, and FcγR binding and signaling was evaluated using BW5147:FcγR-ζ reporter cell lines. We demonstrate that rFIXFc and rFVIIIFc bound C1q as well as activating and inhibitory FcγRs (I, IIA, IIB, IIIA). These therapeutic proteins also signaled via FcγRIIIA, and to a lesser extent via FcγRI and FcγRIIB. Other Fc-fusion drugs (TNFR-Fc; CTLA4-Fc) bound FcγRI (TNFR-Fc also bound FcγRIIIA), but did not result in FcγR signaling. As predicted, a control anti-CD20 monoclonal antibody modified for enhanced FcγRIIIA engagement, bound and signaled via FcγRIIIA, while the parent molecule did not; supporting our use of BW5147: FcγR-ζ reporter cells for the assessment of Fc:FcγR interactions. Our studies show that Fc-fusion drugs have distinct C1q/FcγR binding and signaling properties. Moreover, FcγR binding does not predict signaling. Our studies suggest that Fc-FcγR engagement can influence the immunogenicity to Fc-fusion drugs as both fusion partners influence this interaction. Future investigations will elucidate the underlying molecular mechanisms.
‘Arid5a expression is induced through MYD88-independent pathway in response to TLR4 stimulation in IFNγ-sensitized human macrophages.’
Authors: Metwally Hozaifa , Tanaka Toshio , Kishimoto Tadamitsu
Published: January 2023

Sepsis is caused by infection, especially gram-negative bacteria, followed by an inflammatory cascade leading to cytokine storm. High IL-6 serum level in patients with sepsis is correlated with severity and high mortality. However, the exact pathological mechanism(s) in sepsis regulating high IL-6 expression and how Arid5a, a post-transcriptional regulator of IL-6, is related to exaggerated IL-6 expression are not fully understood yet. We found that Arid5a and IL-6 are more markedly expressed in response to Toll-like receptor 4 (TLR4) than TLR2 stimulation in both human primary monocyte-derived macrophages (MDM) as well as human macrophage cell line (THP-1 cells). TLR4 signals through the cell surface as well as the endosomal compartment activating MYD88-indpendent pathway. Pre-treatment of THP-1 cells with endocytosis inhibitor (MiTMAB) significantly decreases the levels of Arid5a, IL-6 and IFNβ, whereas TNFα and IL-8 are marginally affected. Moreover, MYD88 gene-knockout THP-1 cells showed similar expression level of Arid5a compared to the wild type cells. Furthermore, IFNγ pre-treatment of THP-1 cells as well as MDM results in enhancement of TLR4 endocytosis, hence higher expression of Arid5a, IL-6 and IFNβ upon LPS stimulation, while IFNγ pre-treatment has little effect on Arid5a and IL-6 expression upon TLR2 stimulation. On the contrary, IL-4 pre-treatment results in an opposite effect on TLR4 endocytosis. These results taken together indicate that priming of macrophages with IFNγ or IL-4 determines the responsiveness to LPS and that Arid5a expression is regulated by MYD88-independent pathway.
IQGAP1 inhibits type I interferon production via interactions with NLRC3
Authors: Davis Beckley K , Roberts David Michael , Tocker Aaron M , Troiani Zachary , Jacob Kimberly , Trieschman Kate , Durocher Emily , Rechnitzer Alma , Talento Suzanna Marie
Published: January 2023

Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 is not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen detection. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein, in particular, IQGAP1, acts as a scaffold important in regulating the cytoskeleton, cell adhesion and proliferation. Structure function analysis has localized the domains necessary and sufficient for interacting with NLRC3. Confocal microscopy demonstrates that these two proteins co-localize in transformed human epithelial cells. Functionally, in the absence of IQGAP1, human monocytic cells are hyperactive in response to cytosolic nucleotides, phenocopying NLRC3 deficiencies. These data suggest that NLRC3 can interact with novel proteins to facilitate squelching of cellular responses to cytosolic nucleotides.
Involvement of IKKα in STAT1 phosphorylation in antiviral signaling
Authors: Matsumiya Tomoh , Xing Fei , Shiba Yuko , Hayakari Ryo , Yoshida Hidemi , Imaizumi Tadaatsu
Published: January 2023

RIG-I-like receptors (RLRs) sense non-self RNA, inducing type I IFNs. Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of STAT1. We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IKKα is involved in antiviral signaling induced by dsRNA in the RLR-mediated antiviral signaling; however, its role remains uncertain. In this study, we investigated the role of IKKα in RLR signaling, which is essential for antiviral innate immune responses in non-professional antigen-presenting cells, such as epithelial cells. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation in response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of IRF3 or activation of NFκB. These results suggest a non-canonical role of IKKα in RLR signaling. In addition, STAT1 phosphorylation was inhibited by IKKα knockdown in IFNAR-deficient cells. These results suggest that the dual regulation of STAT1 by IKKα in the RLR-mediated antiviral signaling.
Structural characterization of TLRs with novel agonists
Authors: Su Lijing , WANG YING , Morin Matthew D. , Jones Brian T. , Whitby Landon R. , Surakattula Murali M.R.P. , Huang Hua , Shi Hexin , Choi Jin Huk , Wang Kuan-wen , Moresco Eva Marie Y , Berger Michael , Zhan Xiaoming , Beutler Bruce , Boger Dale , Zhang Hong
Published: January 2023

Small molecule TLR4 and TLR2 agonists have been reported, but no structural data reveals their activation mechanism and detailed interactions with the TLRs. We have developed two small molecule agonists, Neoseptin-3 and Diprovocim, that activate TLR4/MD-2 and TLR1/TLR2 complexes, respectively, with exquisitely specific structure activity relationships. These two molecules bear no structural similarity to the natural ligands, lipopolysaccharide (LPS) and tri-acylated lipopeptide (Pam3CSK4). The crystal structures of Neoseptin-3 in complex with mouse TLR4/MD-2 and Diprovocim in complex with human TLR2 provide the first glimpse of how these TLRs bind to unconventional agonists, revealing unique and unexpected binding modes. Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, and induces an active receptor complex (a dimer of TLR4/MD-2) similar to that induced by lipid A. However, Neoseptin-3 and lipid A form different molecular contacts with TLR4/MD-2 to achieve receptor activation. Diprovocim forms a symmetrical dimer and interacts with the same hydrophobic pocket of TLR2 as Pam3CSK4, inducing homodimerization of TLR2 that has a different conformation than the active TLR1/TLR2 heterodimer. Diprovocim binds to TLR2 through an extensive intermolecular hydrogen bonding network that is not observed in the Pam3CSK4/TLR2/TLR1 structure. These two structures are now guiding us in optimization of TLR4/MD-2 and TLR1/TLR2 agonists and antagonists for clinical applications.
PKC driven DC differentiation utilizes the NFκB and ERK signaling pathways
Authors: Chavel Colin A , Lee Kelvin P
Published: January 2023

A key activator of adaptive immune responses is the dendritic cell (DC), which is a professional antigen presenting cell that has multiple roles in immunity from combating infections to recognizing tumor cells in order to activate T-cell mediated immune responses. Our lab has previously shown that the PKC isoform PKCβII is required for DC differentiation and that tumor derived factors can inhibit PKCβII expression, which in turn inhibits DC differentiation. However, how PKCβII induces DC differentiation and the pathways activated downstream of PKCβII remains to be characterized. In order to study this, we used the K562 cell line, which our lab and others have shown to be capable of differentiating into DCs with the addition of PMA, a known chemical activator of PKCβII. We have shown previously that PMA specifically activates PKCβII in K562 cells and not the other conventional PKC kinases. We found that both the ERK and NFκB pathways are activated by PKCβII in K562 cells. Chemical inhibition of either pathway yielded DCs with a reduced capacity to stimulate T-cell proliferation. Both the canonical and non-canonical NFκB pathways are activated by PKCβII and knockdown of PKCβII shows inhibition of both pathways. The canonical NFκB pathway is activated in an atypical fashion that has previously not been shown for PKCβII. PKC signaling also induced the activity of specific components of the ERK signaling cascade, including the modulation of downstream ERK effectors. Active Ras levels increased with PKC signaling, indicating GEF induced activity not previously associated with PKCβII. Taken together, these results show that PKCβII utilizes the NFκB and ERK signaling pathways and initiates these pathways in a novel manner to induce DC differentiation.
Expression pattern of TRIM genes in bovine macrophages stimulated with PAMPs
Authors: Toka Felix N , Dunaway Kiera , Mielcarska Matylda , Smaltz Felicia , Bossowska-Nowicka Magdalena
Published: January 2023

In mammals innate immune mechanisms form the first line of defence against invading pathogens. Detection of viruses early in infection relies on intracellular receptors that sense microbial molecular patterns, subsequently leading to gene transcription that eventually produces IFN type I and II. Type I and II IFNs act to prevent replication of viruses. Tripartite Motif-containing (TRIM) proteins belong to a superfamily of RING-domain E3 ubiquitin ligases. They represent a novel class of antiviral molecules involved in innate immunity. These enzymes function in a wide variety of important cellular processes, particularly in innate antiviral response mechanisms. We studied the expression profile of 46 TRIM genes in a bovine macrophage cell line BoMac using RT2 PCR. PAMPs were used to imitate 2 important groups of pathogens. PolyI:C was used in place of viral dsRNA, LPS was used in place of Gram-negative bacteria, Pam3CSK4 was used in place of Gram-positive and negative bacteria, PolyI:C-LyoVec was used in place of viral sRNA and CpG was used in place of bacterial and viral DNA. Of the 46 TRIM genes, only 8 bovine TRIM genes were upregulated following stimulation of BoMac with individual PAMPs. PolyI:C induce upregulation of TRIM21, TRIM25 and TRIM56. All three TRIMs are known for their antiviral activity in human cell lines and mice. Pam3CSK4 and LPS, both upregulated TRIM10. PolyI:C-LyoVec upregulated TRIM9, TRIM40 and TRIM55. TRIM40 is an anti-inflammatory molecule. CpG upregulated TRIM40 and TRIM29. High expression of TRIM29 is regarded as a poor prognostic in many tumors, but no antimicrobial role has been described yet for TRIM29. Data will be discussed in the context of antiviral role of TRIMs in bovine viral infections.
IL-21 maintains CD62L expression during NKT-cell ex vivo expansion and enhances antitumor activity of NKT cell therapy in vivo
Authors: Ngai Ho , Tian Gengwen , Courtney Amy N , Marinova Ekaterina , Huang Wei , Guo Linjie , Metelitsa Leonid S
Published: January 2023

Vα24-invariant Natural Killer T cells (NKTs) have potent antitumor properties and are being developed for cellular immunotherapy of cancer. Such therapy requires extensive ex vivo expansion of NKTs while preserving their longevity and function. In a recent report from our group, CD62L+ subset of NKTs has been shown to have longer persistence in vivo and stronger antitumor activity than CD62L− counterpart. However, the requirements for the preservation of CD62L+ NKTs during ex vivo expansion remain largely unknown. The gene expression analysis comparing CD62L+ and CD62L− NKTs revealed a significantly higher expression level of IL-21R mRNA in the former. FACS analysis confirmed the finding at the cell surface protein level. These led us to hypothesize that IL-21 preferentially supports CD62L+ NKTs. To test this hypothesis, we expanded primary human peripheral blood NKTs using in vitro stimulation with their cognate antigen, α-galactosylceramide. The culture was supplemented with IL-2, IL-21, or both cytokines. We found that in contrast to IL-2, IL-21 alone failed to support NKT-cell expansion. A combination of IL-2 and IL-21 led to similar absolute numbers of NKTs compared with IL-2 alone. However, the proportion of CD62L+ NKT-cell subset was significantly higher in the culture with IL-2/IL-21 combination compared with IL-2 alone after both primary and secondary expansions. Furthermore, after transfer to NSG mice, IL2/IL-21-expanded NKTs persisted significantly longer and had higher therapeutic efficacy in a lymphoma model compared with IL-2-expanded NKTs. Our results suggest that inclusion of IL-21 in the NKT-cell expansion protocol, which currently uses IL-2 alone, would increase antitumor potential of NKT cell therapy.