bluuebunny/crossref_metadata_2025 huggingface dataset:
CD8 T cell exhaustion is dynamically controlled by the chromatin regulator factor BRD4
Authors: Mullins Genevieve N , Green Jarred Michael , Green William David , Milner J Justin
Published: January 2023

In the context of chronic viral infections and cancer, the protective potential of CD8 T lymphocytes is often dramatically blunted and characterized by a functionally exhausted cell state. T cell exhaustion contributes to pathogen persistence and can limit responsiveness to promising immunotherapy strategies. Therefore, defining the signals controlling the development and maintenance of an exhausted T cell state has critical implications for global public health. While it has become apparent that T cell exhaustion is a transcriptionally and epigenetically driven process, critical epigenetic mechanisms mediating T cell exhaustion remain unresolved. To this end, we sought to define how the ‘chromatin reader’ BRD4 controls an exhausted state in a CD8 T cell-intrinsic manner during chronic viral infection. Utilizing inducible deletion mouse models and RNAi, we found that BRD4 is a critical regulator of T cell differentiation and function. BRD4 was essential for driving a terminally exhausted CD8 T cell state. Mechanistically, BRD4 was found to bind near and modulate expression of key genes that influence T cell differentiation and exhaustion. These findings provide insights into the epigenetic underpinnings of T cell exhaustion and have important implications for enhancing immunotherapy efficacy.

Supported by grants
Differential localization and kinetics of antiviral CD8 T cell responses to chronic and acute murine norovirus infections from initiation onwards
Authors: Hardman Blair K , Osborne Lisa C
Published: January 2023

Recent global events have highlighted the need to better understand mucosal antiviral immunity. Murine Norovirus (MNV) is small non-enveloped positive sense RNA virus that can be used as a model of intestinal viral infection. In this work we use two highly related MNV strains with distinct biological behaviors; CR6 establishes chronic colonic infection, while CW3 establishes systemic infection initiated in the small intestine and is cleared by day 8, correlating with the peak of cell-mediated adaptive immunity. Using flow cytometry and quantitative immunofluorescent microscopy (QIM) of transgenic P14 CD8 T cells specific to recombinant CR6gp33 and CW3gp33, we characterized CD8 T cell responses to CR6 and CW3 from initiation to clearance of CW3. Our data indicate that initiation of CD8 T cell division in response to CR6 infection is delayed, less robust, and largely limited to the mesenteric lymph nodes (MLNs) with less splenic division compared to CW3 infection. Further, the biogeography of P14 T cell priming differs, as early T cell expansion was restricted to distinct nodes of the MLN complex. These early differences propagate out to the time of CW3 clearance. By day 8, there is differential localization of P14 cells in the GI tract to the site of highest viral load and a larger systemic population of P14 cells following CW3 infection. Infection of MLN-deficient mice revealed a critical role for MLN surveillance in targeting P14 T cells to the GI tract following CR6 infection while the spleen plays a compensatory role in response to CW3. These results show how distinct infectious locations of a continuous mucosal surface influence the responses of antiviral CD8 T cells without confounding effects of antigenicity.

This work is supported by the Canadian Institutes for Health Research (CIHR) and a UBC 4 Year Fellowship awarded to Blair K Hardman
Early heterogeneity of IL2R□ expression influences the programming of exhausted CD8T cells during chronic infection
Authors: Toumi Ryma , Xiao Hanxi , Deo Rucha , Reed Steven James , Sarkar Surojit , Kalia Vandana
Published: January 2023

IL-2 signaling at the time of CD8 T cell activation is a critical factor dictating effector and memory CD8 T cell differentiation. However, how IL-2 signaling impacts exhausted CD8 T cell fate outcomes remains poorly defined. Here, we address the role of IL-2 signaling in regulating the differentiation of the stem-like and terminally exhausted CD8 T cells. Prolonged expression of the high affinity IL-2 receptor (IL-2Ra, CD25), and resultant increase in IL-2 signaling drives terminal differentiation of effector cells during acute infection. We hypothesized that curtailing this signal would prevent terminal differentiation and promote the formation of less exhausted stem-cell like TCF-1+CD8T cells in chronic antigenic setting. Our studies show that blocking IL-2 signals at the early phase of chronic infection significantly increased the proportions of TCF-1+CD8T cells. Similarly, knocking down Il2ra augmented the development of TCF-1+CD8T cells. The therapeutic success of PD-1/PD-L1 checkpoint blockade immunotherapy (CBI) relies primarily on stem-cell like cells for vigorous expansion and effector responses. Thus, we next investigated how FACS-purified CD25Hi and CD25Lo cells adoptively transferred into infection-matched chronically infected recipients respond to PD-1 CBI. In contrast to CD25Hi cells, CD25Lo cells underwent vigorous proliferation after PD-1 CBI and produced high amount of effector molecules (GzmB, TNF-α and IFN-γ). Collectively, these data highlight the role of IL-2 signaling in the regulation of CD8 T cell exhaustion, which may be therapeutically exploited to optimize treatments of chronic infections and cancers with immune checkpoint inhibitors.

Supported by NIH R21AI154363 NIH R01AI32819
Localization of terminal memory CD8 T cells in the lungs during respiratory infection
Authors: Suarez-Ramirez Jenny E , Chandiran Karthik , Cauley Linda S
Published: January 2023

Cytotoxic CD8 T cells (CTLs) play an important role in recovery from respiratory virus infection and provide protective immunity by targeting epitopes in well-conserved viral proteins that are shared by different strains of the virus. To provide immunity, CTLs migrate to different anatomical compartments in the lungs and destroy infected cells by injecting lytic molecules into the cytoplasm. Several adhesion molecules aid migration to the infected tissues and cytolysis. These molecules include killer lectin like receptor G1 (KLRG1), which is a membrane bound adhesion molecule that is expressed on terminally differentiated CD8 T effector (Teff) cells. Recently, several studies identified some long lived “terminal memory CD8 T cells (TTM)” that express KLRG1. The goal of our study is to determine the location of these TTM cells within the different compartments of the lungs during a respiratory infection. Our previous work showed that SMAD4 regulates several homing receptors on activated CTLs including KLRG1. We used transfer experiments to examine the distribution of WT cells that express SMAD4 and mutant CTLs that lack SMAD4 in the lungs. At 30 days after intranasal infection with Listeria monocytogenes, there were larger numbers of mutant CTLs in the airways (BAL fluid), while WT CTLs were primarily located in the lung tissue. Interestingly, the localization of the WT cells correlated with the expression of KLRG1. We are using in situ imaging to visualize these transferred CTLs during respiratory infection to better understand their role in protective immunity. Intravascular staining will be used to determine whether these CTLs remain in the blood vessels or enter the tissues of the infected recipient mice.

This work was supported by NIH Grant AI123864.
The Protective Role of IL-36/IL-36R Signal in Con A-Induced Acute Hepatitis
Authors: Sun Jiaren , Wang Xiaofang , Soong Lynn , Liang Yuejin
Published: January 2023

The IL-36 family, including IL-36α, IL-36β, IL-36γ, and IL-36 receptor antagonist (IL-36Ra), belongs to the IL-1 super-family. Although IL-36 is known to play a role in autoimmune diseases, it remains unclear as how IL-36 regulates inflammation in the liver. To determine the role of IL-36/IL-36R signaling pathways, we established an acute hepatitis mouse model by intravenous injection of the plant lectin concanavalin A (Con A), and found that the levels of IL-36 were increased in the liver following Con A injection. We also demonstrated that intrahepatic leukocytes, but not hepatocytes, were the main source of IL-36. Moreover, further research verified that infiltrated neutrophils were the main source of IL-36γ in the liver. Using the IL-36R knockout mouse model (H-2b), surprisingly, we found that the absence of IL-36 signals led to aggravated liver injuries, as evidenced by increased mortality, accompanied by elevated serum ALT and AST levels and severe pathological changes in the liver. Further investigations demonstrated that a lack of IL-36 signaling induced intrahepatic activation of CD4+ and CD8+ T lymphocytes and increased the production of inflammatory cytokines. In addition, IL-36R knockout mice had reduced Treg cell functions and chemotaxis in the liver. Together, our results suggest a vital role of IL-36 in regulating T cell function and homeostasis during liver injury and inflammation.

Supported by grants from NIH (R01 EY028773, AI132674, R21 AI153586)
Interferon-gamma controls pathogenic T-helper 17 and B cells in a new Aquaporin-4 induced experimental autoimmune encephalomyelitis
Authors: Arellano Gabriel , Loda Eileah , Chen Yanan , Popko Brian , Balabanov Roumen , Miller Stephen D
Published: January 2023

Background

Neuromyelitis Optica (NMO) is an autoimmune disease of the Central Nervous System (CNS) mediated by Th17 cells and antibody response to the water channel protein Aquaporin 4 (AQP4), leading to chronic damage of the optic nerves and spinal cord. NMO animal models are substandard inducing disease inconsistently and weakly.

Objectives

Characterize a new NMO mouse model, which best represents the human disease in terms of incidence and chronicity of the disease, severity of symptoms and underlying molecular mechanisms.

Methods

C57BL/6 (WT) and IFN-γ receptor knockout (R-KO) mice were immunized subcutaneously with the AQP4201–220 peptide in Complete Freund Adjuvant and treated with pertussis toxin at day 0 and +2. WT were treated weakly with neutralizing anti-IFN-γ antibodies. mRNA expression and flow cytometry analysis of infiltrating CNS cells were performed. CNS histopathology was determined by immunofluorescence. In vivo B-cell depletion was made using anti-CD20 antibodies.

Results

WT mice treated with anti-IFN-γ and RKO mice showed ascendant paralysis starting from the tail to complete hind limb paralysis. Mice showed increased disease incidence and symptoms were more severe and chronic. CNS IL-17 expressing T cells and Th17 associated genes were upregulated in the absence of IFN-γ. CSN histological analysis showed an increased presence of T and B cells in lesion sites, associated with astrocytes and myelin loss. B-cell depletion in AQP4 primed RKO mice, diminished the incidence and severity of the disease confirming B cell pathogenicity.

Conclusions

AQP4 mouse model is strictly regulated by IFN-γ and its signaling, which control the disease progression and severity, by restraining pathogenic Th17 and B cells.

Supported by grant from NIH (R21 Grant 12878760)
Role of type l IFN in lacrimal gland-protective Treg dysfunction in nonobese diabetic mouse model of Sjögren’s disease
Authors: Allred Merri-Grace , Lieberman Scott M
Published: January 2023

Sjogren’s disease is a chronic autoimmune disease of the lacrimal and salivary glands characterized by foci of inflammation in the tissue composed of aggregations of lymphocytes. Male non-obese diabetic (NOD) mice spontaneously develop autoimmunity of the lacrimal glands. Tregs from these mice are dysfunctional. Disruption of the type I interferon (IFN) signaling pathway protects NOD mice from developing lacrimal gland disease. Whether type l IFN signaling drives lacrimal gland protective Treg dysfunction is not known.

Here we investigated the role of type l IFN in modulating Treg phenotype and function. Through adoptive transfer studies comparing cells from wild-type or IFNAR1-deficient donor mice, we found that recipients of IFNAR1-deficient cells had fewer foci of inflammation. IFNAR1 expression was correlated with a population of CD44loCD62Lhi Tregs. Co-adoptive transfer of both WT and IFNAR1-deficient cells determined that this altered Treg phenotype was due to intrinsic type l IFN signaling. In vitro studies suggest that Treg phenotype can be shaped by IFNα in a dose dependent manner. Ongoing in vivo studies will interrogate the impact of IFNAR1 expression on Treg function.

Type l IFN signaling plays a role in shaping Treg phenotype and may antagonize Treg function. These studies will help define the role of type l IFN signaling in early pathogenesis of Sjögren’s disease and may provide insight into other systemic autoimmune diseases.

Supported by grants from NIH (R01EY027731-01A1)
C57BL/6 Mouse Transgenic for Human MeCP2 as a Murine Model of Neuropsychiatric Lupus
Authors: Wu Tianfu , Li Yaxi , Zhang Shu
Published: January 2023

Introduction

Systemic Lupus Erythematosus (SLE) impacts the central nervous system (CNS), leading to severe neurological and psychiatric manifestations known as neuropsychiatric lupus (NPSLE). To dissect the underlying disease mechanism and to discover novel drug targets of NPSLE, a robust animal model of NPSLE is highly desirable.

Methods

C57BL/6 mice transgenic for human MeCP2 (B6.MECP2Tg1) were phenotyped, including autoantibody profiling, analysis of cellularity and activation of splenic immune cells, proteinuria, as well as neuropsychiatric functions.

Results

The protein expression and phosphorylation of MeCP2 were significantly elevated in the brain of a spontaneous lupus mouse model MRL/lpr, compared to normal B6 mice. The B6.MECP2Tg1 transgenic mice exhibited elevated proteinuria, increased proinflammatory cytokines, altered autoantibody profiles, augmented IgG deposition and macrophage infiltration in the kidney, compared to B6 mice. Effector memory T cells, germinal center B cells and plasma B cells were markedly increased in the female B6.MeCP2Tg1 mice. In addition, B6.MECP2Tg1 mice exhibited anxiety and depression-like behavior, as well as cognitive disorders as early as 9 weeks of age. The abnormal behaviors of the B6.MECP2Tg1 mice are associated with the aberrant neurogenesis and inflammation in the hippocampus, immune cell infiltration in the choroid plexus, as well as the disrupted blood-brain barrier (BBB) and activated microglia, as demonstrated by immunofluorescence microscopy.

Conclusion

Collectively, this work demonstrates that B6.MECP2Tg1 mice exhibit lupus-like phenotypes as well as robust CNS dysfunctions, suggesting it may represent a new animal model for NPSLE.
Murine Epidermis Harbors Functionally Distinct Langerhans Cell Subsets
Authors: Dimitrion Peter Michael , Adrianto Indra , Pawlitz Michael , Yao Yi , Yin Congcong , Peng Hongmei , Zhou Li , Mi Qing-Sheng
Published: January 2023

Epidermal Langerhans cells (LCs) derive from embryonic myeloid progenitors at the steady-state and monocyte progenitors under inflammatory conditions. LCs have the capacity to induce both immunity and tolerance in the skin, but how a single population of LCs mediates both these functions has perplexed researchers for decades. We hypothesized that LCs in murine epidermis have functionally heterogenous subpopulations. We employed single-cell RNA sequencing (scRNAseq) and scATACseq to identify transcriptional and epigenetic heterogeneity in LCs during late embryonic development, adult steady-state and inflamed-state. We found three transcriptionally distinct clusters in adult at steady-state: ATF3hi CD207lo (LC1), ATF3lo CD207hi(LC2), and CD207+ cells expressing keratinocyte (KC) genes (kLCs). Ingenuity pathway analysis showed LC1 had downregulated immunostimulatory pathways and LC2 had upregulated immunostimulatory pathways. LCs from ATF3 knockout mice promoted Th1/2/17 immunity in co-culture experiments, confirming the immunotolerant function of LC1s. LC1 and LC2 clusters had corresponding scATACseq clusters but kLCs did not, suggesting that kLCs may acquire cellular material (i.e. mRNA) through interactions with KCs. scRNAseq analyses of E18.5 pre-LCs and 3 weeks post UVC-treatment also identified ATF3hi and ATF3lo clusters, but kLCs were neither present at E18.5 nor after UVC treatment. Overall, our single cell analyses uncover murine epidermal LC subsets with distinct functions during late embryonic development, steady-state and inflamed-state.

Supported by NIH/NIAMS, R01AR078688-01A1
IL-7 Receptor marks entire embryo-derived Mast Cell
Authors: Kobayashi Michihiro , Cornelius Samuel , Valiente Noemi , Cheng Haizi , Yoshimoto Momoko
Published: January 2023

Mast cell (MC) is an unique immune cell displaying wide variety of functions. Recent break through concluded MC generation is mostly provided by embryonic Yolk Sac (YS) and AGM, and post-natal BM HSC does not supply MC (HSC-independent) as our last presentation. Because YS erythro-myeloid progenitor (EMP) provide earliest tissue resident macrophage (brain microglia) and MC together as a common progenitor, and one report shows part of tissue resident macrophages are marked by IL-7 receptor (IL-7R) as an exception of lymphoid essential lineage marking, we hypothesized that MC could be also marked by IL-7R. We tested by utilizing IL-7Rcre/flox-dTom model and found that newborn to young peritoneal cavity (PerC) and skin MCs, surprisingly, exhibited more than 90% dTom positivity (90.8 ± 3.1%). IL-7R protein was not expressed on the mouse PerC, skin MCs and E12.5 Fetal Liver MC progenitors. To confirm functional IL-7R involvement for the MC development, we measured MCs in IL-7Rcre/+(Het) and IL-7Rcre/cre (KO) mice. Despite marked reductions of T/B cell counts, PerC MC count was comparable (WT: 2.4 ± 1.6 vs KO: 4.1 ± 2.3 × 10e4), suggesting IL-7R is temporarily expressed in early EMP stage in an only short period.

We also tested MC differentiation from the adult BM dTom-negative Lin− Sca+ Kit+ (LSK) cells. MC production was comparable between Het and KO and interestingly enough, both WT/KO LSK derived MCs showed least dTom positivity (3.4± 2.5 vs 4.7 ± 3.1 %) whereas more than 70% of macrophages turned dTom positive, suggesting embryonic and adult MC differentiation used different program respectively. Consistently, %dTom in PerC MC was reduced (79.2 ± 8.1 %) in old mice (> 1year), indicating that post-natal de novo MC production is minimum without IL-7R use.

Supported by R01AI121197-01A1 NIAID R01AI147685-01 NIAID
Cell autonomous metabolic remodeling governs regeneration of the thymus
Authors: Kinsella Sinéad , Evandy Cindy Anggelica , Cooper Kirsten , Hopwo Kayla , Iovino Lorenzo , deRoos Paul , Smith Colton , Granadier David , Dudakov Jarrod A
Published: January 2023

Competent T cell development relies on efficient functioning of the thymus, which is extremely sensitive to acute and chronic insults, but has a remarkable ability to regenerate. We identified that cell-specific metabolic remodeling triggers key molecular mechanisms driving this endogenous regeneration process, namely the transcription of Foxn1 by thymic epithelial cells (TECs). Firstly, following acute damage (sub-lethal total body irradiation) murine CD4+CD8+ (DP) thymocytes, which undergo high levels of death, have a disrupted glycolytic flux triggering a switch to OXPHOS, with reduced mitophagy and increased mitochondrial ROS, an effect not observed in TECs, which are relatively damage-resistant, suggesting metabolic stability confers protection. Furthermore, DPs preferentially undergo pyroptotic cell death after damage facilitated by increased pyruvate-induced TCA cycling, evidenced by an acute surge in caspase 1 cleavage compared to caspase 3. Importantly, pyroptotic thymocytes induce Foxn1 transcription in cortical TECs (cTECs) and in vitro screening revealed the purinergic receptors P2Y2 as a critical molecular target. Moreover, we translated this therapeutic strategy in vivo and demonstrated enhanced thymic regeneration after damage in mice treated with a P2Y2 agonist. Together these data describe an innovative mechanism of immunometabolic regulation of T cell development and tissue regeneration and provides a novel therapeutic strategy to enhance immune function.
VEDOLIZUMAB REDUCES DENDRITIC CELLS AND NAÏVE LYMPHOCYTES IN THE COLON MUCOSA
Authors: Lord James D , Kongala Ramya , Juarez Julius
Published: January 2023

Vedolizumab blocks integrin alpha4/beta7 interaction with MAdCAM-1 on intestinal vascular endothelial cells. It is believed to treat inflammatory bowel disease (IBD) by preventing alpha4/beta7+ cells in blood from entering the intestinal mucosa. By CyTOF and flow cytometry, we found high expression of alph4/beta7 on nearly all myeloid dendritic cells in the blood. Flow cytometry was also performed on colon biopsies from 71 IBD patients on vedolizumab matched 1:1 with similar biopsies from 71 matched IBD patients not on vedolizumab. In the colon, vedolizumab use was associated with significantly fewer intramucosal myeloid dendritic cells (CD1c+, CD14−, CD11c+, HLA-DR+, Wilcoxon p=0.000001) in patients responsive to vedolizumab, but not in non-responders, relative to their respective controls. Fewer intramucosal naïve B (IgD+, Wilcoxon p=0.0001) and CD4 T cells (CD45RA+, Wilcoxon p=0.001) were also observed, regardless of treatment efficacy. No differences were observed in the frequency of other T or B cell subsets, including CD8 T cells, effector T cells, plasma cells and memory B cells expressing different immunoglobin isotypes, suggesting that they have an alternative means of recruitment not present among naïve lymphocytes, such as integrin alpha4/beta1, which binds VCAM-1. Thus we find no evidence that that vedolizumab functions by preventing effector lymphocytes from entering the intestinal mucosa. We instead associated vedolizumab use with fewer colonic naïve lymphocyte and myeloid dendritic cells. The latter is of particular interest, as it alone correlates with clinical response to this therapy.

Supported by a collaborative research agreement with Takeda Pharmaceuticals
Peripheral IgE+ plasmablasts as a biomarker of clinical allergy onset and severity
Authors: Larson Elisabeth M , Babasyan Susanna M , Wagner Bettina
Published: January 2023

IgE-mediated allergies depend on the continuous production of allergen-specific IgE to maintain allergic sensitization. While IgE+ plasma cells are the primary source of this IgE, IgE+ plasmablasts can provide a snapshot in peripheral blood of the current response to allergen. We developed a novel approach to characterize IgE+ plasmablasts in peripheral blood and determined the relationship between allergen exposure, IgE+ plasmablasts and clinical disease. We used a unique large animal model of naturally occurring allergy, equine Culicoides hypersensitivity, which allowed all individuals to have similar timing and frequency of allergen exposure and allowed longitudinal collection of samples from the same allergic and healthy individuals over time. We found that peripheral IgE+ plasmablasts spontaneously secrete IgE, express high levels of the IgE receptor CD23 on their surface, and positively correlate with clinical disease severity. In conclusion, IgE+ plasmablasts are critical in the mechanism of allergy through IgE secretion and serve as a quantitative biomarker of allergic disease development and severity.

This work was supported by the Harry M. Zweig Memorial Fund for Equine Research at Cornell University; the USDA/NIFA [#2005-01812, #2015-67015-23072, and #2019-67015-29833]; and National Institutes of Health [#1S10RR025502 through Cornell University’s Biotechnology Resource Center].
Ligation of Siglec-9 inhibits FcɛRI-dependent mediator release from human mast cells
Authors: Molina Irina Miralda , Samanas Nyssa Becker , Piliponsky Adrian Martin
Published: January 2023

Mast cell activation is critical for the development of allergic diseases. Ligation of Sialic acid-binding immunoglobin-like lectins (Siglecs) such as Siglec-7 and 8 has been shown to inhibit mast cell activation and is considered one of the main approaches to reduce mast cell contribution to allergic disease. Recent high throughput studies provided evidence that human mast cells express Siglec-9, an inhibitory receptor that is mainly expressed by innate immune cells such as neutrophils, monocytes and eosinophils. Based on this evidence, we aimed to examine Siglec-9 expression and function in human mast cells. Flow cytometry analysis showed that Siglec-9 is expressed in the human mast cell lines LAD2, LUVA, and HMC-1, and in peripheral blood-derived cultured human mast cells (PBCMCs). The expression of Siglec-9 in PBCMCs peaks at week 5 of culture and correlates positively with the expression of the high affinity receptor for IgE (FcɛRI). Pre-treatment of human mast cells with the Siglec-9 agonists glycophorin A or high molecular weight hyaluronic acid (HMW-HA) followed by FcɛRI-dependent stimulation has an inhibitory effect on mast cell degranulation. SIGLEC9 gene disruption by CRISPR/Cas9 editing resulted in a significant reduction in Siglec-9 expression in LAD2 cells that also became impervious to inhibition by Siglec-9 agonists. Together, our data shows that human mast cells express Siglec-9 and that engaging this inhibitory receptor can reduce mast cell degranulation.
Elucidating the Immunogenic Mechanisms of Merck’s Lipid Nanoparticle (LNP) Adjuvants
Authors: Bass Antonia R , Kimball Sonya , Jenkins Anjelica , Burton Aimee , Trostle Katherine , Corcoran Halea , Allen Meredith , Gallagher Catherine , Harms Jacque , Bett Andrew , Sullivan Nicole
Published: January 2023

Adjuvants are crucial components of subunit vaccines, as they help induce immune responses to poorly immunogenic antigens. Merck has developed a variety of lipid nanoparticles (LNPs) that contain different cationic lipids, a critical component that is mainly responsible for the reactogenicity induced by the LNP. LNP A that incorporates cationic lipid A results in high antigen expression, robust antigen-specific humoral and cellular responses, and some dose dependent reactogenicity when combined with subunit proteins. In contrast, LNP B, which incorporates cationic lipid B which has a shorter half-life than cationic lipid A, results in high antigen expression, slightly lower immunogenicity, and improved tolerability. Previous studies have shown that mouse TLR2 may be involved in the mechanism of action of LNP A. To determine the human innate signaling pathways triggered by LNP A or LNP B, we stimulated human TLR-expressing HEK-Blue cell lines (Invivogen) with the LNPs followed by measuring SEAP activity. We did not see any activation of the human TLRs despite stimulating with high amounts of LNP. Next, we evaluated whether these LNPs generated different cytokine/chemokine profiles, indicative of differential cellular recruitment in BALB/c mice in vivo. We found that LNP A stimulation results in significant upregulation of monocyte and neutrophil-specific cytokines in the serum and quadricep muscle injection site, while LNP B leads to a lower response. Furthermore, we plan to investigate the recruitment of cells at the injection site and draining LNs by flow cytometry and imaging. Together, these experiments will help mechanistically determine how these fundamentally different LNPs can activate innate immune responses.

Supported by Merck & Co.
Engineering iPSC-derived neutrophils to fight infection
Authors: Giese Morgan , Bennin David , Johnson Chad , Klemm Lucas , Yung Ho Sun , Nett Jeniel , Slukvin Igor , Huttenlocher Anna
Published: January 2023

Neutrophils are the initial responders to infection and injury and are critical for host survival. Immunocompromised patients are prone to recurrent bacterial and fungal infections which may require granulocyte transfusion. However, these transfusions have minimal efficacy as donor material is limited and highly heterogeneous. Induced pluripotent stem cells (iPSCs) are a promising option for generating a defined therapeutic that can improve patient outcomes. Therefore, we aimed to engineer iPSC-derived neutrophils to improve recruitment and antimicrobial function. Prior research in the field has shown that iPSC-derived neutrophils have limited function compared to primary human neutrophils, likely due to inhibitory signaling pathways. To generate iPSC-derived neutrophils with enhanced capacity, we utilized CRISPR/Cas9 to delete the protein tyrosine phosphatase 1B (PTP1B), a negative regulator of neutrophil function. PTP1B−/− neutrophils displayed increased cellular migration and inflammatory cytokine signaling in response to bacterial stimuli versus WT cells. Additionally, upon selection of cells with mature neutrophil markers, these PTP1B−/− neutrophils show similar fungal killing ability and improved phagocytosis compared to WT. Thus, these data indicate that deletion of PTP1B can improve the antimicrobial function of iPSC neutrophils and highlights the value of our system for genetically engineering iPSC-derived neutrophils.

Supported by grants from the NIH (5R01AI134749-03 and Microbes in Health and Disease T32 NIAID: T32AI055397)
C-type lectin receptor MGL-1 in SARS-CoV-2 disease pathogenesis
Authors: Huante Matthew B , Gonzalez Orozco Maria J , Naqvi Kubra F , Martinez-Martinez Yazmin Berenice , Menachery Vineet , Endsley Janice J , Rajsbaum Ricardo , Stephens Robin , Endsley Mark
Published: January 2023

Pattern recognition receptors, such as the C-type lectin receptor (CLR) family, play an important role in the generation of the innate immune response against a variety of pathogens. The CLR Macrophage galactose-type lectin (MGL), also known as CLEC10, is one member of this family which we and others found can bind SARS-CoV-2. MGL is known to increase in expression during SARS-CoV-2 infection and, in concert with other CLRs, is associated with elevated proinflammatory immune responses. Dexamethasone, a clinical treatment commonly used to mitigate the hyperinflammatory response in individuals with COVID-19, is also a positive stimulus for MGL expression. We therefore sought to investigate the role of MGL in disease pathogenesis using MGL-1 receptor deficient mice. Mice were intranasally infected with mouse adapted SARS-CoV-2 strain CMA3p20 and euthanized 3- or 7-days post infection. Following acute infection at 3 days, MGL-1 deficiency did not affect the viral burden in the lungs of mice. This was supported by multiplex cytokine and chemokine data which revealed similar acute inflammatory responses in the lung of infected WT and MGL-1 knockout (KO) mice. Interestingly, MGL-1 KO mice did display a significant increase in the lung viral burden when compared to WT mice 7 days post-infection. These results suggest a role for MGL-1 in resolution of SARS-CoV-2 at later stages of infection. Further investigations to determine differences in cellular recruitment and lung pathology in the absence of MGL-1 are warranted. In addition, assessment of a role for the MGL-2 orthologue expressed by murine myeloid cells may identify roles for MGL pathways in SARS-CoV-2 disease severity and pathogenesis.

Supported by UTMB Institute for Human Infection and Immunity COVID-19 Pilot Grant
Innate training induced by subcutaneous BCG administration in pre-weaned calves
Authors: Samuel Beulah Esther Rani , Maina Teresia , McGill Jodi L L
Published: January 2023

Innate immunity, initially thought to lack immunological memory, has been shown to have the capacity to be trained or to mount a heightened immune response towards an unrelated second challenge. The Bacillus Calmette Guerin vaccine, administered to prevent human and bovine tuberculosis, is one treatment that can induce innate immune memory. Here we evaluated the efficacy of BCG-induced innate training on the outcome of a respiratory virus challenge in pre-weaned calves. Groups of calves were given BCG Danish strain subcutaneously (or PBS as control). PBMCs and CD14+ monocytes were re-stimulated in-vitro with E. coli LPS (1μg/mL) or Pam3CSK4 (10μg/mL) at 2- and 4-weeks post-BCG respectively, and pro-inflammatory cytokine production was measured by ELISA. Calves were challenged via aerosol inoculation with BRSV at five weeks post-BCG. PBMCs from BCG calves showed enhanced IL-1b production upon in-vitro stimulation with LPS, and CD14+ monocytes from BCG calves showed increased IL-1b and IL-6 secretion. Alveolar macrophages from BCG calves showed enhanced cytokine production when re-stimulated with LPS. No significant changes were observed in clinical scores or viral burden between groups post BRSV challenge. Subcutaneous BCG administration can train bovine innate immune cells to exhibit a “memory-like” phenotype. Current efforts are focused on the characterization of epigenetic reprogramming in trained innate immune cells.

Supported by grants from USDA, NIFA. R01 HD099104-01
The Cdkn2a gene product p19ARF (alternative reading frame) is a critical regulator of IFNβ-mediated Lyme arthritis
Authors: Li Jinze , Ma Ying , Paquette Jackie K , Richards Amanda C , Mulvey Matthew A , Zachary James F , Teuscher Cory , Weis Janis J
Published: January 2023

Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, with a spectrum of clinical disorders. Infected C3H mice develop severe arthritis while B6 mice display mild arthritis, allowing analysis of host genetic contribution to disease. B6 mice introgressed with the C3H allele of Bbaa1 on Chr4, B6.C3-Bbaa1, display increased severity of arthritis, and heightened expression of Type I IFN. IFNβ, was identified as the key effector for arthritis severity, acting through the muscle regulatory protein myostatin. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing in macrophages. The Cdkn2a gene encoded protein p19ARF was identified as the modulator of IFNβ expression. B6 Arf−/− mice reconstituted with cells expressing the C3H allele of p19ARF developed severe arthritis, whereas mice reconstituted with cells expressing the B6 allele developed mild disease. IFNβ induction by p19ARF is inducible by other pathogens and a variety of PAMPs. P19ARF regulation of IFNβ involved the tumor suppressor p53 and transcription repressor BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Similar responses in B6 Rag1−/− mice, indicate dependence on myeloid cells expression of BCL6. Thus, we have identified novel involvement of p19ARF in modulating IFNβ expression in Lyme arthritis development.

Supported by R01 AR043521
Autocrine prostaglandin feedback governs the kinetics of bacterial induced type I interferon by driving autophagic targeting of the TLR4/TRIF complex.
Authors: Perkins Darren , Schlenker Catherine
Published: January 2023

TLR4 is unique in its capacity to initiate two qualitatively distinct transcriptional programs from two cellular locations. Cell surface TLR4/MyD88 complexes drive pro-inflammatory cytokine transcription through NF-kB, while endosomal TLR4/TRIF complexes activate IRF3 and a type I interferon response. Feedback circuits to limit signals from both locations are necessary to balance the inflammatory response to Gram negative bacteria, yet how endosomal TLR4/TRIF signals are terminated remains an open question. It has been reported previously that engagement of TLR4 triggers activation of autophagic machinery and that macrophages defective in core autophagy genes display selective hyper activation of the TRIF versus MyD88 pathways in response to LPS. Previous studies from our lab have shown that TLR4 activation induces rapid synthesis and secretion of the immunomodulatory host lipid Prostaglandin E2 (PGE2) which selectively diminishes endosomal TLR4/TRIF signaling. In this ongoing study we find that, mechanistically, autocrine PGE2 signals downstream of TLR4 significantly enhance activation of autophagic processes to likely negatively regulate the persistence of endosomal TLR4/TRIF complexes and define the kinetics of TRIF signaling through directed autophagy.

Supported by a grant from the NIH R21AI151352
PD-L1 back signaling manipulates IL-6 signaling to coordinate innate immune responses
Authors: Shirley Jamie L , Forward Tadg , Schafer Johnathon B , Lucas Erin D , Tamburini Beth
Published: January 2023

Programmed death ligand-1 (PD-L1) interaction with PD-1 on T cells limits immune activation to prevent immunopathology in healthy tissues and the development of autoimmunity. While ligation of PD-1 by PD-L1 leads to T cell-intrinsic PD-1 signaling, PD-L1 back signaling into the ligand-bearing cell remains poorly defined. We recently outlined a major role for PD-L1 back signaling in regulating dermal dendritic cell (DC) migration to the lymph node following a type I interferon inducing stimulus or infection. Similar to prior studies in IFNβ treated cancer cells, we noted increased pSTAT3 post DC activation that was unchanged with addition of CCL21. Increased pSTAT3 occurred in Pdl1−/− DCs and in DCs from mice in which 3 cytoplasmic residues were mutated to alanine (Pdl1CM). Since STAT3 propagates signalling through the IL-6 receptor, we hypothesized that increased IL-6 signaling may explain elevated pSTAT3 in Pdl1−/− and Pdl1CM DCs. To test whether PDL1 back signaling contributed to STAT3-associated cytokine responses, we generated bone marrow derived DCs from Pdl1−/− and Pdl1CM mice and observed significantly higher levels of IL-6 compared to WT controls following activation. We next asked if PD-L1 back signaling is important for proper innate activation as PD-L1 is upregulated on many innate immune populations early after systemic L. monocytogenes (LM) infection. We found loss of PD-L1 back signaling following infection led to differences in circulating levels of many innate immune cells and reduced IL-6 in the serum. These findings demonstrate that PD-L1 back signaling in innate immune cells may assist in coordinating IL-6 signaling and proper activation of early innate immune responses during systemic LM infection.

Supported by grants from NIH (R01 AI155474, T32 AI074491 - “Molecular Mechanisms of Immune Tolerance”)
High-throughput Quantification of Antibody-Dependent Phagocytosis using Live-Cell Analysis
Authors: Trigg Jasmine E , Lovell Gillian , Bevan Nicola , Dale Timothy
Published: January 2023

The uptake and clearance of tumor cells can be promoted with monoclonal antibodies (mAbs) via antibody-dependent phagocytosis (ADCP) or through the blockade of “don’t-eat-me” signals, such as CD47. These mechanisms hold immunotherapeutic promise and are studied extensively in drug development.

High-throughput assays were conducted using pHrodo®, a pH-sensitive fluorophore, that enables live-cell imaging and kinetic quantification of phagocytosis. pHrodo® labeled target cells were incubated with mAbs and added to effector cells in 96-well plates. Images were acquired using the Incucyte® Live-Cell Analysis System and fluorescence automatically quantified with integrated software.

In line with known pro-phagocytic effects, anti-CD47 promoted phagocytosis of CCRF-CEM tumor cells by blocking CD47 “don’t-eat-me” signals. Clinical anti-CD20 mAbs Truxima® and Rituximab both promoted ADCP of Ramos target cells by primary macrophages. In addition, the ADCP response of different Rituximab isotypes were compared. Anti-CD20-IgG1 mAb and Fc mutated anti-CD20-IgG1fut (non-fucosylated) exhibited concentration-dependent responses (EC50 values of 25.1 ng/mL and 42.8 ng/mL, respectively), however anti-CD20-IgG1NQ (non-glycosylated) showed no response. ADCP was also examined in adherent target cells with varied HER2 profiles. Anti-HER2 (Trastuzumab) induced an ADCP response in HER2-positive AU565 cells but not in HER2-low MDA-MB-231 cells, consistent with established correlations between HER2 expression and ADCP response.

These data exemplify that live-cell analysis is a powerful approach that enables functional quantification of ADCP and is amenable to antibody screening for therapeutic candidates.
Myeloid cell interleukin-10 is essential for atherosclerosis protection in Apoe−/− mice.
Authors: Orecchioni Marco , Wolf Dennis , Suryawanshi Vasantika , Winkels Holger , Kobiyama Koji , Makings Jeffrey , Kiosses William B , Ley Klaus
Published: January 2023

Atherosclerosis, the disease underlying stroke, myocardial infarction and ischemic heart failure, is initiated by the subendothelial retention of lipoproteins and cholesterol, which then triggers a non-resolving inflammatory process driving plaque progression in the artery wall. Myeloid cells and in particular macrophages are the primary driver of this process. IL10 is a prototypic anti-inflammatory cytokine made by several immune cells such as macrophages. IL10 is also essential for the protection against atherosclerosis. However, it is not known which cells are responsible for the IL10 production mediating protection from atherosclerosis. Here, we crossed the VertX mouse model, in which an IRES-enhanced green fluorescent protein (eGFP) fusion protein was placed downstream of exon 5 of the IL-10 gene, to the atherosclerosis-prone Apoe−/− background. We found that myeloid cells express high levels of IL10 GFP in VertX-Apoe−/− mice. By scRNAseq we found that macrophages especially inflammatory macrophages in plaques are the main producers of IL10 in atherosclerosis. To address whether IL10 secreted by myeloid cells is essential for the protection, we developed a LyzMCre IL-10flox/flox mouse crossed in the Apoe−/− background and confirmed that macrophages were unable to secrete IL-10. We discovered that the LyzMCre IL-10flox/flox Apoe−/− mice developed significantly more atherosclerosis than LyzMCre IL-10+/+ Apoe−/−, both on chow and lipid-rich diet. Flow cytometry data and cytokine measurements suggest that the depletion of IL-10 in myeloid cells increases Th17 cells with elevated IL-17a, IL-17f and CCL20 in blood plasma. These data underscore the critical roles of macrophage-derived IL-10 in limiting atherosclerosis.

Supported by grants NIH HL 115232, 145241, and HL088093.
Hypoxia-inducible factor regulation of NK cell dysfunction in lupus
Authors: Feldman Harrison Alexander
Published: January 2023

Natural Killer (NK) cells are innate antiviral and antitumor effector cells that have emerged as potent regulators of adaptive immune responses. However, NK cells are known to be dysfunctional in many diseases including systemic lupus erythematosus (lupus), where inability to restrain autoreactive T and B cells leads to potentially irreversible organ damage. To determine mechanisms driving NK-cell dysfunction in autoimmunity, we performed transcriptomic analysis of these cells in pediatric patients with lupus. Analysis of the differentially expressed genes revealed a putative gene regulatory node controlled by the heterodimeric pairing of the transcription factors EPAS1 and ARNT that is active in NK cells from patients with lupus, but not controls. Cytokines linked to lupus promote up-regulation of the protein encoded by EPAS1 in NK cells. Small molecule inhibitors of EPAS1 (PT2385) enhanced the capacity of NK cells to kill K562 cells. These results suggest that a hypoxia-related transcriptional program contributes to functional restraint of NK cells in lupus.

Supported by T32GM063483, AR073228, AR47363 (Flow Cytometry Core), DK78392 (Flow Cytometry Core), DK90971 (Flow Cytometry Core)
A critical role of energy sensor AMPKα1 in rapamycin dependent memory CD8+ T cell survival
Authors: Ara Anjuman , Xiang Jim
Published: January 2023

Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa), an inhibitor of mTORC1, supports T-cell memory. However, the underlying pathway, the exact mechanisms, and the role of AMPKα1 in Rapa-induced T-cell memory is not well understood. Using the Listeria monocytogenes (rLmOVA) infection model and genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice post-rLmOVA infection. When T-cells stimulated in vitro in the presence of IL-2 without rapamycin (IL-2/T) or with rapamycin (IL-2+Rapa /T) and transferred into mice, it differentiated into short-term effector T (TE) [IL-7R− CD62L− KLRG1+] and long-lived memory T (TM) [IL-7R+ CD62L+ KLRG1−] cells, respectively. We determined that rapamycin-treated T cells activated transcriptional factors, FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555), and ATG7 molecules and promoted mitochondrial biogenesis and oxidative phosphorylation (OXPHOS). Using Seahorse-real time metabolic analyzer, we found that rapamycin-treated AMPKα-deficient TM cells up-regulated transcription factor HIF-1α and induced a metabolic switch from OXPHOS to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Altogether, our data provide a mechanistic explanation for enhanced T-cell survival after rapamycin treatment and suggest that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis. And AMPKα1 plays a critical role in rapamycin-induced increased survival and metabolic switching of CTLs during the transition of effector to memory T cells.

Supported by Canadian Institutes of Health Research grant (409228). Saskatchewan Health Research Foundation (SHRF) postdoctoral fellowship.
Increased proliferation of epidermal gamma delta T cells and expression of the transmembrane protein, BST2, in Alopecia areata
Authors: Hagan Yosiris , Gonzalez Alex , Kasler Antolette , Macedo Matilde , Rincon Anthony , Jameson Julie
Published: January 2023

Alopecia areata is an autoimmune disease characterized by patchy non-scarring hair loss in affected individuals. Typically, research has focused on hair follicle infiltration and disruption via CD8+ and CD4+ T cells. Our research attempts to elucidate the role of resident epidermal γδ T cells in disease. Analysis of publicly available bioinformatics data shows a statistically significant downregulation in a number of immune-related genes after patients were treated with a JAK inhibitor, Tofacitinib. We have focused on one of these proteins, bone marrow stromal antigen 2 (BST2), in vitro and in vivo using the C3H mouse model of alopecia areata. We have identified populations of epidermal γδ T cells expressing BST2 in the follicle using immunofluorescence microscopy. In addition, BST2 expression is upregulated in unaffected skin from mice with active alopecia areata suggesting BST2 expression may precede loss of hair follicle immune privilege. Furthermore, epidermal γδ T cell number is increased in skin affected by alopecia areata. Together our findings suggest that BST2 is expressed by epidermal T cells prior to hair loss and is followed by an increase in epidermal γδ T cell number. Our lab is focused on how these changes in epidermal γδ T cell number and BST2 expression impacts disease.

Supported by grants from NIH Supported by grants from NSF
The sexually dimorphic histone demethylase promotes optimal T cell responses during infection
Authors: Krawczyk Connie , Zhai Lukai , Guak Hannah , VanderArk Alexander , Davidson Paula , Weiland Matthew , Corrado Mario
Published: January 2023

Females have stronger immune responses than males. This can be a protective benefit for immune responses to infection and cancer, but also a detriment for the development of autoimmune disease. The genetic contributions to sexually dimorphic immunity are not well understood. We have identified the X-linked histone demethylase KDM5C as an important regulator of T cell immunity. KDM5C escapes X inactivation so is expressed more in females than males. KDM5C demethylates H3K4, participating in gene silencing, however also has a role in promoting gene expression. Mice deficient in KDM5C in immune cells have reduced ability to control acute and chronic infection. CD8 and CD4 T cells have reduced expression of T cell effector molecules and increased expression of PD-1. We find that KDM5C promotes optimal T cell responses through epigenetic and metabolic programming. These data implicate KDM5C as an important promoter of T cell immunity and a mechanism underpinning sexual dimorphic immune responses to infection.