bluuebunny/crossref_metadata_2025 huggingface dataset:
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Infection in a Bat Gastrointestinal Organoid Model
Authors: Hashimi Marziah , Sebrell Thomas , Hedges Jodi , Snyder Deann Teresa , Lyon Katrina , Cherne Michelle D , Robison Amanda , Sidar Barkan , Wilking James , Walk Seth , Schountz Tony , Jutila Mark A , Bimczok Diane
Published: January 2023

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19, is thought to have originated in bats, since multiple viruses with a high level of genetic similarity have been identified in bats. In addition to respiratory symptoms, COVID-19 frequently involves gastrointestinal symptoms such as diarrhea and vomiting, indicating the SARS-CoV-2 can target the gastrointestinal (GI) tract. However, there is no robust in vitro model for assessing the SARS-CoV-2 infection in the bat GI tract. Here, we established gastrointestinal organoid cultures from Jamaican fruit bats (JFB), (Artibeus jamaicensis), which replicated the characteristic morphology of the gastrointestinal epithelium and showed tissue specific gene expression patterns and cell differentiation. To analyze whether JFB intestinal epithelial cells are susceptible to SARS-CoV-2, we performed in vitro infection experiments. While increased SARS-CoV-2 RNA was detected in both cell lysates and supernatants from the infected organoids after 48 h, there was no evidence of active viral replication, and no infectious virus was produced. However, the JFB distal organoids significantly upregulated anti-viral and pro-inflammatory genes in response to SARS-CoV-2. Unexpectedly, SARS-CoV-2 infected JFB organoids had a decreased incidence of apoptotic cell death. Collectively, our data suggest that primary intestinal epithelial cells from JFBs are resistant to SARS-CoV-2 infection and cell damage, likely because they are able to mount a strong antiviral interferon response.
Early post-vaccination gene signatures correlate with the magnitude and function of vaccine-induced HIV envelope-specific plasma antibodies in infant rhesus macaques
Authors: Kandiyil Vidya Vijayan Karuvan , Curtis Alan D , Cross Kaitlyn , Van Rompay Koen K A , Pollara Justin , Fox Christopher , Tomai Mark , Hanke Tomas , Fouda Genevieve , Hudgens Michael , Permar Sallie R , De Paris Kristina
Published: January 2023

Systems vaccinology approaches are important tools in rational vaccine design. Our goal was to determine whether early innate immune responses to the vaccine prime in infant rhesus macaques, immunized with two different HIV envelope (Env) vaccine regimens, were associated with functional antibody responses in the memory phase.

We compared plasma cytokine levels and molecular signatures of a 3M-052-SE adjuvanted HIV Env protein vaccine (n=10) to a regimen combining the adjuvanted HIV Env protein and MVA-HIV Env (n=10) at days (D) 0, 1, and 3 post the vaccine prime. Whole blood transcriptional profiling applying NanoString technology was employed to identify differentially expressed genes (DEG). Innate immune responses were correlated with vaccine-induced adaptive immune responses at weeks 14, 20, 32, and 34.

The vaccine prime induced a rapid, but transient, increase in inflammatory plasma cytokines and changes in mRNA expression that peaked at D1. In the HIV protein group, we identified 31 DEG with increased mRNA levels, whereas a single, downregulated, DEG was identified in the MVA-Env plus Protein group. Day one signatures were positively correlated with week 14 Env-specific IgG responses, and, at week 34, with Env-specific follicular T helper cells and Env-specific antibody-dependent cytotoxicity function, but negatively correlated with Env-specific CD8+ T cell responses. A protein-protein interaction network confirmed that several of the DEG-encoded proteins have predicted interaction partners that are important for B cell activation.

These results support the idea that vaccine-induced HIV-specific antibody and T cell responses can be optimized through the modulation of the vaccine prime.

Supported by National Institutes of Health grants R01 DE028146, P01 AI117915, T32 5108303, the Office of Research Infrastructure Programs/OD P510D011107 (CNPRC), and the Center for AIDS Research award P30AI050410
A Lupus-Associated Variant in IRF7 amplifies IFN-α Production in response to TLR stimulation
Authors: Virolainen Samuel Juhani , Keddari Yasine , Dunn Katelyn , Forney Carmy , Yin Cailing , Donmez Omer , Parameswaran Sreeja , Javier Ellen , Porollo Alexey , Waggoner Stephen , Weirauch Matthew , Kottyan Leah
Published: January 2023

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by chronic immune activation, loss of self-tolerance and organ destruction. More than 90 genetic risk loci are implicated in the etiology of SLE, yet the mechanisms connecting these loci to disease risk are not well understood. We focus on a SLE-associated coding variant (rs1131665) at the IRF7 risk locus that results in an arginine (R, non-risk) to glutamine (Q, risk) amino acid change in the auto inhibitory domain of interferon regulatory factor-7 (IRF7), a key transcriptional regulator of the expression of and response to type I interferon (IFN). The majority of SLE patients exhibit elevated levels of circulating IFN, a feature that correlates with disease activity. We generated cell lines expressing the risk and non-risk variants of IRF7 and isolated plasmacytoid dendritic cells from mice with CRISPR-mediated introduction of a mouse homologue of the risk variant of IRF7. In both experimental systems, the rs1131665 lupus risk variant drives increased toll like receptor-7 (TLR-7) ligand-induced IFN-α expression at both the mRNA and protein levels compared to the non-risk variant of IRF7. Additionally, we performed a covariance analysis that predicts that amino acid residues at the 84, 140, 142 positions interact with the variant position in the three-dimensional structure of IRF7. Mutation of the amino acid at the 84 position from a glutamic acid (E) residue to a threonine (T) residue results in amplified IFN production in response to TLR-7 stimulation. Our findings suggest a genotype-dependent mechanism for lupus risk variants in IRF7 in promoting exaggerated IFN production in SLE.

These experiments would not have been possible with out NIH NIAMS R01 AR073228.
Nutraceutical Apigenin regulates DC function in a RelB-dependent manner during neuroinflammation.
Authors: Jain Pooja , Ginwala Rashida , McTish Emily , Moore Patrick , Revuri Nikil , Raman Chander , Singh Narendra , Nagarkatti Mitzi , Nagarkatti Prakash , Kranz Victoria A , Houle John D , Khan Zafar
Published: January 2023

Apigenin, a natural flavonoid, found in several plants is known to have anti-oxidant and anti-inflammatory properties indicated by its use for centuries as a medicinal approach to treat inflammatory disorders. However, there are significant gaps in knowledge regarding its effect on dendritic cell (DC) function in maintaining an immune balance in immunospecialized locations like the central nervous system (CNS). In order to establish the potential utility of Apigenin as a therapeutic agent against neuroinflammatory diseases, we tested and found that Apigenin treatment ameliorated severity of disease progression and relapse after onset of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 and SJL mouse models of multiple sclerosis. An increased retention of DCs and other myeloid cells in the periphery correlated with decreased immune cell infiltration and reduced demyelination in the CNS in treated mice. Mechanistically, Apigenin treatment reduced RelB expression in presence of LPS in human peripheral blood DCs, which is central to DC maturation, its antigen presentation capabilities and DC-mediated T cell activation. IL-12A and IL-23, downstream pro-inflammatory targets of RelB were reduced upon Apigenin treatment in these cells. Further, RelB causes a metabolic switch in immune cells upon inflammation, which was seen as a decrease in glucose uptake and lactate production (glycolysis), and an increase in mitochondrial activity when LPS-induced DCs were treated with Apigenin. These results indicate a protective role of Apigenin against DC-regulated neurodegenerative effects through a probable RelB mediated pathway thus implicating a potential therapy for neuroinflammatory disease.

Supported by grants from NIH: 1R01NS0971-47
High-potency synthetic STING agonists rewire myeloid stroma in the tumour microenvironment to amplify immune checkpoint blockade efficacy in refractory pancreatic cancer.
Authors: Boda Akash R , Ager Casey R , Rajapakshe Kimal , Lea Spencer T , Curran Michael A
Published: January 2023

Pancreatic ductal adenocarcinoma (PDAC) is clinically unresponsive to immune checkpoint blockade (ICB) immunotherapy. Dense immunosuppressive myeloid stroma (MS) and consequent T cell exclusion from the tumour microenvironment renders PDAC resistant to immune-based therapies. Innate immune activation of the MS via cyclic dinucleotide (CDN) agonists of the STING (Stimulator of Interferon Genes) pathway can trigger T cell infiltration into cold tumours leading to robust anti-tumour immunity. Despite proven therapeutic efficacy in preclinical models, the cellular mechanisms of how CDNs reprogram the suppressive MS to sensitise tumours to ICB is poorly understood.

Using multi-omic profiling of MDSCs and M2 Macrophages of human and murine origin, we show that the synthetic STING agonist, IACS-8803, rewires these populations from immunosuppressive to immune-permissive phenotypes in part through inhibition of c-Myc signaling, energy metabolism modulation, and antagonism of cell cycle. Furthermore, dimensionality reduction analyses of multiparameter flow cytometry data and survival studies in a KPC-derived model of PDAC show a proinflammatory remodeling of the MS and an increased T/NK cell infiltration and function by synthetic CDN STING agonists into the tumour that underlies an amplified therapeutic response to ICB.

We are the first to report that synthetic CDN STING agonists affect MDSC and M2 macrophage repolarization through altering energy metabolism, cell cycle dynamics and c-Myc signalling. This study uncovers molecular and cellular mechanisms by which high-potency STING agonists drive a proinflammatory conversion of the tumour MS to overcome resistance to ICB in an aggressive orthotopic tumour model of PDAC.

Supported by grants from PanCan (206100-90-100300-54-00006617-11111)
Unique and shared molecular features of human B and T lymphocyte memory differentiation
Authors: Singh Amit K , Roy Roshni , Kaileh Mary , Sarantopoulou Dimitra , Hernandez Dena , Arepalli Sampath , Bektas Arsun , Kim Jaekwan , McKelevy Julia , Zukely Linda , Dunn Christopher , Nguyen Cuong , Wallace Tonya , Wood William , Piao Yulan , De Supriyo , Sen Jyoti Misra , Weng Nan-ping , Ferrucci Luigi , Sen Ranjan
Published: January 2023

Long-term immunity against infections and effective response to vaccination are dependent on remarkable and unique feature of lymphocytes referred as “memory generation”. Despite having multiple mice studies focused on naïve to memory transition of lymphocytes our understanding of human naïve and memory properties is limited. In this study we explored epigenetics (DNA methylation and accessible chromatin), transcriptomics and activation induced gene expression of FACS sorted naïve and memory subsets of B, CD4 T and CD8 T lymphocytes of healthy donors from GESTALT cohort. Shared and specific methylomes between B and T lymphocyte subsets were identified. The relationship between DNA methylation, chromatin accessibility and transcriptome through ATAC-Seq and RNA-Seq data from the same donors is established. Through in-silico analyses and by utilizing data from Roadmap Epigenomics project and ChromHMM, we further identified differentiation-specific connections between DNA methylation, chromatin accessibility, and histone modifications. Transcription factor motif analyses identifies presence of ARNT, OCT and NF-κB motifs around sites hypomethylated in B memory while ETS motifs are in memory-associated hyper-methylated sites. Interestingly, OCT motifs were also significantly enriched in B memory specific accessible chromatin. To understand the extent to which these epigenetic changes effected lymphocytes, we activated naïve and memory subsets of B and CD4+ T lymphocytes in vitro and assayed early and late gene expression changes. These findings will provide the foundation for future studies related to dysregulation of memory cell differentiation in aging and infections.

Supported by Intramural Research Program of the National Institute on Aging.
The SWI/SNF canonical BAF complex and c-Myc cooperate to promote early fate decisions in CD8+ T cells
Authors: Guo Ao , Huang Hongling , Zhu Zhexin , Chen Mark J , Shi Hao , Sharma Piyush , Liedmann Swantje , Haydar Dalia , Yang Mao , Beere Helen , Krenciute Giedre , Roberts Charles W M , Chi Hongbo , Green Douglas R
Published: January 2023

The identification of mechanisms to promote the generation of memory T cells (TMEM) has important implications for vaccination and anti-cancer immunotherapy. Using a CRISPR-based screen for negative regulators of TMEM generation in vivo, we discovered many components of the mammalian canonical SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, also called canonical Brg1/Brg-associated factor (cBAF). cBAF is essential for the differentiation of activated, naïve CD8+ T cells into T effector (TEFF) cells, and loss of cBAF promotes TMEM formation of CD8+ T cells in vivo upon infection with Listeria monocytogenes and influenza A virus (IAV). During the first division of activated CD8+ T cells, we found that cBAF and c-Myc frequently co-assort asymmetrically to the two daughter cells. Daughter cells with high c-Myc are enriched for cBAF expression and display a cell fate trajectory towards TEFF cells, while those with low c-Myc have reduced cBAF, and preferentially differentiate towards TMEM cells. Moreover, the cBAF complex and c-Myc physically interact, and act in concert to establish the chromatin landscape in activated CD8+ T cells. Treatment of naïve CD8+ T cells with an inhibitor of cBAF during the first 48 hours of activation, prior to the generation of chimeric antigen receptor T (CAR-T) cells, markedly improves anti-cancer immunity in a solid tumor model. Our results establish cBAF as a key negative determinant of TMEM cell fate and suggest that manipulation of cBAF early in T cell differentiation can improve adoptive immunotherapy for cancer treatment.

Supported by AI123322
Expansion and maintenance of long lived effector CD8 T cells is modulated by inflammation
Authors: Lucas Erin D , Huggins Matt , Thefaine Claire , Pierson Mark , Hamilton Sara
Published: January 2023

Long-lived effector T cells (LLEC) are a circulating population of KLRG1 and CX3CR1 expressing memory CD8 T cells. LLEC were first described by the Hamilton Lab and are highly effective at clearing systemic infections compared to other memory T cells. However, LLEC express lower levels of IL-7 and IL-15 receptor chains and are poorly proliferative in specific pathogen free (SPF) mice as compared to central and effector memory T cells, which leads to a slow attrition of their numbers. The factors supporting LLEC expansion and persistence are unknown, and the goal of this study is to understand what internal and external factors contribute to LLEC expansion and maintenance. Data from models of chronic inflammation suggest pro-inflammatory factors contribute to the expansion and/or survival of LLEC. Specifically, we have found that LLEC populations are increased in young mice that have been exposed to a diverse mixture of microbes through co-housing with pet-shop mice. This co-housing model chronically increases systemic inflammation, and LLEC specific for model antigens not only expand more, but also persist longer than in SPF mice. Similarly, we also find increased LLEC in aged mice, another model associated with chronic inflammation. In preliminary work, we find that LLEC division is increased during cohousing, and that IL-18R expression is elevated, further suggesting that inflammatory cytokines not typically associated with T cell persistence may support LLEC proliferation. Ongoing studies will determine which specific inflammatory markers support LLEC expansion and maintenance, whether these factors are detrimental to other memory subsets, and if this process can be altered to improve T cell responses to infection and vaccination.

Supported by grants from NIH (R01 AI143828).
Plasmodium infection elevates risk of severe secondary bacterial disease by altering the immunological landscape of the lung
Authors: Reed Jenna , Cornwall Douglas , Jacobs Nathan , Evavold Brian D , Lamb Tracey J
Published: January 2023

Lower respiratory infections cause an estimated two million sepsis deaths yearly, with >50% attributed to the opportunistic pathogen Streptococcus pneumoniae. Sub-Saharan Africa, the site of most severe Streptococcus burden, is also ravaged by the malaria-causing Plasmodium parasite family, which afflicted 241 million people and killed 627,000 in 2020. Compared to healthy children, those with asymptomatic malaria have an elevated risk to severe bacterial infections, but the underlying mechanism(s) remain elusive. To fill this knowledge gap, we infected C57BL/6J mice with P. berghei NK65-NY either 7, 14, or 21 days prior to inoculation with S. pneumoniae 6304. Coinfected mice exhibited a 40–60% fatality rate, with death beginning 1–4 days post-bacterial infection. In contrast, singly infected groups survived the duration of the experiment. Coinfected mice exhibited equivalent parasitemia but significantly higher bacterial burden in the lung compared to mono-infections. Neutrophil recruitment and ROS production was altered in coinfected mice, which we predict is due to the formerly described effects of the Plasmodium metabolic product hemozoin. As such, we hypothesize that neutrophil control of S. pneumoniae is impaired. Since malaria has been shown to increase blood brain barrier permeability, we used Evans blue dye to test for a similar effect at the alveolar-capillary interface. Lung weight significantly increased as early as 6 days post-infection, while permeability trended towards a slight increase, peaking at day 9. Thus, we hypothesize that Plasmodium infection increases pathogenicity of Streptococcus infection due to a combination of impaired bacterial control and increased vascular leak in the lungs.

This work was supported by the NIH (ROl AI123425-01A1).
Host-targeted self-attenuated influenza virus as a potential therapeutic influenza vaccine
Authors: Ieremia Lanaiah M , Wen Ke , Wang Haiyan , Chen Yanping , Yang Huixiao , Zheng Zhichao , Yan Yongyong , Pena Adilene Realivazquez , Zeng Mingtao
Published: January 2023

Influenza is still one of leading causes of morbidity and mortality around the world, despite of annual influenza immunization in many countries. New strategies are urgently needed for developing effective vaccines and therapeutics against influenza viruses. In this study, we have constructed new recombinant influenza viruses, named host-targeted self-attenuated influenza viruses (SAIVs), which can express functional mammalian species-specific artificial microRNAs (amiRNAs). The expression of these amiRNAs can inhibit expression of some host factors critical for influenza replication, and therefore the resultant recombinant influenza viruses are replication restricted and attenuated in the host cells. One of these SAIVs, which can express an amiRNA that inhibits expression of the host cellular Cdc2-like kinase 1 (CLK1), was produced in embryonic chicken eggs and evaluated in a mouse model of influenza infection. It elicited robust antibody responses against influenza virus and demonstrated significantly protective efficacy against lethal infection with wild type influenza virus H1N1 PR8 after single dose of intranasal vaccination. Additionally, post-exposure treatment with this CLK1-targeted SAIV showed therapeutic effect against PR8 lethal-dose infection. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further developed to a therapeutic vaccine for prophylactic and therapeutic use against influenza.

Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).
Role of inflammation in the pulmonary hemorrhage induced by a snake venom metalloproteinase
Authors: Castro Ana Cristina , Gutiérrez José María , Escalante Teresa , Rucavado Alexandra
Published: January 2023

Snakebite envenoming (a neglected tropical disease that affects millions of people worldwide every year) is characterized by a complex pathophysiology. In cases of viperid envenomings, local and systemic hemorrhage is one of the major systemic effects that manifest in patients. This work focuses on studying pulmonary hemorrhage caused by a type P-III snake venom metalloproteinase (SVMP) extracted from Crotalus simus venom, and the extent to which inflammation affects the outcome of this hemorrhage. In order to determine the role of inflammation in pulmonary hemorrhage, we created a new automated image analysis methodology using the CellProfiler platform to quantitatively assess hemorrhage under different conditions, in conjunction with a statistical analysis program to process and analyze said image analysis results. With this toolset, we subsequently analyzed hemorrhage under the effects of treatment with three anti-inflammatory agents (a COX inhibitor, a TNF-ɑ inhibitor, and anti-neutrophil antibodies) in combination with the toxin on a murine model. Our findings conclude that inhibiting inflammation reduced lung hemorrhage significantly, which shows promise as a potential novel avenue for the treatment of snake envenomings. Moreover, we propose new hypotheses for how the toxin may enter lung capillaries and subsequently cause basement membrane damage which eventually leads to lung hemorrhage.

This study was supported by Vicerrectori?a de Investigación, Universidad de Costa Rica (Project 741-B4-524), and by a fund for support of graduate studies of the Sistema de Estudios de Posgrado (Universidad de Costa Rica)
Imaging Mass Cytometry identifies structural and cellular composition of the mouse tissue microenvironment
Authors: Lowrie Kerry , Raza Qanber , Cohen Michael , Kala Smriti , Awong Geneve , Quong Andrew , Loh Christina
Published: January 2023

Imaging Mass Cytometry™ (IMC™) is a vital tool to deeply characterize the complexity and diversity of any tissue without disrupting spatial context. The Hyperion™ Imaging System utilizes IMC, based on CyTOF® technology, to assess up to 40 individual structural and functional markers in tissues, providing unprecedented insight into the organization and function of tissue microenvironment. We have previously demonstrated the application of IMC in combination with Maxpar® panel kits on human tissues. Here, we showcase Maxpar OnDemand Antibodies for IMC application including 11 new highly relevant markers to construe cellular and molecular composition of mouse tissues.

We analyzed a normal mouse tissue microarray using IMC spatial proteomic analysis. Tissues were stained with a 20-marker panel designed to highlight tissue architecture and major immune lineage markers. We generated a detailed spatial map of the diverse tissue architecture and successfully identified immune, epithelial, and stromal cell populations in various mouse tissues. Additionally, we classified the activation state of immune cell populations, adhesion state of epithelial cells, and molecular composition of the extracellular matrix.

This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers in the mouse tissue microenvironment. Future studies utilizing IMC in combination with Maxpar OnDemand Antibodies will enable in-depth phenotypic characterization of the tissue microenvironment in various mouse models of development and disease, and thus provide the basis for the use of high-multiplex imaging in preclinical investigations.

For Research Use Only. Not for use in diagnostic procedures.
Robust human regulatory T cell expansion with fusion proteins HCW9302 and HCW9213 circumvents need for magnetic-bead or feeder cell approaches for adoptive cell therapy
Authors: Wong Hing C , Shrestha Niraj , George Varghese , Dee Michael J , Chaturvedi Pallavi , Zhu Xiaoyun , Spoundis Catherine , Liu Bai , Kong Lin , Echeverri Christian A , You Lijing , Egan Jack O , Jiao Jin-An , Rhode Peter R
Published: January 2023

Regulatory T cells (CD4+CD25+FoxP3+) (Tregs) are a subset of CD4 T cells that suppress the activities of other immune cells and have applications in the treatment of autoimmune and inflammatory diseases. Their use as an adoptive cell therapy has been limited by the practicality of expanding and purifying clinically sufficient numbers of cells. HCW9213 and HCW9302 are fusion proteins based on HCW Biologics’ TOBI™ technology platform, consisting of anti-CD3/anti-CD28 antibody domains and IL-2 domains, respectively. When used in combination, these fusion proteins were capable of expanding human Treg cells in vitro without the use of anti-CD3/CD28 magnetic beads and/or feeder cells, improving the overall yield, process efficiency and overcoming regulatory hurdles in manufacturing. Tregs generated with these molecules displayed similar phenotypes and suppressive cytokine production as Tregs expanded with recombinant human IL-2. Using a proprietary anti-CD39 antibody to isolate CD39+ Tregs, we have also been able to generate a Treg population with twice the suppressive activity against CD4+ T responder cells as traditional CD4+CD25+CD127lo Tregs. Thus, using its novel fusion proteins, HCW Biologics has been able to develop a superior Treg cell product ideal for the use in adoptive cell transfer. Additionally, this Treg platform can potentially be further optimized with addition of disease-targeted chimeric antigen receptors (CAR).
Characterizing arterial lesions in cardiac allograft vasculopathy rejected grafts using NanoString GeoMx digital spatial profiling
Authors: Nevarez-Mejia Jessica , Pickering Harry C , Sosa Rebecca A , Lau Ryan P , Fishbein Gregory A , Baldwin William M , Fairchild Robert L , Reed Elaine F
Published: January 2023

Cardiac allograft vasculopathy (CAV) is the leading cause of graft failure following heart transplantation. Donor specific antibodies (DSA) contribute to CAV by triggering vessel inflammation and endothelial cell injury. This results in thickening of the arterial intima and vessel occlusion. In this study, we aim to define the innate and adaptive immune cells mediating inflammation and vessel neointima formation using CAV+DSA+ rejected cardiac explants (N=3) and GeoMx digital spatial profiling (DSP). Arterial regions containing macrophages (CD68+CD163+) within vessels (CD34+) were selected for whole genome sequencing and a 76-protein panel. Arteries were scored as containing ‘low’ or ‘high’ neointima based on H&E. A total of 41 proteins including markers of inflammation and apoptosis were found to be expressed in all arteries. The expression of CD8, CD56, CD20, CD127, and CD11c exhibited a significantly positive correlation with RNA counterparts. A total of 15 differentially expressed proteins (e.g., T-cell makers: CD8, CD45RO, and CD127), and high expression of genes related to inflammation (ITGAX), and apoptosis (TP63), along with pathways for antigen presentation were elevated in arteries with low neointima. High neointima arteries showed higher SMA protein expression and increased genes related to cell growth (LECT2), complement regulation (CFHR3) and anti-inflammatory factors (FNDC4). Enriched pathways in high neointimal arteries included platelet activation/degranulation and cell migration. Our results provide insight into the mechanisms mediating CAV progression seen by inflammatory profiles in arteries with low neointima followed by pro-fibrotic/reparative phenotypes in high neointima arteries.

This work is funded by the Ruth L. Kirschstein National Research Service Award (NRSA) T32HL069766 (UCLA Vascular Biology Training grant), the Eugene V. Cota Robles Fellowship and NIH Grant 441450 ER 29762
Loss of IFNgR1 signaling glioblastoma drives resistance to CAR T cell binding avidity and cytotoxicity due to lower downstream expression of ICAM-1
Authors: Larson Rebecca , Kann Michael , Bailey Stefanie , Haradhvala Nicholas , Stewart Kai , Bouffard Amanda , Scarfo Irene , Leick Mark , Berger Trisha R , Jan Max , Joung Julia , Ouspenskaia Tamara , Law Travis , Regev Aviv , Getz Gad , Maus Marcela
Published: January 2023

Though CAR T cell therapy has had success treating hematologic malignancies, these treatments have shown only modest efficacy in solid tumors. Little is known about the necessary molecular components required for CAR T cell cytotoxicity, especially in the context of solid tumors. We investigated the role of IFNgR signaling on solid tumors and discovered across tumor types (including glioblastoma, pancreatic, ovarian and lung) loss of IFNgR1 signaling resulted lower CAR T cell cytotoxicity in vitro and that this was irrespective of target antigen (including endogenous EGFR, IL13Ra2, mesothelin, and exogenous CD19). We also validated this in orthotopic xenograft models of glioblastoma and pancreatic cancer in vivo. CAR T cells exposed to wildtype (WT) or IFNgR1 KO tumor had similar transcriptional profiles, but the tumor cells had different signatures in response to CAR T cell co-culture. Further investigation showed marked upregulation of cell adhesion pathways in WT cells compared to IFNgR1 KO. We utilized microscopy with acoustic force and discovered CAR T cells had lower cell binding avidity to cells lacking IFNgR1 compared to WT cells. Following CAR T cell exposure, Intercellular Adhesion Molecule 1 (ICAM-1) was strongly upregulated at both the transcriptional and protein levels in WT cells but not IFNgR1 KO cells. We overexpressed ICAM-1 on IFNgR1 KO tumor cells and observed that CAR T cell binding avidity and cytotoxicity was restored to that of WT levels. This work highlights the importance of CAR T cell binding avidity and adhesion for optimal cytotoxicity. Better understanding of CAR T cell function will inform future construct design for successful therapies against solid tumors.

RCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.
ZBP1 is a crucial regulator for tumor necroptosis during tumor development.
Authors: Baik Jin Young , Choksi Swati
Published: January 2023

Tumor necrosis happens commonly in advanced solid tumors. We reported that necroptosis plays a major role in tumor necrosis. By studying TNF-induced necroptosis, it is now known that receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3) and the mixed lineage kinase domain-like (MLKL) constitute the core of the necroptosis machinery. Two other proteins, Z-DNA-binding protein 1 (ZBP1) and TIR-domain-containing adapter-inducing interferon-β (TRIF) are known to function upstream of RIPK3 and interact with RIPK3 to mediate necroptosis in response to viral infection or TLR signaling respectively. Our study shows that it is the ZBP1, and not RIPK1 nor TRIF, that mediates tumor necroptosis during tumor development in preclinical cancer models. We found that ZBP1 expression is dramatically elevated in the later stages of both the MVT-1 breast cancer model and the MMTV-PyMT, a genetically engineered mouse model (GEMM) of breast cancer, when necrosis happens. Next, we generated CRISPR ZBP1 knockout (ZBP1 KO) MVT-1 cells to examine whether ZBP1 is needed for tumor necroptosis during MVT-1 mammary tumor development. ZBP1 deletion blocks tumor necroptosis during primary tumor development, more importantly, we observed decreased lung metastasis comparing to wildtype. We showed that glucose deprivation (GD) triggers ZBP1-depedent necroptosis in tumor cells. GD causes mitochondrial DNA (mtDNA) release to the cytoplasm and the binding of mtDNA to ZBP1 to activate MLKL in a BCL-2 family protein, NOXA-dependent manner. Therefore, our study reveals ZBP1 as the key regulator of tumor necroptosis and provides a potential drug target for controlling tumor metastasis.
Abrogating regulatory T cells overcomes tumor-specific T cell exhaustion and prevents metastatic pancreatic cancer
Authors: Schmiechen Zoe C , Burrack Adam L , Miller Ebony , Rollins Meagan , Wang Iris X , Cruz Eduardo , Patterson Michael , Stromnes Ingunn
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths and has a dismal 5-year survival rate of 10 percent. PDA lethality is attributed to late diagnosis, early metastasis, and therapeutic resistance. Metastasis can occur before the development of histologically detectable tumors and is a leading cause of cancer-related deaths. We identified that pancreatic tumor cells derived from mice that resist immunotherapy (e.g., tumor escape variants, TEV) and re-implanted into the pancreas of syngeneic and immunocompetent mice rapidly metastasize, reflecting the pathogenesis of human PDA. We show that TEVs retain the targeted tumor antigen, and despite a defect in IFNgamma-inducible MHC class I upregulation, TEVs remain sensitive to tumor antigen specific T cell-mediated lysis in vitro, suggesting TEVs may confer unique qualities in vivo to resist T cell killing. Using a peptide:MHC tetramer to identify the tumor-specific CD8 T cells, we identified that intratumoral T cells in EV tumors have increased Granzyme B production and a reduction in prototypical exhaustion markers PD1, Lag3, and Tox, perhaps due to reduced MHC class I signaling. Notably, primary tumors from TEVs were significantly enriched for Foxp3+ Tregs as compared to parental tumors. Using a genetic model, we identified that Treg depletion resulted in a drastic reduction in tumor burden and metastasis and improved tumor-specific T cell function in TEV tumors. Tumor-cell intrinsic changes driving Treg accumulation in TEVs will be discussed. In summary, Tregs are key drivers of both T cell exhaustion and immunosuppression in pancreatic cancer and may prove a valuable clinical target for tumors that evade immune checkpoint blockade.

Supported by grants from NIH (R01 CA249393)
Tumor immunity is enhanced in mice expressing the pro-autoimmune allele of tyrosine phosphatase Ptpn22, but not in Ptpn22 knock-out mice
Authors: Orozco Robin C , Marquardt Kristi , Mowen Kerri A , Sherman Linda A
Published: January 2023

The 1858C>T allele of the tyrosine phosphatase PTPN22 (causing amino acid substitution R620W in encoded protein Lyp) is present in 5–10% of the North American population and is strongly associated with numerous autoimmune diseases. Although much research has been done to define how this allele potentiates autoimmunity, the influence PTPN22 and its pro-autoimmune allele has in tumor immunity is poorly defined. To interrogate the role this allele has during the anti-tumor immune response, we used CRISPR/Cas9 to generate mice in which the ortholog of Lyp, PEP, is mutated at position 619 to produce the relevant pro-autoimmune mutation (PEP-619WW) or lack Ptpn22 expression (PEP-null). Using the B16 and other tumor models, we tested the hypothesis that pleiotropic effects of the  PTPN22  pro-autoimmune allele enhances tumor immunity. Results of this study show that PEP-619WW mice, but not PEP-null mice, resist tumor growth as compared with wildtype mice. Consistent with these results, tumors from PEP-619WW mice have more CD45 infiltrates containing; more activated CD8 T cell and CD4 T cells, more cDC1 cells, and less MDSCs than WT animals. Interestingly, the tumor infiltrating PEP-619WW cDC1s have lower PD-L1 expression compared to cDC1s from PEP-WT mice. Furthermore, using single-cell RNA sequencing we show that intra-tumoral myeloid cells from PEP-619WW mice have a more cytokine responsive transcriptional signature compared to PEP-WT cells. Our results suggest that a pro-autoimmune allele can be beneficial by promoting a strong anti-tumor immune response and may have a protective effect in this disease. Also, these data highlight an important difference between the PEP-null and PEP-619WW murine models during the tumor response.

Supported by grants from NIH (UO1 AI130842 and T32 AI007354 27).
Evaluating plasma antibody repertoires in breast cancer patients with progesterone receptor positive and negative tumors
Authors: Chapoval Andrei I , Podlesnykh Stepan V , Kolosova Eugenia A , Koltysheva Polina I , Tishchenko Ekaterina D , Shoikhet Yakov N , Lazarev Aleksandr F , Johnston Stephen Albert
Published: January 2023

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Early cancer detection and tumors molecular characterization are the key measures for effective therapy. Molecular changes associated with tumor development are detected by the immune system and reflected with specific antibody production. We used microarrays containing 120K of peptides with random amino acid sequences to analyze the repertoire of circulating antibodies in the blood plasma of patients with early stages of breast cancer (BC). Previously we have shown that healthy donors and BC patients have distinct circulating antibody repertoires. Further analysis reviled that BC patients with various progesterone receptor (PR) expression levels also have circulating antibodies reacting with different peptides (mimotopes) on microarrays. In this study we selected 61 peptides from 120K peptides presented on microarrays that show statistically significant difference for the interaction with the circulating antibodies of blood plasma from PR+ and PR- BC patients. Peptides interacting with PR+ and PR- patients plasma have common amino acid motifs suggesting that patients with tumors expressing different PR levels mount specific immune response reflecting molecular changes associated with various tumor subtype. Therefore, the panel of peptides that specifically interact with the immunoglobulins of patients with different PR expression can be used for the development of non-invasive test systems. Thus, peptide microarrays can be used to create a new tool for studying the repertoire of circulating antibodies for different diseases evaluation. Supported by grant FZMW-2020-0007.

Supported by grants from the Ministry of Science and Higher Education of the Russian Federation (FZMW-2020-0007)
Analysis of expression and function of LLT1 (CLEC2D) in Ewing sarcoma
Authors: Mathew Stephen O , Buller Casey W
Published: January 2023

Ewing Sarcoma (EWS) is a pediatric bone cancer that is characterized by a chromosomal translocation giving rise to a neomorphic gene fusion. Although treatment of EWS has a 5-year incident free survival rate of 66%, treatment has plateaued since the 1980’s. Natural killer (NK) cells are an important innate immune cell due to their ability to recognize and lyse virally infected and cancerous cells. Cancerous cells often employ strategies such as the downregulation of major histocompatibility complex (MHC) I, or upregulation of inhibitory ligands enabling the escape of T-cells and NK cell mediated killing. Previously, we showed that LLT1 is an inhibitory ligand that is expressed on triple negative breast cancer (TNBC) and prostate cancer cell lines. The expression of LLT1 and its function in Ewing Sarcoma (EWS) cell lines has yet to be performed. Our results have demonstrated a higher expression of LLT1 on EWS cell lines. We have shown that blocking LLT1 and NKRP1A interaction with anti-LLT1 antibodies increases NK cell cytotoxicity against EWS target cells. These results indicate that LLT1 is a potential immunotherapy target that needs to be further evaluated in an in vivo model.

Supported by intramural funds from UNT Health Science Center
CD4 T cell memory restoration upon antiretroviral initiation in people living with HIV.
Authors: Sponaugle Alexis Thieme , Abad-Ferenandez Maria , Clutton Genevieve , Weideman Ann Marie K , Hudgens Michael G , Davy-Mendez Thibaut , Adimora Adaora A , Ramirez Catalina , Floris-Moore Michelle , Kuruc JoAnn , Margolis David M , Gay Cynthia , Eron Jospeh J , Goonetilleke Nilu
Published: January 2023

HIV infection depletes CD4 T cells and dysregulates formation of long-lived memory CD4 T cells. Studies show that antiretroviral therapy (ART) in people with HIV (PWH) more effectively restores CD4 T cell memory phenotypes when initiated early (< Fiebig V). We used a 31 marker mass cytometry panel to assess CD4 T cell phenotype in PWH treated in acute infection (AHI) (median 2.3 years post ART initiation) and chronic infection (CHI) (median 5.8 years post ART initiation) compared to healthy donors (HD) (n=10 per group). Markers of activation (HLA-DR, CD25), exhaustion (PD-1), survival (Bcl-2) and memory (CD127) were examined. CD4 T cell memory phenotypes of AHI clustered closely with HD whereas CHI had fewer central memory CD4 T cells. We also examined IL-7 signaling in CD4 T cells, measuring STAT5 phosphorylation (pSTAT5) in response to IL-7. CHI exhibited significantly lower pSTAT5 than HD in contrast to AHI who exhibited restored IL-7 signaling. This suggests ongoing and underappreciated functional defects in CD4 T cells in CHI. To examine this further, we are completing detailed analysis of CHI before and after ART initiation (pre-ART, day 2, 7, 10, 14, 28, month 5, 9 and 18). Data will be compared to our recent work showing that CD4 T cell memory subsets, activation and exhaustion markers are highly stable in durably suppressed CHI on ART over 19–27 months. New work is examining CD4 T cell function in immunological non-responders (INR) who fail to restore CD4 counts following ART. Altogether these studies will inform how HIV impacts formation of both CD4 memory and the HIV reservoir (which is largely harbored in long lived memory CD4 T cells) following ART initiation, potentially identifying novel therapeutic targets to accelerate HIV cure in CHI.

This work was made possible by the National Institute of Allergy and Infectious Diseases U01 AI131310, Collaboratory of AIDS Researchers for Eradication (UM1 AI164567) and UNC WIHS (U01 AI103390).
Altered cytokine and chemokine profiles in the lungs of vitamin A deficient mice with respiratory syncytial virus infection
Authors: Hong Suyeon , McGill Jodi L
Published: January 2023

Human respiratory syncytial virus (RSV) is a leading cause of severe acute lower respiratory tract disease worldwide in infants and young children. Epidemiologically, low levels of vitamin A (VA) are known to increase susceptibility to severe respiratory infection. However, little is known regarding how VA impacts lung immune function, specifically during RSV infection. Here, we investigated the impact of VA deficiency (VAD) on innate immunity in the lungs following RSV infection using a BALB/c mouse model. Groups of VA sufficient (VAS) and VAD, 6 to 8-week-old mice were intranasally infected with RSV, and lung tissues were collected at 3, 4, and 8 days post-infection for evaluation of vitamin A status, flow cytometry for innate immune populations, and Luminex bead-based multiplex assays for inflammatory cytokines. Acute RSV infection negatively impacted lung VA status in vitamin-replete mice. Although no differences in morbidity were observed between treatment groups, VAD mice had more neutrophils recruited to the lungs. VAD also altered the phenotype of γδ T cell populations in the lung, resulting in significantly increased frequencies of CD27+ γδ T cells, and fewer CD27 negative γδ T cells compared to VAS mice. Moreover, cytokine and chemokine production was altered in the lung of VAD animals after RSV infection, with reduced production of IL-1β, IL-6, IL-17, and KC (CXCL1). These results suggest that VAD alters the inflammatory environment in the lung, which may contribute to disease susceptibility. Future experiments will focus on further changes to the innate and adaptive immune systems in the lungs during VAD after RSV infection.

Supported by Roy J. Carver Charitable Trust Grant #20-5306
Bclaf1 promotes hematopoietic stem cell repopulating capacity and self-renewal
Authors: Crowley Stephanie , White Lynn S , Li Yanan , Yang Wei , Magee Jeffrey A , Bednarski Jeffrey J
Published: January 2023

Hematopoietic stem cells (HSC) balance differentiation and self-renewal to promote maintenance of mature blood cell lineages. The mechanisms that regulate HSC expansion during fetal hematopoietic development and adult HSC maintenance remain significant knowledge gaps. BCLAF1 (Bcl2-associated factor 1), a transcriptional regulator, is highly expressed in hematopoietic cells. We recently defined its function as a modulator of ETS-family transcription factor activity in developing B cells. Our objective in this study is to determine the molecular mechanism of BCLAF1 in regulation of fetal and adult HSCs. We used mouse models with selective deletion of Bclaf1 in hematopoietic cells to test whether BCLAF1 regulates HSC development and function. We find that loss of Bclaf1 results in significantly reduced numbers of fetal HSCs. The reduced HSC numbers persist in adult mice; but there is no additional decline or development of bone marrow failure with aging. In competitive transplant experiments, Bclaf1-deficient fetal and adult HSCs have defective repopulation and self-renewal capacity resulting in reduced reconstitution of all hematopoietic populations. Bclaf1-deficient HSCs do not have abnormalities in emergence, proliferation, cell death, or homing activity. Using single cell RNA-sequencing of fetal HSCs, we find that loss of BCLAF1 results in reduction of a subpopulation of HSCs and upregulation of the early response genes Jun, Fos, and Egr1. Collectively, these findings suggest BCLAF1 restrains activity of immediate early genes to preserve HSC self-renewal capacity and promote HSC expansion during both fetal development and repopulation of hematopoiesis after HSC transplant.

Supported by grants from Children’s Discovery Institute, St. Louis Children’s Hospital Foundation, Siteman Cancer Center, and NIH (T32 AI007163).