bluuebunny/crossref_metadata_2025 huggingface dataset:
Protease activated receptor-1 signaling: its potential implication in HIV driven immune activation, inflammation/coagulation
Authors: Chen Hui , Smith Mindy , Herz Jasmin , Gossa Selamawit , Karpova Tatiana , McGavern Dorian , Catalfamo Marta
Published: January 2023

Persistent immune activation remains the hallmark of HIV infection even in the context of successfully suppressed viremia. Its clinical significance is suggested by increased risk to develop non-AIDS defining illnesses such as cardiovascular disease. We previously have shown that human CD4 and CD8 T cells with effector memory and terminal effector memory phenotype expressed the protease activated receptor-1 (PAR1) and that expression was increased in CD8 T cells from HIV infected patients. Thrombin, in a PAR1 dependent fashion, enhanced CD8 T cell function measured by increased secretion of IFN-g and chemokinesis. PAR1 is the main receptor of thrombin and is also expressed on platelets and endothelial cells suggesting a cross-talk between the coagulation system and T cells at the site of vascular injury. To address the role of PAR1 in vivo, we used an infection model with the hepatotropic strain of lymphocytic choriomeningitis virus (LCMV-WE). To evaluate the role of PAR1 signaling in this model, we analyzed the T cells of littermate wild type control and PAR1−/−mice eight days following infection with LCMV-WE (2×105 pfu i.v.). In this model, we observed a similar expansion of total CD8 T cells in the spleens and livers of both WT and PAR1−/−mice at 8 days post infection. Despite similar expansion, PAR1 deficient mice showed impaired viral clearance at day 8 of infection. This lack of viral control was associated with a significant reduction in cytokine secretion by gp-33-specific CD8 T cells in the livers and spleens. These results suggest that PAR1 deficiency leads to an impair viral control that is in part mediated by a decrease ability to secrete cytokine by virus specific CD8 T cells.
Mechanisms of MCMV immune evasion provide insight into MHC-I folding and assembly.
Authors: May Nathan A. , Boyd Lisa , Margulies David H.
Published: January 2023

As part of its strategy to evade detection by the host immune system, murine cytomegalovirus (MCMV) encodes three proteins that modulate cell surface expression of major histocompatibility complex class I (MHC-I) molecules: the MHC-I homolog m152/gp40 as well as the m02–m16 family members m04/gp34 and m06/gp48. We have explored the interaction between recombinant m06 and both full-length and truncated forms of the MHC-I molecule H2-Ld using in vitro binding assays. Previous work using solution NMR has mapped the interaction footprint of the m06 protein on MHC-I, revealing that the targeted surface area overlaps with that of the MHC-I chaperone molecule Tapasin. Furthermore, we observe enhanced binding between m06 and peptide-free forms of MHC-I, suggesting a possible chaperone-like interaction between m06 and MHC-I. Here, we express recombinant m06 in mammalian cells to explore the interplay between the viral protein and its potential targets through immunofluorescence localization experiments as well as well as binding assays. This study provides insight into the mechanism of the interaction of m06 with MHC-I, suggesting a structural manipulation of the target MHC-I molecule at an early stage of the peptide-loading pathway.
Understanding how AKT phosphorylation of hnRNPA1 modulates T cell fate and function
Authors: White Theresa L , Gable Matthew , Jin Ye , Morel Penelope
Published: January 2023

T regulatory cells (Treg) play a significant role in maintaining self-tolerance and preventing autoimmune diseases. We and others have shown that low dose favors Treg and T helper (Th) 2 cell differentiation, while high Ag dose stimulation activates the PI3K/Akt/mTOR pathway, favoring inflammatory Th1 and Th17 cell differentiation (Tconv). Differences in PI3K/Akt/mTOR signaling not only affects T cell fate but our research shows that Akt phosphorylation of the RNA-binding protein, (RBP) heterogeneous nuclear ribonucleoprotein (hnRNP) A1, is dependent on TCR signal strength. RBPs such as hnRNPA1 are emerging as regulators of RNA processing and stability in immune cells, and the effect of RBP on T cell differentiation is a growing subject of interest. We have shown hnRNPA1 is required for optimal Treg differentiation by performing knockdown experiments, and our present research is focused on identifying a role for Akt phosphorylation in hnRNPA1 function. HnRNPA1 is known to have a single Akt phosphorylation site at S199 and our lab has generated a new mutant mouse model, hnRNPA1-S199A (mA1). This mutation affects the ability of Akt to phosphorylate hnRNPA1 in all immune cells. Using Cytek’s Aurora flow cytometer we characterized the immune cell populations of mA1 at steady state. Preliminary data do not indicate any major changes in innate immune and B cells populations at steady state. We observed modest changes in CD4 T cell population frequencies at steady state. Current work using In vitro skewing suggests that Akt phosphorylation of hnRNPA1 influences Treg fate. This project suggests a novel mechanism by which Akt modulates T cell fate.
AID deficiency greatly improves survival and diminishes renal pathology in the BXSB mouse model of SLE
Authors: Leeth Caroline McPhee , Zhu Jing , Potter Ashley , Hasham Muneer , Richwine Madison , Roopenian Derry C
Published: January 2023

Eight years have passed since the approval of belimumab, a monoclonal antibody directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus. erythematous (SLE). While well tolerated, the efficacy of belimumab remains limited and is not recommended for patients suffering severe nephritis, a life threatening complication of SLE. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB model is a long established model of human SLE developing elevated antinuclear antibodies and immune complex nephritis along with other manifestations of autoimmune disease. We used CRISPR-Cas9 to inactivate Activation Induced Cytidine Deaminase (Aicda, AID) directly in the BXSB background and found greatly improved survival. While mice continued to develop plasma cells, follicular structure was restored and renal pathology was reduced. While antinuclear antibody concentrations were reduced in knockout mice, renal immune complex deposition was grossly the same as wild type while complement deposition was very limited in the knockout mice. Mice develop expanded germinal center B cell populations as in other models of AID deficiency without concurrent expansion of follicular T cells. The prolonged survival in these mice appears to be attributed to the reduced renal pathology warranting further exploration as current therapeutics targeting lupus nephritis are limited and thus in great demand.
CD300c is a negative regulator of T cell immunity
Authors: Lai Laijun , Cui Cheng , Su Min , Lin Yujun
Published: January 2023

Immune responses are tightly controlled by T cell regulators in which B7 family members are of central importance. The importance of the B7 family molecules has been highlighted by the FDA approval of several drugs for the treatment of cancer and autoimmune disease by targeting the ligands or their receptors. The unprecedented success has attracted considerable interest in identifying additional T cell regulators. In this study, we identify CD300c as a novel T cell co-inhibitory molecule that shares significant sequence homology with existing B7 family members. CD300c protein is expressed on professional antigen-presenting cells, including B cells, monocytes, macrophages and dendritic cells. The putative CD300c receptor is expressed on CD4 and CD8 T cells, and the expression levels are upregulated upon activation. Soluble human and mouse CD300c-Fc fusion proteins significantly inhibit the proliferation, activation, and cytokine production by CD4 and CD8 T cells in vitro. Administration of CD300c-Fc protein attenuates graft-versus-host disease (GVHD) and experimental autoimmune encephalomyelitis (EAE) in mice. Our results suggest that therapeutic interaction with the CD300c inhibitory pathway may represent a new strategy to modulate T cell-mediated immunity for the treatment of GVHD and autoimmune disease. [This work was supported by grants from NIH (1R01AI123131), and Connecticut Regenerative Medicine Research Fund (16-RMB-UCONN-02)].
Peripherally induced tissue resident memory CD8 T cells in the brain mediate protection against CNS infections
Authors: Urban Stina Lisa , Jensen Isaac J , Pewe Lecia L. , Badovinac Vladimir P , Harty John T
Published: January 2023

The central nervous system (CNS) is classically viewed as an immune privileged site; however recent advances in the field highlight the importance between the interaction of the peripheral immune system and the CNS in maintaining tissue homeostasis. Tissue resident memory (Trm) CD8 T cells have been described in almost every organ and are shown to provide a first line of defense against secondary invading pathogens. Trm cell generation generally involves active infection at the site of tissue residence. Using multicolor flow cytometry, we describe an increased representation of antigen-specific (Ag) CD8 T cells in the CNS after peripheral systemic immunizations or infections. Utilizing unbiased SPADE analysis, the intravascular (IV) stain negative Ag-specific tissue residing cells isolated from the CNS were phenotypically distinct from the IV stain positive cells in the vasculature. Further characterization indicated peripherally-induced CD8 T cells residing in the brain adopted a Trm phenotype and were resistant to systemic antibody-mediated depletion. The increased representation and phenotype of CD8 T cells in the brain after systemic immunization was similarly found in the meninges but not in peripheral organs including liver or lungs. 2-photon microscopy revealed peripherally-induced Trm are highly dynamic in the brain. CD8 T cells surveyed the tissue with multiple mechanisms including moving along the blood vessels, traveling long distances in apparently random fashion or remaining stationary. Most importantly, systemically-induced Trm cells in the CNS provided protection against neurotropic challenges by reducing bacterial and viral loads and limiting morbidity and mortality associated with the infections.
BAFF and VCAM-1 cooperate on the Interaction between B lymphocytes and fibroblast-like synoviocytes
Authors: Moon Eun Yi , Yoon Sung Sik
Published: January 2023

Synovium and joint in rheumatoid arthritis (RA) is expanded and destructed by chronic and systemic inflammatory disorders. Fibroblast-like synoviocytes (FLSs) play a key role in the pathogenesis of RA by producing inflammatory cytokines leading to cartilage destruction. RA-FLSs develop a unique aggressive phenotype that increases invasiveness into the extracellular matrix and further exacerbates joint damage. Tumor necrosis factor-α(TNF-α), a major pro-inflammatory cytokine triggers the expression of inflammatory molecules such as B cell activating factor(BAFF) and cell adhesion molecules such as vascular cell adhesion molecule-1(VCAM-1). Here, we investigated whether cell-cell contact between B cells and FLSs may be significant in the patho-physiology of RA through BAFF and VCAM-1 using WIL-2NS and MH7A synovial cells. TNF-α treatment increased the number of B cells bound FLSs as judged by MTT assay, cell count, cell staining and flow cytometry analysis. TNF-α treatment enhanced the expression of BAFF and VCAM-1 as measured by RT-PCR, promoter assay and western blot analysis. Small interference (si) RNA of BAFF or VCAM-1 inhibited the number of B cells bound FLSs. TNF-α induced JNK phosphorylation, which might mediate the interaction between e B cells and FLSs. Taken altogether, data suggest that TNF-α-induced BAFF and VCAM-1 could be related with the interactions between MH7A cell and WIL-2NS leading to the aggravation of RA.

This work was supported by the R&D program for Society of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (Grant from Mid-career Researcher Program : #2016R1A2B4007446 and # 2018R1A2A3075602), Republic of Korea.
Sclerostin depletion may induce inflammation in the bone marrow
Authors: Donham Cristine , Loots Gabriela , Manilay Jennifer
Published: January 2023

The interactions between long-term hematopoietic stem cells (LT-HSCs) and cells in the bone microenvironment result in changes in stem cell behavior that are incompletely understood. Our goal is to define the molecular mechanisms that guide the behavior of LT-HSCs after exposure to an irregular bone marrow (BM) niche. Sclerostin (Sost) is a secreted Wnt antagonist that is important for bone homeostasis. We hypothesized that that loss of Sost would influence LT-HSC differentiation and migration. To test this, we performed hematopoietic progenitor transplantation assays to track donor-derived hematopoiesis in wild-type (WT) and Sost-knockout (KO) recipients. We observed increased frequencies of donor-derived myelopoiesis as well as decreased lymphopoiesis in WT to KO chimeras possibly due to an inflammatory microenvironment. We are currently investigating LT-HSC inflammatory response genes, such as C/EBP-beta and PU.1, as well as BM serum inflammatory cytokine levels. As the SostKO mice age, we observe extramedullary hematopoiesis (EMH) in the spleen compared to age matched controls. We hypothesize that changes in the BM microenvironment as well as cytokine levels in the spleen induce LT-HSC migration to the spleen. We are characterizing the EMH LT-HSC splenic niche by investigating LT-HSC localization as well as examining cytokines, such as CXCL12, known to induce LT-HSC migration to the spleen. Taken together, our data indicates that depletion of Sost in the bone regulates LT-HSC differentiation and migration. Our results could have significant impact on the tracking of hematopoietic function in human patients treated with SOST-depleting antibodies for bone diseases such as osteoporosis.
Metabolic pathways that mediate expansion and homeostasis of hematopoietic lineages
Authors: Pizzato Hannah A , Lam Wing Y , Finck Brian N , Wolfgang Michael J , Bhattacharya Deepta
Published: January 2023

Due to the rarity of hematopoietic stem cells, their differentiation into progenitors then on to mature cells must occur with proliferation, leading to an expansion of each downstream population. This process, called transit amplification, is required to produce a sufficient mature cell population and must be balanced with survival to maintain homeostasis and prevent hematological malignancies. How transit amplification and subsequent homeostasis is controlled is not well understood but may involve metabolic switches. Various groups have demonstrated that hematopoietic expansion requires a switch from glycolysis to oxidative phosphorylation, but what induces this switch and the specific carbon sources that fuel it remain unclear. To address these questions, we employed mice conditionally deficient in mitochondrial pyruvate import, fatty acid oxidation, or glutamine hydrolysis and examined the requirements of these pathways in hematopoietic homeostasis. Ablation of carnitine palmitoyltransferase II (Cpt2) or glutaminase (Gls) had no effect on lineages examined, suggesting fatty acid oxidation and glutaminolysis are not required for the production or survival of various immune cells. In contrast, upon deletion of mitochondrial pyruvate carrier 2 (Mpc2), peripheral myeloid and lymphoid cell numbers declined dramatically. Over time after MPC2 ablation, a progressive recovery of mature cells was observed. This recovery was associated with increased proliferation seen in Mpc2−/− progenitors relative to their wild type counterparts immediately following genetic deletion. Further studies will seek to elucidate if cells switch to another metabolic pathway to recover from mitochondrial pyruvate ablation.
Inhibiting host DNA release prevents mixed neutrophilic/eosinophilic airway inflammation in severe asthma phenotype
Authors: Chung Sangwoon , Karpurapu Manjula , Christman John W.
Published: January 2023

Asthma pathophysiology and treatment strategies are predicted by inflammatory phenotypes. Unlike the relatively well-defined mechanisms that result in eosinophilia, those leading to noneosinophilic asthma, particularly non-type 2 neutrophilic/eosinophilic asthma, remain relatively poorly understood.

We recently discovered that leukocytes released extracellular traps (ETs) comprising DNA fibers and granule proteins in the BAL fluid of patients with mild asthma who were experimentally challenged with allergen according to the subsegmental bronchoprovocation to allergen (SBP-AG) protocol. Key aspects of ETs in the immune response are sufficient to recapitulate many features of asthma pathophysiology which results in mucus hypersecretion and airway remodeling in the airways of asthmatics.

To test hypothesize that inhibiting ET-associated host dsDNA release prevents neutrophilic/eosinophilic airway inflammation in severe asthma (SA) phenotype, we set up the SA mouse model, that mirrors the complex immune response identified in human SA. We showed that host dsDNA and neutrophil elastase were greatly increased in the airways and short noncoding miR-155 contributes to the release of host dsDNA, which exacerbates many features of allergic inflammation. In accordance with what we observed in high miR-155 expression in alveolar macrophages, we also noted that there was markedly less ETs formation in the airways of miR-155fl/flLysMcre mice subjected to the SA model compared with their WT counterparts. In sum, miR-155 engages in the release of host dsDNA that contributes to airway damage and thus promotes mixed granulocytic inflammation.
Effect of different adjuvants on various immune cells and cytokines in allergic asthma mouse model
Authors: Shin Hyun soo , Chun Hye Rin , Na Hye Young , Sohn Moah , Ryu Seul Hye , Choi Wanho , In Hyunju , Park Ji Soo , Park Chae Gyu
Published: January 2023

Allergic asthma is a complex chronic disease which displays combined traits referred to as Th2 mediated eosinophilic allergy, ILC2 mediated allergy, and Th17 cell mediated neutrophilic allergy. In our current study of allergic asthma model, the populations of immune cells related to as well as outside of the Th2 immune response were analyzed for the ratio of cytokines in IL-4, IL5, IL17A and IFN-γ expressing CD4+ T cells utilizing of BALB/c mice sensitized with ovalbumin and a different adjuvant such as Alhydrogel (alum), papain, lipopolysaccharide (LPS), and CpG. In lung and bronchoalveolar lavage fluid, the percentage of eosinophils was lower in groups injected with LPS and papain than those with no adjuvant. In contrast, the injection of LPS and papain increased the percentage of neutrophils when compared to the no adjuvant group. The number of B cells in alum-injected group was higher than those of other groups. Although the percentage of innate lymphoid cells (ILCs) increased slightly for ILC1 and ILC2 in the group injected with papain, the other adjuvants did not cause any differences. After PMA/ionomycin stimulation, the ratio of IL-4 expressing CD4+ T cells in the papain-injected group increased significantly while the other adjuvant groups showed little or no changes. The production of IL-17A in CD4+ T cells increased in LPS-injected group and that of IFN-gamma also increased in CpG-injected group. Our results suggest that many distinct allergic asthma mouse models can be established by specific sensitization protocols exploiting different adjuvants, thus representing the complexity of clinical symptoms.
Role of Ets Transcription factors in the development of TH9 cells
Authors: Kharwadkar Rakshin P , Ulrich Benjamin J. , Fu Yongyao , Kaplan Mark H
Published: January 2023

T cells are critical regulators of inflammatory diseases and immunity. CD4+ T helper cells develop into various subsets that are specialized in the secretion of particular cytokines and mediate restricted types of inflammation. One of these subsets recently identified to play an important role in development of allergic inflammatory response, is the IL-9 producing TH9 subset. TH9 cells differentiate in the presence of IL-4 and TGFβ, which activate a network of transcription factors downstream of STAT6 and SMAD signaling pathways to regulate IL-9 secretion. Previous studies showed that PU.1, an Ets transcription factor, can bind at the Il9 promoter region to regulate TH9 function during pro-allergic inflammation. Furthermore, members of the Ets family of transcription factors Etv5 and PU.1 appear to share overlapping functions to promote IL-9 production and TH9 development. We screened for additional ETS factors that might play a role in IL-9 regulation. Jabeen et. al. showed the Ets transcription factor termed Erg (Ets-related gene) is expressed preferentially in TH9 subset. Accordingly, we determined that siRNA knock down of Erg in TH9 cells decreased IL-9 production in vitro. Moreover, chromatin immunoprecipitation assay demonstrated that Erg binding at the Il9 promoter and enhancer region is enriched in TH9 cells. Furthermore, ectopic expression of Erg induced IL-9 secretion in TH2 cells. In addition to Erg, we identified Fli-1, another Ets transcription factor, that can enhance IL-9 secretion in TH2 subset and whose expression is altered by Erg-deficiency. Thus, multiple Ets transcription factors regulate Il9 expression in TH9 cells, suggesting that Erg and other Ets transcription factors may cooperate to promote TH9 function.
Highly multiplexed single-cell spatial analysis of tissue specimens using CODEX
Authors: Mistry Sejal , Dakshinamoorthy Gajalakshmi , Singh Jaskirat , Nikulina Nadya , Kim Joseph , Bashier Roya , Gallina Maria Elena , Kennedy-Darling Julia
Published: January 2023

Characterizing the complexities of the tumor microenvironment is elemental to understanding disease mechanisms. The spatial relationships between infiltrating immune cells and the remodeling of the cellular matrix is widely recognized as a key component to defining tumor heterogeneity. Here, we present a novel multiplexed imaging technology, termed CODEX, (CO-Detection by indEXing) that combines the high parameter capabilities of single-cell methodologies with the associated spatial dimension. Over 50 parameters are measured within a single tissue through fully-automated, iterative cycles of adding and removing dye-conjugated Reporters to corresponding nucleic acid based Barcodes. The CODEX technology, developed by Akoya, is comprised of a fluidics instrument that interfaces with existing microscope hardware, a suite of reagents and associated control and analysis software. Over 100 antibody clones have been validated for this platform with more than ten tissue types analyzed, including both FFPE and fresh-frozen from human and mouse samples. The CODEX technology can be used to ascertain complex cellular niches and spatial associations between multiple cell types based on their staining pattern. CODEX data from various normal and cancer tissue types are shown here with corresponding single-cell analysis of key tissue features. Overall, the CODEX platform is an accessible and versatile technology for high parameter, spatial profiling of tissue specimens.
Generation of a B-1 lymphocyte-specific conditional knockout mouse model Bhlhe41tdTomato-Cre
Authors: Wang Hui , Hong Li , Tang Yangyang , Xu Meizhen , Pan Shuai , Wang Qingling , He Cheng
Published: January 2023

B-1 lymphocytes play indispensable roles in defensing a wide range of pathogens prior to adaptive immune response arising. Although several transgenic mouse models targeting different stages of B lymphocytes have been established e.g. Cd19-Cre and Mb1-Cre, a B-1 lymphocyte specific conditional knockout mouse model for illuminating the role of a gene of interest in B-1 lymphocytes is still missing. In this study, we aimed to generate a B-1 lymphocyte-specific knockout mouse model by expression of Cre recombinase under the control of the promoter of Bhlhe41, which is preferentially expressed in B-1 lymphocyte and microglia in immune system. A tdTomato-2A-Cre-polyA cassette was inserted to the locus of Bhlhe41 by CRISPR-CAS9 technology. Bhlhe41tdTomato-Cre/+Rosa26EYFP/+ mice were generated for determining Cre-mediated recombination efficiency. The accuracy of insertion of tdTomato-2A-Cre-polyA cassette to Bhlhe41 locus was confirmed by significantly reduced peritoneal B-1a lymphocytes in Bhlhe41tdTomato-Cre/tdTomato-Cre mice. Flow cytometric analysis of tdTomato in immune cells from Bhlhe41tdTomato-Cre/+confirmed the specific expression of BHLHE41 in B-1 lymphocytes rather than other B subsets. Further analysis of EYFP in immune cells from Bhlhe41tdTomato-Cre/+Rosa26EYFP/+ mice revealed larger than 90% recombination in splenic and peritoneal B-1 lymphocytes while less than 10% recombination in other B subsets. Hence, we generated a B-1 lymphocyte-specific conditional knockout mouse model Bhlhe41tdTomato-Cre, which can be used for studying the function of a specific gene in B-1 lymphocyte-mediated immune responses.
Integration of multi-omics data from re-wilded laboratory mice to identify key parameters that determine immune heterogeneity and activation
Authors: Lin Jian-Da , Yeung Frank , Devlin Cooper , Chen Ying-Han , Mccauley Caroline , Cronkite Alex , Leung Jacqueline M. , Graham Andrea L. , Cadwell Ken H. , Loke P’ng
Published: January 2023

Current research on immunology are largely based on clean laboratory mice kept in specific pathogen free (SPF) environments. They have the advantage of being inbred, genetically homogeneous and can be manipulated genetically in disease models. However, humans as with all free-living mammals face a more complex environment and are exposed to various food resources, microbial experiences, thermogenic changes, and must constantly respond to environmental challenges. We have established a unique experimental system in which laboratory mice are “re-wilded” through release into an outdoor enclosure facility. Here, we use multi-parameter assays including 16S/Metagenomic sequencing, intestinal RNA-sequencing, and 34 parameters of flow cytometry to explain heterogeneity of immune responses from exposure to the wild environment. Increased immune activation of re-wilded mice occur in the absence of viral, bacterial or parasitic pathogen exposure in this enclosure, including increase of CD4+ and CD8+ memory T cells, effector T cells, innate lymphoid cells (ILCs), circulating neutrophils, reduction in naïve T cells, and increased expression of costimulatory molecules on antigen presenting cells. When we examined cytokine production in response to ex vivo stimulation of mesenteric lymph node cells with 8 bacterial and fungal stimuli, the mesenteric lymph node cells from re-wilded mice are poised for different responses via robust induction of cytokines than laboratory mice. By using machine learning models, we are integrating these multi-omic datasets to better predict the association between environmental and host genetic factors on driving heterogeneity of immune responses in the re-wilded mice.
Scalable and comprehensive characterization of antigen-specific CD8 T cells using multi-omics single cell analysis
Authors: Boutet Stephane C , Walter Dagmar , Stubbington Michael J T , Pfeiffer Katherine A , Lee Josephine Y , Taylor Sarah E B , Montesclaros Luz , Lau Julia K , Riordan Daniel P , Barrio Alvaro Martinez , Brix Liselotte , Jacobsen Kivin , Yeung Bertrand , Zhao Xinfang , Mikkelsen Tarjei S
Published: January 2023

Understanding the antigen binding specificities of lymphocytes is key to the development of effective therapeutics for cancers and infectious diseases. Recent technological advancements have enabled the integration of simultaneous cell-surface protein, transcriptome, immune repertoire and antigen specificity measurements at single cell resolution, providing comprehensive, scalable, high-throughput characterization of immune cells.

Using the 10x Genomics Single Cell Immune Profiling Solution with Feature Barcoding technology with 14 oligo-conjugated antibodies and 50 Immudex peptide-MHC I Dextramer reagents (pMHC) panels spanning different CMV, EBV, Influenza, HIV and Cancer antigens, we performed multi-omic characterization of ~100,000 CD8+ T cells from four MHC-matched donors. The multi-omic combination of gene expression, paired alpha/beta T cell receptor (TCR) repertoire, cell surface proteins and pMHC binding specificity allowed the identification of CD8+ T cell subpopulations with specificity for pMHCs within our panel. We observed multiple TCRs that bound the same pMHC and identified enriched amino acid motifs within TCR sequences that shared specificities. We compared the CDR3 amino acid sequences of the pMHC-specific TCR clonotypes with previously reported sequences with the same binding specificities to show that we could identify new and known CDR3 sequences. This analytical framework provides a systematic and scalable method for deciphering TCR–pMHC specificity combined with cellular phenotype identity which is critical for developing a better understanding of the adaptive immune response to cancer and infectious diseases and will be key in the development of successful immunotherapies.
Expression of Interferon Genes is Influenced by Sex Hormones in SLE
Authors: Singh Ram Pyare , Hahn Bevra H
Published: January 2023

Environmental factors, genetic defects, miRNA dysregulation, and hormonal factors can influence susceptibility to SLE. Estradiol has been shown to increase the expression of interferon genes and pathways and thus could promote susceptibility to the disease in women. Recent evidence suggests the existence of a nexus between inflammatory pathways and 17b-estradiol, resulting in increased interferon stimulated genes (ISGs), autoantibodies and dysregulation of immune cells in SLE. The mechanisms by which 17b-estradiol interacts with IFN and various immune cells in SLE is poorly understood.

The goal of this study is to determine the role of estradiol in the regulation of candidate target genes, miRNAs, and mechanisms of regulation of autoantibody production in SLE. Women may be more susceptible than men to SLE and other autoimmune diseases in part because many healthy women have higher interferon-regulated genes. We found that plasma estradiol levels positively correlated with levels of serum/plasma IL-6 and 21/IL-23 in SLE patients. Plasma 17b-estradiol levels are significantly increased in female SLE patients compared to healthy females (p<0.01). Furthermore, we found that estradiol increases pro-inflammatory cytokines, and level of estradiol positively correlated with expression of miR21, 25, and 186 in SLE patients. Expression of miR21, 25, and 186 are positively correlated with SLEDAI score in SLE.

We also found that expression of interferon genes (IL-12, 17, 21) is influenced by sex hormones in SLE. Understanding the role of 17b-estradiol in the regulation of pro-inflammatory pathways, ISGs, and microRNAs will further advance our understanding of SLE disease pathology and may identify additional novel therapeutic targets.
Characterization and Optimization of SLE Mouse Models for the Evaluation of Immunomodulation Therapeutics
Authors: Case Laure , Knowlton Hannah , Christianson Greg , Vann Lewis , Lutz Cathleen
Published: January 2023

Systemic Lupus Erythematosus is a chronic autoimmune disease that causes systemic damage throughout the body and is characterized by a broad spectrum of clinical manifestations. The pathogenic mechanisms underlying the disease remain unclear, but are thought to be the result of gene-by-environment interactions that lead to the breakdown of self-tolerance in susceptible individuals. Mouse models of SLE have been instrumental in our understanding of the genetic and cellular requirements for SLE induction, and represent a valuable resource for testing SLE therapies and drug candidates in vivo. No one mouse model completely recapitulates the complexity of the human disease, thus it is crucial to evaluate the mode of action and therapeutic efficacy of targeted therapeutics in multiple SLE mouse models. Here we demonstrate an effective program for screening therapeutic candidates using two well-established SLE mouse models, NZBWF1/J mice and MRL/MpJ-Faslpr/J mice, which mimic the genetic and pathophysiological aspects of the human disease. We characterize the disease course in each model in order to optimize the treatment schedule for either prophylactic or therapeutics drug efficacy studies. Using the immunosuppressant drug cyclophosphamide, we longitudinally compare the clinical measures of the disease to immune cell profiles during both in-life and at study terminus. This program provides a valuable resource for the investigation into the specific effects novel therapeutics have on immune homeostasis.
MAP4K3/GLK-induced AhR-RORγt complex is a novel therapeutic target for GLKhighIL-17Ahigh subpopulation of SLE patients
Authors: Chuang Huai-Chia , Tan Tse-Hua
Published: January 2023

The cytokine IL-17A plays critical roles in the pathogenesis of autoimmune diseases. The frequencies of MAP4K3 (also named GLK)-overexpressing T cells are correlated with disease severity of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and adult-onset Still’s disease. T-cell specific GLK transgenic mice develop spontaneous autoimmune responses. GLK signaling selectively stimulates IL-17A production in murine T cells through inducing AhR-RORγt complex formation. Here, we investigated whether GLK-induced AhR-RORγt complex in T cell is a therapeutic target for human SLE or RA. The population of GLK+IL-17A+ T cells was enhanced in the peripheral blood from SLE patients compared to that of healthy controls using flow cytometry. The ROC curve analyses showed that increased GLK+IL-17A+ T cell population in peripheral blood reflected an active stage of SLE. In addition, peripheral blood T cells from SLE or RA patients displayed induction of RORγt phosphorylation and the AhR-RORγt complex. Moreover, we identified a small-molecule GLK inhibitor that inhibited GLK kinase activity and AhR-RORγt interaction. The GLK inhibitor suppressed the disease severity in autoimmune mouse models and IL-17A production in T cells from SLE or RA patients. Collectively, GLK-induced AhR-RORγt complex is a therapeutic target for the GLKhighIL-17Ahigh subpopulation of SLE or RA patients.
Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis
Authors: Raffin Caroline , Muller Yannick , Barragan Jessica , Zhou Yu , Piccoli Luca , Lanzavecchia Antonio , Tareen Semih U , Fontenot Jason D , Bluestone Jeffrey A
Published: January 2023

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damage. Regulatory T cells (Tregs) are defective in RA patients while adoptive transfer of expanded autologous Tregs efficiently reverse disease in collagen-induced arthritis, an animal model of RA. Moreover, providing an antigen (Ag) specificity to Tregs render them more potent to suppress abnormal inflammation by increasing specific activity and promoting selective migration to the Ag-expressing tissue, reducing the risk of pan-immunosuppression in the meantime. These studies strongly suggest that Treg therapy may be effective in the treatment of RA patients by reducing joint inflammation and inducing immune tolerance and may be improved by using Ag-specific Tregs. Therefore, we aimed to engineer Ag-specific Tregs from polyclonal Tregs using a chimeric antigen receptor (CAR) specifically targeting an Ag present in the joint of RA patients. We generated a CAR directed against a post-translationally modified intermediate filament protein, citrullinated vimentin (CV), an Ag found, abundantly and almost exclusively, in the extracellular matrix of the inflamed synovial tissue of the RA patients. CV-specific CAR was transduced into human Tregs that were efficiently activated, expanding and suppressing following CAR-mediated stimulation. Importantly, CV-CAR Tregs were shown to react with CV expressed in RA patient synovial fluid. Studies are underway to demonstrate the functional activity of these CV-CAR Tregs in in vivo mouse models, a validation step in the development of a promising therapeutic tool to treat RA and potentially other autoimmune disorders.
Phenotypical and functional analysis of natural killer cells from primary human head and neck squamous cell carcinomas
Authors: Korrer Michael J , Kim Young
Published: January 2023

The purpose of this study was to determine if natural killer (NK) cells from primary human head and neck squamous cell carcinomas (HNSCC) expressed altered phenotypical and functional capabilities compared to matched NK cells from the periphery. NK cells are a critical part of the host defense against cancer through direct cytotoxicity of transformed cells and the release of pro-inflammatory cytokines. To facilitate the targeting of NK cells for immunotherapy we phenotyped human HNSCC NK cells using flow cytometry and RNA-SEQ analysis of matched flow-sorted PBMC and tumor infiltrating NK cells.

We found that tumor infiltrating NK cells of HNSCC patients, significantly downregulated activating receptors NKG2D, DNAM-1, NKp30, CD16 and 2B4 compared to matched blood NK cells (n=13). Surprisingly, the majority of inhibitory KIR receptors on tumor-infiltrating NK cells were decreased. However, the inhibitory receptors NKG2A and PD-1 were found to be highly overexpressed in tumor-associated NK cells. In addition, RNA-SEQ analysis of flow sorted NK cell populations from matched blood and TIL of HNSCC patients (n=9) demonstrated significant differential expression of multiple immune pathways. Functional assays confirmed that tumor infiltrating NK cells have reduced cytotoxicity and produce significantly less IFNγ after ex-vivo stimulation (n=10).

We believe that this study provides the first in-depth look at NK cells from primary HNSCC tumors. We found that tumor associated NK cells express a profoundly different phenotype than NK cells from the blood of the same patients, with multiple activating receptors downregulated and the inhibitory receptors NKG2A and PD-1 overexpressed.
Exosomal communication by metastatic osteosarcoma cells contributes to the modulation of alveolar macrophages to an M2 tumor-promoting phenotype
Authors: Wolf-Dennen Kerri K , Gordon Nancy , Kleinerman Eugenie S
Published: January 2023

Osteosarcoma (OS) metastasizes to the lung and there is a link between tumor-promoting M2 macrophage content and poorer patient survival. Conversely, high M1 content correlates with improved survival. M2 macrophages are immunosuppressive, contribute to tumor progression and metastasis, and can be induced by various stimuli in the tumor microenvironment, including exosomes (Exo). Exo are small 40–150 nm vesicles that are involved in inter-cellular communication, and contribute to tumor progression, increased tumor survival and immune evasion. Because of the link between M2 content and inferior patient survival, we evaluated the effect of Exo from non-metastatic K7 OS cells and the metastatic subline K7M3 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophage cells with K7M3 Exo induced mRNA expression of IL10, TGFB2, IL1rα, and CCL22, (markers of M2 macrophages), and decreased phagocytosis, efferocytosis and macrophage-mediated tumor cell killing. In contrast, Exo from non-metastatic K7 or 3T3 normal fibroblast cells did not inhibit phagocytosis, efferocytosis or macrophage-mediated tumor cell killing. Our data suggest that the Exo from metastatic OS cells can modulate cellular signaling of tumor associated macrophages, thereby promoting the M2 phenotype and inhibiting their phagocytic and cytotoxic abilities. Identifying the factors within Exo from metastatic OS cells and the mechanisms responsible for Exo communication between metastatic OS cells and macrophages could reveal new therapeutic targets and opportunities to improve the treatment of patients with metastatic disease in the lung.
Frequency of Myeloid Derived Suppressor Cells in Spleen and Peripheral Blood from Cancer Patients
Authors: Talmadge James E , Cole Kathryn , Ly Quan , Hollingsworth Michael , Cox Jesse
Published: January 2023

The peripheral blood (PB) of cancer patients is frequently neutrophilic and lymphopenic with few studies focused on tissue immunity. In this study we examined eight paired sets of PB and spleen populations to assess the frequency and sub-populations of myeloid derived suppressor- (MDSC) and T-cells. These studies observed a significant decrease in the frequency of granulocytic (G), monocytic (M) and immature MDSC’s in the spleen vs PB (4.8±1.6 vs. 54.4±6.1; 0.2±0.1 vs. 0.9±0.4; and 0.6±0.1 vs. 2.3±0.8 % respectively) but not T-regulatory cells (0.8±0.5 vs. 0.1±0.04 percent). The difference in the G vs M MDSC was consistent with arginase and nitric oxide synthase expression. In contrast, the % of splenic T-cells was increased relative to the PB including CD3+ (35±5 vs. 16±4%), CD4+ (19±2 vs. 10±2%) and CD8+ (10±1 vs. 5±2%). The frequency of transitional memory T-cells (Ttm) was significantly higher in the spleen vs. PB (37.5±6 vs. 26±3.0%) as was the frequency of PD1+CD8+ T-cells (8±2 vs 1±0.5% respectively). The increase in Ttm with their heightened proliferation, effector potential and tumor specific cytotoxicity of PD1+CD8+ cells support the superiority of spleen vs PB cells for adoptive cellular therapy. In support of this both the PD1+ cells were LAG3− and TIM3−, and thus not exhausted. In summary, the spleen cells of cancer patients have an increase in tumor specific effector cells with proliferative potential supporting their utility in therapeutic protocols.
Non-small cell lung cancer tumor microenvironment induces Arginase1 in neutrophils via the Toll-like receptor 2 signaling pathway
Authors: Zhang Huajia , Kargl Julia , Zhu Xiaodong , Wang Xiaoxia , Cao Jianhong , Houghton A. McGarry
Published: January 2023

Neutrophils dominate the tumor microenvironment of non-small cell lung cancer. Previous studies show neutrophils suppress T cells and promote immunosuppression. The objective of the current study is to identify how tumor associated neutrophils (TAN) adopt the T-cell suppressive entity after being recruited into the tumor microenvironment.

In the murine lung squamous cell carcinoma autochthonous tumor model (AdCre-Lkb1fl/fl, Ptenfl/fl; LP) and subcutaneous tumor model (Lewis lung carcinoma cell line; LLC), we found that Arginase 1 (Arg1) mRNA level in TAN is drastically higher than neutrophils from the bone marrow, spleen and peripheral blood. Arg1 suppresses T cell through Arginine depletion. Whole tumor lysates from LP tumor and LLC tumor induce Arg1 level in bone marrow-derived and peripheral neutrophils from healthy B6 mice in vitro, suggesting that the tumor microenvironment alters neutrophil properties. To identify the putative molecule(s) that induce Arg1 in TAN, we used fast protein liquid chromatography (FPLC) and mass spectrometry to analyze the whole tumor lysates from LP tumor and LLC tumor. Annexin A1 and 2 are among the common molecules that appear in Arg1-inducing FPLC fraction in both LP and LLC tumor lysates. Annexin A1, A2 are involved in toll-like receptor signaling. Flow cytometry showed more than 70% of Arg1+ TAN are either TLR2 or TLR4 positive. Interestingly, only TLR2 but not TLR4 inhibition blocked Arg1 induction by whole tumor lysates in naïve neutrophils. Taken together, tumor microenvironment induces Arg1 level in neutrophils in a TLR2 dependent manner. TLR2 is a putative therapeutic target for blocking tumor associated neutrophil-mediated immunosuppression in non-small cell lung cancer.
Single cell RNA sequencing reveals distinct gene expression signatures of myeloidderived suppressor cells in breast cancer
Authors: Alshetaiwi Hamad , Pervolarakis Nicholas , McIntyre Laura L. , Ma Dennis , Nguyen Quy , Nee Kevin , Rath Jan , Evans Katrina , Torosian Leona , Silva Anushka , Walsh Craig , Kessenbrock Kai
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells with potent immune suppressive activity. MDSCs regulate anti-tumor immunity by suppressing T cell proliferation. MDSCs can be further classified into granulocytic MDSCs (G-MDSCs) and monocytic MDSC (M-MDSCs). However, there is a lack of MDSC-specific markers since MDSCs, normal neutrophil granulocytes, and monocytes are defined by the same flow cytometry markers (CD11b+Gr1+). We used a breast cancer mouse model (MMTV-PyMT) to define cellular and molecular properties of MDSCs in single cell resolution. To test the capacity of MDSCs to inhibit immune responses, CD11b+Gr1+ cells from PyMT and wild type (WT) mice were sorted by fluorescence-activated cell sorting (FACS) from bone marrow, lung, and spleen, and then subjected to a T cell activation assay in co-culture with T cells. We found that predominantly spleen-derived CD11b+Gr1+ cells from PyMT significantly suppressed CD4 and CD8 T cell proliferation, while CD11b+Gr1+cells from bone marrow and lung showed no effect on T cell proliferation. Thus, we focused on spleen-derived CD11b+Gr1+ cells and utilized single cell RNA sequencing (scRNAseq) to compare breast cancer derived MDSCs to the respective cell populations harvested from normal, and non-tumor bearing hosts. Our computational analysis of 14,646 single cell transcriptomes revealed a separate G-MDSCs cluster from normal neutrophils and a M-MDSCs cluster from normal monocytes and a distinct gene expression signature for the entire MDSCs population. Our studies provide crucial insights into the biology of MDSCs, which may ultimately form the basis for novel marker and therapeutic avenues to improve cancer immunotherapy.
PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleicacid sensing
Authors: Nemazee David , Gavin Amanda L , Huang Deli
Published: January 2023

Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer’s disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins that we recently discovered are nucleases. PLD4-deficient mice have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes are blocked in the absence of TLR9. PLD3−/− mice have a relatively benign phenotype. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which was blocked on a Unc93b-3d/3d background. These data suggest that PLD3 and PLD4 enzymes play important roles in the regulation of endosomal TLR signaling.