bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of Virostatic genes in modulating Immune Checkpoints in Epstein-Barr Virus associated Tumors
Authors: Chakravorty Srishti , Yan Bingyu , Chauss Daniel , Wang Luopin , Canaria D. Alejandro , Jethava Krupal , Chopra Gaurav , Briggs Scott D , Zhao Bo , Olson Matthew R. , Afzali Behdad , Kazemian Majid
Published: January 2023

Epstein-Barr Virus (EBV) is a complex oncovirus that infects around 90% of adults. Most primary infections remain asymptomatic but result in lifelong infection. EBV can drive lethal malignancies of both lymphoid (e.g. Burkitt lymphoma) and epithelial origins (e.g. Stomach Adenocarcinoma). Despite decades of research, the exact molecular mechanisms of EBV and host immune system interactions that lead to tumorigenesis remain elusive.

Through systematic analyses of high-throughput sequencing data from >1000 patients with different cancer types, we identified twelve EBV transcripts, including LMP1 and LMP2, which when overexpressed, significantly suppressed viral reactivation. These ‘virostatic genes’ had frequent missense and nonsense variations in cancer patients, disrupting their ability to restrict EBV reactivation. We also found that viral reactivation by classical chemical inducers up-regulates the expression of Programmed Death-Ligand 1 (PD-L1). Further analyses of the same RNA-seq dataset classified EBV+ tumors into two molecular subtypes based on expression pattern of upstream regulatory immune-related genes and Immune Checkpoint (IC) markers, including PD-L1. Overexpression of virostatic genes in lymphoblastic cells significantly repressed cellular PD-L1 transcription, leading us to hypothesize that a combination of IC and virostatic gene modulation may have synergistic benefits for immunotherapy of EBV-associated cancers.
Depression is more severe among females with autoimmune conditions
Authors: Ramirez Francisco E , Maghelli Giorgia , Nedley Neil
Published: January 2023

Rationale

We explore the relationship between severity of depression on participants with autoimmune conditions and its relationship with gender.

Methods

Participants from a non medical community educational program that finished the intervention and had an autoimmune condition were used in the study. Participants met once a week for 8 weeks for a 2-hour program, which educated them about healthy lifestyle habits like diet, exercise, control of thoughts, etc. Each patient took the Depression and Anxiety Assessment Test (DAAT registration TX 7-398-022) before and after the intervention. The questionnaire measured depression and asked about demographics, patient history and research consent.

Results

From n=5651 people that finished the intervention n=432 participants reported autoimmune disease and were used in the study, n=369 (85%) were female and n=64 (15%) were male.

At baseline regarding depression severity, female participants n=116 (32%) reported severe depression, n=130 (35%) moderate, n=60 (16%) mild, and n=62 (17%) none. Male participants on the other hand reported n=63 (22%) severe depression, n=14 (29%) moderate, n=18 (30%) mild, and n=12 (19%) none. At the end of the program, females reported the depression reported in the group was n=22 (5.9%) severe depression, moderate n=94 (26%), mild n=61 (17%), and n=191 (52%) none. For males, end group depression distribution was n=4 (6.3%) severe depression, n=11 (17%) moderate, n=15 (24%) mild, and n=33 (52%) none.

Conclusions

More women with autoimmune disease seem to have more severe depression than men, but both genders improved at the end of the program. Further follow up is being planned.
Characterizing the TCR repertoire in Neuropsychiatric Lupus
Authors: Moore Erica , Huang Michelle , Putterman Chaim
Published: January 2023

Approximately 40% of lupus patients experience neurological and/or psychiatric disorders. However, the mechanisms contributing to the wide range of neuropsychiatric manifestations (NPSLE) have not yet been fully elucidated. We have demonstrated that the choroid plexus (CP) in the MRL/lpr NPSLE lupus mouse model is heavily infiltrated with effector T cells. We investigated T cell receptor (TCR) usage and repertoire diversity in CP infiltrating T cells, to enhance our understanding of this T cell population and its possible contribution to neuropsychiatric disease. The CP, salivary glands, and spleens were dissected from female MRL/lpr mice (n=9, n=9, n=4 respectively) and the spleens from female MRL/MpJ (n=5) at 17–22 weeks of age, at which time MRL/lpr mice display prominent neurobehavioral deficits. The TCR repertoire was evaluated by isolating genomic DNA, amplifying the CDR3 sequence by multiplex PCR (Adaptive Technologies), and analyzing the sequences with the Adaptive Technologies immunoSeq Analyzer and the R software, LymphoSeq. MRL/lpr CP tissues had increased numbers of T cell sequences, unique productive sequences, and clonality. The top 10 productive rearrangements were more abundant in MRL/lpr CP when compared to the MRL/lpr and MRL/MpJ splenic tissues (p = 0.0673 and p = 0.0003 respectively). The MRL/lpr CP had an increased frequency of TCRBV02, TCRBV04, TCRBV12, and TCRBV26 genes compared to MRL/lpr splenic and salivary gland tissues. Despite the lymphoproliferation found in this lupus strain, the increased oligoclonal expansion and limited TCR V beta gene usage in the MRL/lpr CP T cell repertoire, as compared to other MRL/lpr tissues, implies a distinct and specific infiltration of T cells into the CP.
Modulation of treg and th17 immunity in myeloid leukemia for therapeutic intervention
Authors: Camacho Virginia , Matkins Victoria , Patel Sweta , Anderson Ashlyn , Ferrell Paul Brent , Weaver Casey Todd , Harrington Laurie E , Welner Robert S
Published: January 2023

Non-immune roles of Regulatory T cells (Tregs) have been described across various tissues. However, the mechanisms by which T cells regulate the bone marrow in myeloproliferative neoplasms lack resolution. Recently, we outlined how Tregs in bone marrow condition the stromal cell niche and enhance hematopoietic support via IL-10 secretion. A hallmark of leukemic progression is the increase in pro-inflammatory cytokines and cellular inflammation. In this study, we demonstrate that this inflammatory milieu promotes pathogenic skewing of both regulatory and effector T cells. We performed a comprehensive characterization of the CD4 T cell compartment in myeloproliferative neoplasms such as chronic myeloid leukemia (CML) and we found that Tregs are unstable and dysregulated the leukemic marrow. This contributes to a phenotype of immune activation as well as an increase of pathogenic inflammatory T cells. Specifically, our findings reveal that Treg Th17 ratios are disrupted in leukemia and that Th17 cells influence disease progression. Additionally, we found that Treg-secreted IL10 has beneficial effects on the functions of healthy and diseased mesenchymal stromal cells (MSCs). We demonstrate that restoring Treg function and expanding their IL10 production can be used therapeutically to delay leukemic burden. Our results support the hypothesis that Tregs are compromised during leukemic progression as a consequence of pro-inflammatory cytokines from the myeloid associated disease. These findings highlight how the Treg Th17 balance regulates disease progression. Furthermore, these studies provide an avenue by which Treg secreted IL10 can be used for therapeutic intervention in the context of heme malignancy.
Role of β-Adrenergic Receptor Kinases in the Internalization of C-C Chemokine Receptor Seven (CCR7) in Mammalian T Cells
Authors: Bill Colin A , Sanchez Anahi , Amparan Jailene , Vines Charlotte M
Published: January 2023

Ligand mediated GPCR internalization/signaling is regulated by G protein-coupled receptor kinase (GRK) induced receptor phosphorylation and recruitment of β arrestins. The GPCR chemokine receptor, CCR7, has two ligands, CCL19 and CCL21 that mediate chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct internalization/signaling pathways are activated in T cells depending upon which ligand is bound to the receptor. It remains unclear to what extent the β-Adrenergic Receptor Kinase family, GRK2 and GRK3 regulate CCR7 internalization.

Incubation of primary mouse T cells or human T cell lines, Hut 78 and CEM with 200 nM CCL19, but not CCL21 led to rapid CCR7 internalization. To determine the roles of GRK2 and GRK3 in CCR7 internalization, we co-transfected HEK cells with plasmids expressing either GRK2 or GRK3 along with arrestin-3, which we have previously shown is required for CCR7 internalization. Immunofluorescence microscopy showed that CCR7 co-localized with arrestin-3 in cells co-transfected with GRK2 after 30 minutes incubation with CCL19, but not CCL21. Limited co-localization of CCR7/arrestin-3 was observed in cells co-transfected with GRK3. Further CCL19/CCR7 incubation studies showed that GRK2-dependent CCR7 internalization was dependent upon the expression level of GRK2 with high protein levels being inhibitory. To further assess the role of GRK2, we used a GRK2 kinase dead mutant (GRK2-K220R) that does not facilitate the internalization of CCR7, GRK2-K220R did, however, result in some clustering of CCR7/arrestin-3. Overall, these data suggest that GRK2 plays an important role in CCR7 internalization in mammalian T cells; however, other GRKs are likely involved in this process.
Improvements in Ultra-Sentive Single Molecule Assays to Extend Quantitative Measurement of Cytokines in Plasma and Serum at Attogram per mL Concentrations
Authors: Lambert Jeremy , Duffy David , Rissin David , Kan Cheuk , Tobos Carmen , Wiener Alexander , Meyer Ray
Published: January 2023

The concentration of cytokines in blood are often at pictogram per mL and sub-picogram per mL. Recent advances in the sensitivity of immunoassays, such as single molecule arrays (Simoa), have allowed the detection of many of these cytokines that were previously undetectable in blood. While the limits of detection (LOD) of these new technologies are in the femtogram per mL range, the concentrations of cytokines in many healthy individuals can still not be detected. In fact, the quantitative measurement of several important cytokines are <50% in healthy individuals. We will report on advances to improve the sensitivity of the Simoa technology into the attogram per mL range that improve the quantitative measurement of a broad range of cytokines.
Catalog of ex vivo whole blood stimulation conditions to generate species-specific cytokine controls
Authors: Repic Allison M , Rowse Amber L , Bruder Eric , Frantz Aric M
Published: January 2023

Commercially available reagents for the detection of cytokines in pre-clinical species often rely on cross-reactivity from other species. Species-specific cytokine control materials are ideal for use in determining the suitability of the cross-reactive reagents within the intended pre-clinical species. To generate species specific cytokine positive control materials in a non-invasive manner, a systematic catalog of ex vivo whole blood mitogen stimulation and collection conditions was developed. Since naïve matrix (plasma or serum) is unlikely to have cytokines detectable above the lower limit of quantitation (LLOQ), ex vivo stimulation of whole blood using a variety of mitogens was performed in order to produce measureable cytokine release. Plasma harvested at specific time points from these stimulations was then spiked into naïve (species matched) plasma or serum to achieve analyte concentrations within the analytical measurement range of the assay. This ‘spike-in’ sample serves as a model of the intended test system, and allows for evaluation of the ability to detect species-specific cytokines within both plasma and serum matrices. A range of detection was determined for a number of analytes using a range of concentrations of spike-in using commercially available multiplex assays. By producing this catalog of stimulation conditions including multiple mitogens and durations of challenge for optimized collection per analyte, we are able to quickly generate positive control materials for a wide range of analytes across multiple species and thereby have simplified and streamlined the approach to generating these much-needed materials in the preclinical testing laboratory.
Interleukin-22 alleviates Angiotensin II-induced cardiomyocyte apoptosis by preventing the intrinsic mitochondrial pathway
Authors: Xia Lin , Che Yang , Su Zhaoliang
Published: January 2023

Background

Cardiac apoptosis plays an important role in the development of heart failure, and Angiotensin II (Ang II) plays a central role in the pathogenesis of RAAS-triggered cardiac apoptosis. Interleukin (IL)-22 is an inflammatory cytokine, which is involved in cardiovascular diseases. Recent evidence has demonstrated that there is a close relationship between IL-22 and human hypertension. However, the effects of IL-22 on Ang II-induced cardiomyocyte apoptosis remain unknown.

Methods

MCM cells and primary cardiomyocytes were exposed to Ang II with or without IL-22. The activation of the apoptosis signaling pathways, reactive oxygen species (ROS), supernatant TNF-α, Fas/FsaL, mitochondrial membrane potential (MMP), cytochrome c (Cyc) and the activity of superoxide diamutase (SOD) were detected.

Results

In MCM cells and primary cardiomyocytes, flow cytometry revealed that IL-22 significantly alleviated Ang II-induced apoptosis, and IL-22 receptors (IL-22R1 and IL-10R1) levels were obviously increased. Western blotting revealed that increased cleaved Caspase 3/8 and Bax protein levels were clearly reduced after IL-22 treatment. The Fas/FsaL and TNF-α levels were identical in the cells with or without IL-22 treatment. But ROS levels were markedly reduced after IL-22 treatment via upregulation of SOD activity, and Ang II-induced decrease of MMP was improved by IL-22, reducing the release of Cyc and the levels of cleaved Caspase 9.

Conclusions

IL-22 improved Ang II-induced cardiomyocyte apoptosis via inhibiting the intrinsic apoptosis pathway.
Th17-derived cytokines synergistically enhance IL-17C production by the colonic epithelium
Authors: Swedik Stephanie M , Madola Abson , Levine Alan
Published: January 2023

Tightly regulated communication between the gastrointestinal (GI) epithelium and immune cells in the underlying lamina propria is critical for immune homeostasis. Interleukin (IL)-17C, produced by epithelial cells after exposure to inflammatory stimuli, facilitates cell-to-cell communication by promoting pro-inflammatory responses in Th17 cells. Since Th17-derived cytokines induce the expression of IL-17C, we hypothesize that IL-17C participates in an inflammatory amplification loop with Th17-derived cytokines, contributing to GI disease. We report that a Th17-derived cytokine TNFα induces IL-17C mRNA and protein expression in human colonic epithelial cell lines via an NFκB dependent pathway. Co-stimulation of TNFα-treated cells with Th17-derived IL-17A or IL-22 induces a respective 2,000- and 1,000-fold increase in IL-17C mRNA and protein. All three cytokines stimulate an 11,000-fold increase of IL-17C. While neither IL-17A nor IL-22 alone induce IL-17C synthesis, their presence at culture initiation is necessary to achieve synergy, suggesting that they stimulate the cells independently from TNFα. Inhibition of NFκB activation reduces IL-17C production by 99.99%, confirming that TNFα is the primary stimulant. Inhibition of AP-1 reduces IL-17C mRNA synthesis induced by pairwise or triple cytokine exposure to levels equivalent to that of TNFα alone, suggesting a role for this transcription factor in the synergistic response. IL-17A-mediated RNA stability was shown not to contribute to the mechanism of synergy. Taken together, cooperation between the NFκB and AP-1 signaling pathways in colonic epithelial cells may mediate the synergy observed in this novel pro-inflammatory feed-forward loop in the GI tract.
Detoxified dental healing abutments trigger an inflammatory response in-vitro
Authors: Valverde Araceli Maria , Narvekar Aniruddh Nitin , Naqvi Afsar Raza , Nares Salvador
Published: January 2023

Healing abutment (HA) is a titanium screw used in implant dentistry to replace missing teeth. Although designed for single procedure, HA are frequently reused. Pathogen Associated Molecular Patterns (PAMPs), present on HA can elicit immune responses in vivo and may lead to implant failure. The aim of this study is to test four commonly employed protocols for HA detoxification. Forty HA collected from subjects with prior implant placement were randomly distributed and detoxified using four protocols (A–D, N=10/group). Group A: Autoclave sterilized, Group B: Ultrasonic bath + autoclave, Group C: Debrided by prophy jet and glycine powder + autoclave, Group D: Debrided using a non-scratch scrub sponge + autoclave, Control: Unused. Residual protein quantification (N=3/group) and macroscopic debris evaluation (N=2/group) was assessed by Protein BCA Assay Kit and Phloxine B staining, respectively. Human primary monocyte-derived macrophages (Mϕ) were challenged with HA (N=5/group) and supernatants were collected at 4, 24, 48 and 120 h to analyze ten cytokines by multiplex bead assay. Control group did not show any detectable protein, debris or cytokine production. Among the treatments, protocol C and D were highly effective in decontamination as observed by reduced protein and debris quantification. Protocol C showed reduced cytokine (TNF-α, IL-8, IL-6, IL-10 and CXCL-10) secretion both at early and late time points compared to other decontamination protocols (A, B or D). The most effective detoxification method for HA is Protocol C. Compared to unused HA, none of the protocols completely decontaminated HA. Potent immunogenicity of decontaminated HA suggests that the practice to reuse HA should be reconsidered.
TNFR2 signaling enhances ILC2 survival, function and induction of airway hyperreactivity
Authors: Hurrell Benjamin P , Galle-Treger Lauriane , Jahani Pedram Shafiei , Howard Emily , Helou Doumet Georges , Banie Homayon , Soroosh Pejman , Akbari Omid
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) can initiate pathologic inflammation in allergic asthma by secreting copious amounts of type-2 cytokines, promoting lung eosinophilia and airway hyperreactivity (AHR), a cardinal feature of asthma. We discovered that the TNF/TNFR2 axis is a central immune checkpoint in murine and human ILC2s. TNFa is a pleiotropic proinflammatory cytokine which is elevated in the airways of patients with severe asthma, signaling through two main receptors with opposing functions: TNFR1 and TNFR2. We found that murine ILC2s selectively express and induce TNFR2 upon IL-33 activation, whereas they fail to express – or induce – TNFR1. Strikingly, blocking the TNF/TNFR2 axis inhibits ILC2 survival, cytokine production, ILC2-dependent AHR and airway eosinophilia. The mechanism of action of TNFR2 in ILC2s is through utilizing non-canonical NFkB pathway as a NFkB inducing kinase (NIK) inhibitor blocks the costimulatory effects of TNFa both in vitro and in vivo. Similarly, human ILC2s selectively express TNFR2 and using the model of humanized mice that our laboratory recently developed, we show that TNFR2 engagement in human ILC2s enhances survival and activation, ultimately promoting AHR through a NIK-dependent pathway. These findings highlight the role of the TNF/TNFR2 axis in pulmonary ILC2s, suggesting that targeting TNFR2 or relevant signaling represents a novel strategy for treating patients with ILC2-dependent asthma.
High fat diet facilitate duffy antigen receptor for chemokine (DARC) expression in mononuclear cells by IL-22 dependent manner
Authors: Lee Hyung Keun , Kim Eun Young , Chang Eun Ju
Published: January 2023

Though interleukin-22 (IL-22) is recently suggested to play a role in immune-inflammatory system, however, the specific role of IL-22 for accumulation of adipose tissue in the context of obesity and its metabolic consequences has not been clearly documented. The purpose of this study is determine the role of IL-22 on adipose tissue accumulation and inflammation through mouse and human model. We here identified a previously unknown subset of monocyte-derived Duffy antigen receptors for chemokines (DARC)+ macrophages in epididymal fat adipose tissue and found that they are preferentially recruited into the crown-like structures of adipose tissue in the mouse upon high fat diet-induced obesity. Importantly, DARC+ macrophages highly express the IL-22 receptor (IL-22Ra1). Exposure to recombinant IL-22 shifts macrophages to an alternative M2 polarization pathway and augments DARC expression via a STAT5b signaling axis. STAT5b directly binds to the DARC promoter and a STAT5 inhibitor abrogates the IL-22-mediated induction of DARC. These M2-like DARC+ subpopulations of monocytes/macrophages were elevated in obese db/db mice compared to WT lean mice. Furthermore, subsets of CD14+ and/or CD16+ monocytes/macrophages within human peripheral blood mononuclear cell populations express DARC and the prevalence of these subsets is enhanced by IL-22 stimuli. This suggested that IL-22 is a critical cytokine that promotes the infiltration of adipose tissue macrophages, which regulate inflammatory processes. Taken together, our present findings provide important insights into the molecular mechanism by which IL-22 signal modulates DARC expression in M2-like macrophages.
Artificial thymic organoids reveal an early block in T cell differentiation in patients carrying mutations in IKZF1
Authors: Bosticardo Marita , Pala Francesca , Kuehn Hye Sun , Montel-Hagen Amelie , Crooks Gay M , Rosenzweig Sergio D , Notarangelo Luigi D
Published: January 2023

IKZF1 is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. Heterozygous germline mutations in IKZF1 give rise to distinct clinical phenotypes. IKZF1 haploinsufficiency causes common variable immunodeficiency (CVID) associated with B cell immune deficiency, B-ALL susceptibility and autoimmune manifestations, without clinical T cell defects. Patients carrying dominant negative (DN) IKZF1 mutations have combined immunodeficiency (CID), with an increased proportion of naïve T cells and impaired T cell function. Mouse models of IKZF1 mutations have a distinct phenotype from patients and some models present a more severe defect in T cell development. To address these controversies and gain novel insights into the effects of distinct IKZF1 mutations on human T cell development, we used the artificial thymic organoid (ATO) system, based on the 3D aggregation and culture of a delta-like Notch ligand 4-expressing stromal cell line (MS5-Dll4) with CD34+ cells, to analyze T cell development from CD34+ cells obtained from one patient with IKZF1 haploinsufficiency and one patient with DN IKZF1 mutation. Both patients showed a similar early block in T-cell differentiation at pre-T cell stage. However, the patient with IKZF1 haploinsufficiency showed a more pronounced leakiness, with a residual production of CD3+TCRαβ+ cells, which could account for the milder T-cell phenotype. Interestingly, the DN patient presented an increased accumulation of CD4−CD8β− CD8αα+ cells. These results show an unexpected role for IKZF1 in humans in early stages of T-cell differentiation and indicate IKZF1 as a necessary factor for the induction of CD8β expression in T cells.
The phosphatidinositol-transfer protein Nir3 is a modulator of T cell development
Authors: Helou Ynes , Au-Yeung Byron B , Weiss Arthur
Published: January 2023

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) functions as a critical source of second messengers implicated in T cell activation. In T cells, hydrolysis of PIP2 by the lipid phosphatase PLCγ1 generates the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), resulting in PKC and Ras/MAPK activation as well as calcium increases that contribute to T cell responses. A secondary consequence of PLCγ1 activation is a transient reduction in PIP2 levels. The PI transfer proteins Nir2 and Nir3 have been shown to replenish PIP2 levels at the PM through delivery of the precursor PI from the endoplasmic reticulum (ER) via non-vesicular transport. In particular, Nir3 has been shown to replenish PI levels during basal signaling and in response to weak signaling. Given the importance of weak signaling in thymocyte positive selection, as well as the elevated levels of Nir3 mRNA during the double positive (DP) stage of thymocytes development (Immgen), we hypothesize that Nir3 is a regulator of PI homeostasis during thymocyte development. Using CRISPR-Cas9 technology, we have generated a Nir3 knockout mouse. DP thymocytes from Nir3 knockout mice have reduced basal calcium levels, as well as reduced TCR-induced calcium flux. Furthermore, introduction of the OT1 transgenic TCR into Nir3 knockout mice resulted in elevated numbers of DP thymocytes and reduced numbers of CD8SP thymocytes, suggesting a block in positive selection. Analysis of TCR signaling in Nir3 knockout OT1 thymocytes revealed attenuated calcium flux as well as defective induction of pERK, pAkt, and pS6. These findings are the first to characterize the function of PI transfer proteins and their importance specifically in T cell development.
Molecular cues dictating positive and negative selection in the thymus
Authors: McIntyre Laura Lynn , Steier Zoe , Lutes Lydia , Streets Aaron , Yosef Nir , Robey Ellen A
Published: January 2023

T cells develop in the thymus where CD4+CD8+ double positive (DP) thymocytes undergo positive and negative selection to generate mature CD4+ and CD8+ single positive (SP) T cells. Positive selection allows for the generation of a diverse repertoire of T cells capable of recognizing peptide:MHC (pMHC) complexes, while negative selection ensures that the mature T cell repertoire is self-tolerant. The cell types involved in thymic selection are well understood and recent work has begun to elucidate temporal and spatial aspects involved in selection. However, the intracellular cues regulating development and CD4 vs. CD8 lineage commitment are incompletely understood. Our work focuses on utilizing a single cell RNA sequencing (scRNA-seq) approach to elucidate the molecular cues guiding thymic selection of CD4+ and CD8+ T cells.
Visualizing thymic regulatory T cell development with single-cell RNA sequencing
Authors: Owen David L. , LaRue Rebecca S , Farrar Michael A
Published: January 2023

CD4SP thymocytes that receive moderate to strong TCR signals upregulate either FOXP3 or CD25, generating distinct Foxp3lo or CD25+ Treg progenitor (TRP) populations, respectively. Stimulation of these TRP populations with IL2 induces co-expression of CD25 and FOXP3, completing thymic Treg differentiation. The alterations in gene expression that drive these respective Treg differentiation programs, as well as the identification of agonist selected cells within conventional CD4SP thymocytes remain unclear. To address these questions, we performed single-cell RNA-Seq with paired TCR sequencing of conventional and Treg lineage CD4SP thymocytes. This data set contained cells of the entire range of maturity, from recent DP-CD4SP converts to mature CD4 T cells competent for thymic export. In conventional CD4SPs we were able to identify a subset of cells undergoing agonist selection that differentiates into TRP cells along with cells undergoing negative selection. The transcriptomes of the subsequent CD25+ and FOXP3lo TRP subsets were clearly distinct. For example, we found that the source of IL2 required for conversion of both TRP subsets into mature Tregs was uniquely found within the most immature CD25+ TRP undergoing agonist selection. Finally, TCR analysis demonstrated that some TCRs were enriched in CD4SP receiving agonist stimulation that gave rise to Tregs, or undergoing negative selection, while others were excluded from this pathway. We are currently carrying out spatial transcriptome studies to identify the specific localization of distinct Treg intermediates within the thymus. Interrogation of these single cell and spatial transcriptomic data sets will generate a comprehensive model of thymic Treg development.
Alteration in Three-Dimensional Microenvironment of Themis-deficient Murine Thymus Using Image Cytometry Approach
Authors: Aziz Nazihah Husna Abdul , Tan Yingrou , Brzostek Joanna , Ng Lai Guan , Gascoigne Nicholas R J
Published: January 2023

Themis is a T-cell intrinsic protein involved in selection during thymocyte development in the thymus. Thus, Themis-deficient mice have gravely reduced numbers of T-cells. Despite extensive research on thymocyte signalling, the exact role of Themis remains a mystery. As the thymic 3-dimensional (3D) microenvironment is a critical element in supporting thymocyte development and vice versa, we were interested to investigate the effect of Themis deficiency on thymic components and processes. In this study, we paired 3D imaging with conventional flow cytometry to understand thymic development in normal and Themis-deficient condition. Current conventional 2-dimensional (2D) histology and 3D reconstruction from 2D sections are unsatisfactory for studying thymus morphology. We thus established a novel approach for whole thymus 3D imaging to visualize developing thymocytes and supporting stroma, such as epithelial cells, myeloid cells and vasculature. 3D imaging coupled with quantitative analysis of images using image cytometry allows for objective comparison of the thymic microenvironment between sample groups. Here, we show an altered medullary and vasculature organization in Themis−/− thymus, suggesting a suboptimal 3D niche supporting thymocyte development. Correspondingly, mature single-positive thymocytes which are dependent on the medullary environment are altered phenotypically. In addition, we detected higher levels of apoptosis in Themis−/− cortex and an increase in clonally deleted double-positive thymocytes. Our findings demonstrate how Themis-deficiency affects developing thymocytes and its supporting stromal components.
Homeostatic Rho GTPase activation governs thymopoiesis
Authors: Kinsella Sinéad , Evandy Cindy A , Cooper Kirsten , Iovino Lorenzo , Jain Reema , deRoos Paul , Dudakov Jarrod A
Published: January 2023

The thymus is extremely sensitive to damage, resulting in attenuated T cell reconstitution and failure to elicit appropriate responses to infection, cancer and immunization. Although the thymus has a remarkable regenerative capacity, the mechanisms underlying this endogenous regeneration remain poorly understood. Previously we have identified two critical secreted factors that drive thymic regeneration, including BMP4 from endothelial cells (ECs) and IL-22 from dendritic cells (ILCs), which can be used as therapeutic regenerative strategies. Additionally, we have shown that steady-state CD4+CD8+ thymocytes (DPs) suppress levels of IL-23 (Science 336:91), a key regulator of IL-22; and as developing thymocytes undergo high levels of apoptosis, we hypothesize that apoptotic DPs mediate suppression of regenerative factors under homeostatic conditions, and damage-induced DP depletion removes this suppression, triggering thymic recovery. Here, we demonstrate a DP-specific reduction in cl-caspase 3 levels after acute injury in mice (total body irradiation, 550 cGy), and reduced Bmp4 expression in ECs co-cultured with apoptotic thymocytes, an effect rescued by apoptosis inhibition, using the pan-caspase inhibitor zVAD-FMK. We identified that apoptotic thymocytes mediate their suppression via TAM receptors in ECs and DCs, which subsequently activate Rho GTPases. Importantly, we show that inhibition of Rac1 activation increases Bmp4 and Il23 expression in ECs and DCs, respectively, and in vivo inhibition of Rac1 with EHT1864 enhances thymus cellularity in models of acute injury and age. These data describe a novel regulatory pathway that can be therapeutically targeted to boost T cell reconstitution after damage.
Ischemic Factors Precipitate a Shift in Immune Cell Populations at the Site of Cerebrovascular Blockage
Authors: Ortega Sterling B , Zevallos Cynthia B , Orozco Darko Q , Tranel Daniel , Karandikar Nitin J. , Ortega-Gutierrez Santiago
Published: January 2023

The incidence of stroke has risen over the past decade and will continue to be one of the leading causes of adult disability and death worldwide. Reperfusion therapy or endovascular thrombectomy (EVT) for large vessel occlusion (LVO) remains the only proven stroke therapy during the acute phase. But a select set of patients still exhibit poor recovery of neurological function, despite active cortical reorganization. The mechanisms that underlie this neurological dysfunction is still poorly understood. Subsequent to ischemic injury, a massive inflammatory response is seen for days, which exacerbates neuropathology in stroke models. Within the draining lymph nodes of ischemic patients, innate immune cells are actively phagocytizing brain antigen and presenting them to adaptive immune cells.

The focus of this project is to determine if early changes in immune cells near the site of occlusion can be used as predictors of long-term neurofunctional decline. Acute anterior circulation LVO patients treated with EVT will be enrolled in this study. At the time of thrombectomy, blood was collected proximal and distal to the thrombus prior to recanalization. As a control, blood was also collected from the femoral artery. Deep immune profiling of innate and adaptive immune cells using 13-parameter flow cytometry was performed revealing a specific increase in post-thrombus CD4 T-cells, as compared to pre-thrombus. Cytokine analysis revealed a decrease in pre- and post-thrombus IFN-γ and IL1α. Induction of in vitro hypoxia in healthy peripheral blood mononuclear cells revealed a similar reduction in IFN-γ.

We hypothesize that ischemia precipitates a shift in immune cells at the site of cerebrovascular blockage that modulates stroke recovery.
Chlorovirus Acceleration of Motor Neuron Disease in SOD1G93A Transgenic Mice may Clarify some of the Augmented anti-Chlorovirus Serology in Amyotrophic Lateral Sclerosis Patients
Authors: Petro Thomas M , Pattee Gary L. , Guinn Zachary P. , Agarkova Irina V. , Dunigan David D. , Van Etten James
Published: January 2023

Infections and inflammatory responses may contribute to motor neuron diseases (MND), such as Amyotrophic Lateral Sclerosis (ALS). A portion of familial ALS is caused by polymorphic Superoxide Dismutase (SOD)1 gene (SOD1G93A). As chloroviruses (CV) infect macrophages inducing inflammatory cytokines, they may contribute to ALS. Moreover, ATCV-1 is one of several chlorovirus types that encodes its own SOD1. Thus we evaluated serum from human ALS patients compared with controls for antibody against ATCV-1. In addition, we i.c. injected saline or ATCV-1 into SOD1G93A-transgenic and C57Bl/6 control mice and assessed development of ALS-like MND. Sera from ALS patients and controls had antibody to ATCV-1. However, ALS patients had significantly greater IgG1 anti-ATCV-1 compared with controls. This was accompanied by elevated IL-6, IFN-γ, and anti-Tetanus toxin antibody, but not IL-17. SOD1G93A mice inoculated with ATCV-1 had significantly accelerated development of MND, including tail paralysis, hindlimb tucking, decreased righting reflex, and latency to fall in a hanging cage lid test, compared with saline injected SOD1G93A mice. Splenic T cells of SOD1G93A and control mice showed similar IFN-γ and IL-17 responses to anti-CD3/-CD28, while Foxp3 expression overall was significantly higher in T cells of SOD1G93A compared with control mice. Inoculation with ATCV-1 decreased Foxp3 expression in SOD1G93A T cells compared with mice not given ATCV-1. These results indicate that chloroviruses, ubiquitous in fresh waters worldwide, induce antibody responses in humans that are elevated in ALS and may accelerate MND in the SOD1G93A type of familial ALS. Funding was generously provided by the Stuart Nichols ALS Research Foundation.
In utero exposure to endogenous maternal polyclonal anti-Caspr2 antibody leads to an autism-like phenotype in male mice
Authors: Bagnall-Moreau Ciara N , Huerta Patricio , Volpe Bruce , Diamond Betty , Brimberg Lior
Published: January 2023

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder with a complex etiology. A strong sex bias has been observed in ASD with boys being four times more likely to be diagnosed than girls. The maternal immune system has been repeatedly implicated in the etiology of ASD. Maternal infection with fever during pregnancy has been linked to ASD. Increased levels of maternal cytokines and chemokines during gestation, including interleukin 6, have been associated with ASD in children with intellectual disabilities. Studies, including our own, have consistently supported the hypothesis that maternal brain-reactive antibodies can predispose to ASD by affecting the developing brain during a critical period. We have previously demonstrated that antibodies targeting the neuronal protein Caspr2 are present at high frequency in mothers with brain-reactive serology and a ASD child. Furthermore, male mice but not female mice exposed in utero to a monoclonal anti-Caspr2 antibody cloned from a mother of a child with ASD, develop an ASD -like phenotype. In the present study we use a new model in which female mice are immunized with the extracellular portion of Caspr2, and therefore harbor anti-Caspr2 antibodies throughout gestation to mimic the human condition. Male mice born to dams harboring polyclonal anti-Caspr2 antibodies exhibit abnormal cortical development, decreased dendritic complexity of excitatory neurons and reduced numbers of inhibitory neurons in the hippocampus, as well as repetitive behaviors and impairments in social interactions as adults. These studies will increase our understanding of the triggers of neurodevelopmental impairment and possibly suggest novel approaches to protecting the fetal brain.
Development of a platform for screening patient-derived CD8+ T cell responses against autoantigens
Authors: Johnson Renee K , Clarkson Ben D.S. , Howe Charles L
Published: January 2023

Cytotoxic CD8+ T cells outnumber CD4+ T cells within multiple sclerosis (MS) lesions, yet the pathogenic role for these cells and the relevant target antigen(s) involved in neuropathology remain unknown. We predict that a subset of lesion-infiltrating CD8+ T cells are specific for neuronal antigens and contribute to axon or neuron injury, thereby driving disease progression and accumulation of disability. To test this, we are developing protocols to measure MS patient-derived T cell reactivity against neuronal and control antigens. Peripheral blood mononuclear cells (PBMCs) isolated from whole blood were incubated with GM-CSF and IL-4 to selectively expand dendritic cells (DCs) or with IL-7 to promote survival and maintenance of T cells. After resting, patient T cells were combined 1:1 with autologous DCs in the presence of vaccine antigens or peptides derived from common pathogens. We tested responses against a pool of cytomegalovirus, Epstein Barr virus, and influenza peptides (CEF) and against mumps viral protein extract. We also tested responses in T cells derived from patients with autoimmune neurological disorders following incubation with autologous DCs and disease-specific proteins. Activation profiles were assessed using CD69, CD44, and LFA-1 by conventional flow cytometry. Cellular responses were also assessed using mass cytometry (cytometry by time of flight; CyTOF). Our next steps include testing MS patient-derived T cell responses against neuronal and axonal protein libraries, with the goal of identifying potential autoantigens that underlie disease progression.
Cytotoxic T cells infiltrate the brain and interact with microglia to reduce Alzheimer’s Disease pathogenesis
Authors: Sanchez Jessica Ramirez , Marsh Samual , McIntyre Laura , Davtyan Hayk , Walsh Craig , Blurton-Jones Mathew
Published: January 2023

The role of the adaptive immune system, specifically T cells, remains poorly understood in Alzheimer’s disease (AD) pathogenesis research. Our lab generated an immune-deficient mouse model of AD (RAG2ɣc−/−5xfAD) which showed a dramatic increase in amyloid plaque load, an activated microglial neuroinflammatory state, and a decrease in the ability of microglia to phagocytose beta-amyloid. Restoration of adaptive immunity via bone marrow transplantation led to a corresponding decrease in amyloid plaques, indicating a fundamental role for adaptive immunity in AD pathology. Further adoptive transfers into these aged immune-deficient mice showed a significant increase in memory CD8+ T cells and microglia in the RAG2ɣc−/−5xfAD brain parenchyma via flow cytometry. Interestingly, T cells were observed adjacent to plaque-associated microglia in these brains via Immunohistochemistry. To determine whether T cell infiltration also occurs in immune-intact AD mice, flow cytometry was conducted on 5xfAD mice that develop amyloid plaques, PS19 mice that develop neurofibrillary tangles, PS19-5xfAD bigenic mouse model, and wild-type mice. This analysis validated the immune-deficient observations, demonstrating a significant increase in memory CD8+ T cells in 5xfAD mice brains but also revealed a further significant increase in the bigenic PS19-5xfAD model. Therefore, I hypothesize that cytotoxic T cells respond to the inflammatory AD environment by infiltrating the brain and directly interacting with microglia to decrease amyloid plaques. My initial data clearly demonstrate that further examination of the crosstalk between adaptive and innate immunity is needed to determine the mechanisms by which T cells influence AD pathogenesis.
Single-Cell RNA Sequencing Identifies a Novel Population of CD4+CD8+ T Cells that are Regulated by COX-2 During Allergic Lung Inflammation
Authors: Li Hong , Bradbury J Alyce , Xu XiaoJan , Edin Matthew L , Li Jianliang , Katen Kevin , Gruzdev Artiom , Graves Joan P , Duval Caroline N , Zeldin Darryl C
Published: January 2023

To better understand the relative contributions of different cell types during allergic lung inflammation and the role of cyclooxygenase (COX)-derived eicosanoids in this process, we compared vehicle (control) and ovalbumin (OVA)-exposed lungs from wild type (WT) and COX-2−/− mice by single-cell RNA sequencing (scRNA-Seq). Twenty-two unique cell clusters were identified in allergic lungs based on cell signature markers. In addition to known cell types, we identified 2 clusters of CD4+CD8+. Both populations were mitotically active (high % in S/G2/M phases) and exhibited a unique molecular identifier (UMI) index. FACS analysis confirmed the presence of these two distinct cell populations (CD4+HCD8+H and CD4+LCD8+H) that were increased after OVA-induced allergic lung inflammation. Interestingly, isolated CD4+HCD8+H T cells stimulated with phorbol ester expressed IL-4, IL-5 and IL-13, but not IFNγ. In contrast, isolated and stimulated CD4+LCD8+H T cells primarily expressed IL-5. Mass cytometry (CyTOF) using a 37-antibody panel confirmed the increase in CD4+CD8+ T cells after OVA sensitization/exposure, as well as the induction of IL-4, IL-5 and IL-13. Importantly, CD4+CD8+-1 and CD4+CD8+-2 cell populations identified by scRNA-Seq were increased in lungs from OVA-treated COX-2−/− mice compared to lungs from OVA-treated WT mice. Furthermore, COX-2−/− CD4+CD8+ T cells preferentially produced more IL-4 and IL-5 than WT CD4+CD8+ T cells. Thus, using scRNA-Seq, CyTOF and FACS analyses, we identified two populations of CD4+CD8+ T cells that produce immunoregulatory cytokines and are regulated by COX-2. These cells may play an important, previously unappreciated role in the pathogenesis of allergic lung disease.
Blockade of RANKL/RANK interaction prevents allergic airway inflammation in a mouse model of asthma
Authors: Maslanka Tomasz , Gregorczyk Izabela , Jasiecka-Mikolajczyk Agnieszka
Published: January 2023

W are constantly looking for new therapeutic options for asthma treatment. In this study, we investigated whether the signaling pathway of the receptor activator of nuclear factor (NF)-κB (RANK), RANK ligand (RANKL), and its decoy receptor osteoprotegerin (OPG) may be considered as a novel drug target for therapy of allergic asthma. Therefore, we tested the effect of blockade of RANK-L with OPG on the development of ovalbumin (OVA)-induced airway allergic inflammation (AAI) and accompanying immune response. The absolute counts of CD11chighCD11b+CD103− (CD11b+) dendritic cells (DC), ST2+Foxp3−CD4+ (Th2) cells and BrdU+(i.e., proliferating)CD4+ T cells were determined in the mediastinal lymph nodes (MLNs) and lungs of untreated non-immunized mice, untreated OVA-immunized mice and OPG-treated OVA-immunized mice. Immunization with OVA induced a significant increase in the mean scores of lung histologic lesions and administration of OPG completely prevented this effect. The absolute counts of CD11b+ DC, Th2 cells and BrdU+CD4+ T cells were increased in the MLNs and lungs of untreated OVA-immunized mice. Treatment with OPG attenuated, but did not abolish, the OVA-induced increase in the count of CD11b+ DC in the MLNs. In turn, administration of this agent prevented the OVA-induced increase in the count of Th2 cells and BrdU+CD4+ T cells in the MLNs and lungs. In conclusion, blockade of RANK/RANKL interaction prevents OVA-induced AAI and this effect is mediated through inhibition the allergen-induced clonal expansion of CD4+ T cells in the MLNs. Clinical implication: the local inhibition of RANK/RANK-L interaction within the respiratory system can be considered as a novel therapeutic strategy in treatment of allergic asthma.
IgM regulates airway hyperresponsiveness in allergic asthma through acetyl choline receptors
Authors: Hadebe Sabelo , Khumalo Jermaine , Ngomti Amkele , Mangali Sandisiwe , Scibiorek Martyna , Brombacher Frank
Published: January 2023

Allergic asthma is a disease driven by T helper 2 (TH2)-type cells secreting interleukin (IL-) 4, 5 and 13. It is characterized by eosinophils, airway hyperesponsiveness (AHR) and IgE secreting B cells. B cells play a role in allergic asthma in an allergen load dependent manner. IgM isotype secreted by naïve B cells is important for class switching. It is currently unclear how IgM isotype contributes in the development of allergic asthma. We investigated the role of IgM isotype in a house dust mite (HDM)-induced TH2 allergic asthma model. We sensitised wild type (wt), IgM-deficient (IgM−/−) and B cell-deficient (uMT−/−) mice with high (>10 ug) and low (<3 ug) dose of HDM extracts. We found IgM to be essential in antibody class switching and in AHR, but not in TH2 airway inflammation or eosinophilia. RNA seq suggested that IgM regulated AHR through modulating muscarinic receptor genes, particularly M3 and this was independent of microbiota. Transfer of wild type serum, but not B cells into IgM−/− mice could restore class switching but not AHR. These unprecedented findings suggest that IgM has a unique function in allergic asthma and regulates AHR by interacting with non-immune genes.