bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeted expansion of tissue-resident CD8+ T cells to boost cellular immunity in the skin
Authors: Hobbs Samuel J , Nolz Jeffrey C
Published: January 2023

Tissue-resident memory (TRM) CD8+ T cells are positioned within environmental barrier tissues to provide a first-line of defense against pathogen entry. Whether these specialized T cell populations can be readily boosted to enhance tissue-specific protective immunity has not been thoroughly investigated. Here, we show that cutaneous immunization with Vaccinia virus establishes rare, but highly functional antigen-specific TRM CD8+ T cells from the endogenous T cell repertoire. Repeated activation of endogenous TRM CD8+ T cells using only topical application of antigenic peptide caused delayed-type hypersensitivity (DTH) and expanded the antigen-specific TRM CD8+ T cell population in the challenged skin by ~15-fold, resulting in significantly increased anti-viral protective immunity. Rather than the result of local proliferation, expansion of TRM CD8+ T cells required circulating memory T cells to be recruited into the skin to become tissue-residents. Although circulating memory CD8+ T cells of other antigen-specificities trafficked into the skin during TRM-mediated DTH, they failed to differentiate into CD69+ TRM cells. However, circulating memory CD8+ T cells of a different antigen-specificity could be coerced to become tissue-resident using a dual peptide challenge strategy, where one peptide causes TRM-mediated DTH and the second peptide ‘captures’ only the desired antigen-specific memory CD8+ T cells in the skin. Overall, our study demonstrates that established TRM CD8+ T cells can be rapidly expanded to enhance tissue-specific cellular immunity, but also reveals how pathogenic T cells specific for commensal or environmental antigens could continually accumulate in the skin following repeated exposures.
Mycobacterium avium subspecies paratuberculosis (MAP) drives an innate Th17-like T cell response and upregulation of inhibitory signals
Authors: DeKuiper Justin Lee , Coussens Paul M
Published: January 2023

Johne’s disease (JD) is a chronic inflammatory gastrointestinal disorder of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Our work suggested that MAP may drive a non-classical Th17-like response in PBMCs. Our objectives were to determine if Th17 cell activation by MAP required the presence of antigen presenting cells (APCs) and to determine how epithelial cells might influence Th17 activation. Monocytes were isolated by adhering and washing protocols and then cultured for 5 days to allow differentiation into macrophages. T cells and B cells were isolated using magnetic-activated cell sorting (MACS) by positive selection of CD3+ and sIgM+ cells, respectively. MACS cells were then cultured or cocultured with macrophages. MDBK cells were used as an epithelial cell model. qPCR was used to determine mRNA expression levels of MDBK, T cells alone, and T cells cultured with macrophages or B cells. All T cell cultures increased IL-17a and IL-23 mRNA expression with MAP-antigen stimulation when compared to untreated cells. APC containing cultures expressed significantly more IL-17a than T cells alone. IL-22 expression was only observed in T cell cultures stimulated with live MAP. T cell exhaustion and tolerance markers (PD-1, PD-L1, LAG-3) were increased in T cell only cultures when stimulated with MAP, but repressed in the presence of APCs. The presence of MDBK cells upregulated IL-23 following stimulation with MAP, suggesting that epithelial cells exposed to MAP may also help drive Th17 differentiation.
Sargassum horneri mitigates particulate matter induced inflammatory response in MLE-12 cells via MAPK pathways
Authors: Mihindukulasooriya Suyama Prasansali , Herath Kalahe Hewage Iresha Nadeeka Madushani , Kim Areum , Kim Hyo Jin , Ko Mi-Ok , Jeon You-Jin , Youngheun Jee
Published: January 2023

Rise of ambient particulate matter (PM) exhibits positive correlation with increased respiratory diseases since PM triggers an inflammatory response in lung. As approaches aiming at limiting PM exposure provide only partial solutions, we investigated a therapeutic approach to mitigate harmful effects of PM in this study. Specifically, we examined the anti-inflammatory potential of the 80% ethanol extract of Sargassum honeri (SHE) against PM exposed murine lung epithelial cells (MLE-12). PM exhibited a cytotoxic effect on MLE-12 from rather low concentrations (concentrations > 15.6 μg/mL; IC50 44.1 μg/mL), and the cytotoxic concentration of PM utilized in this study was 125 μg/mL. But, SHE (0–125 μg/mL) was not cytotoxic on MLE-12 cells, and concentrations 62.5 and 125 μg/mL were tested in this study. SHE reversed the PM induced lactate dehydrogenase (LDH) release and cell proliferation loss of MLE-12 cells. SHE also decreased the PM induced secretion of pro-inflammatory cytokines (IL-1β, TNF-α), pro-inflammatory mediator COX-2, anti-inflammatory cytokines (IL-6, IL-8) and inflammatory chemokines (MCP-1, CCL5). Furthermore, SHE decreased PM induced phosphorylation of p38 mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun NH (2)-terminal kinase (JNK). Taken together, we conclude that SHE’s anti-inflammatory effect was regulated probably via the inhibition of p38, ERK and JNK MAP kinase signaling pathway in MLE-12 cells. This research is a part of the project, “Development of functional food products with natural materials derived from marine resources”, funded by the Ministry of Oceans and Fisheries, Korea in 2017 (Project NO. 20172085).
CCR2 mediates transendothelial migration of human pathogenic Th17 cells
Authors: Parween Farhat , Kathuria Noshin , Zhang Hongwei , Farber Joshua M
Published: January 2023

Leukocyte extravasation is characterized by rolling and arrest on the endothelium followed by transendothelial migration (TEM). Leukocyte arrest and TEM typically require chemokines and their receptors. Previously, we showed that CD4+CCR5+CCR2+ T cells are highly differentiated, readily activated memory cells in human blood that exhibit diverse effector capabilities. We and others have also shown that CCR6 is the signature chemokine receptor for Th17 cells. Here we describe studies of the migratory capabilities and effector profiles of CD4+CCR6+CCR2+ as compared with CD4+CCR6+CCR2− and CD4+CCR6−CCR2− effector-memory T cells from human blood. Using flow chamber assays with TNFa-activated human umbilical vein endothelial cells, we found that among the CCR6+ cells, only the CCR2+ subset was able to cross the endothelial monolayer efficiently. We found that CCR6 and CCR5 were important for arrest whereas CCR2 was critical for subsequent TEM. Our RNA-Seq data and intracellular staining data of ex vivo activated T cells showed that the CCR6+CCR2+ cells were distinguished by their expression not only of the cytokines typical of Th17-cells, but also of cytokines, including IFNg and GM-CSF, and transcription factors characteristic of the pathogenic Th17 cells that mediate tissue injury in mouse models of autoimmune disease. Taken together, our studies suggest that chemokine receptors on memory Th cells play non-redundant roles in the egress from blood, that providing T cells with a combination of CCR6 CCR5 and CCR2 could enhance their trafficking into tissue, and that, on the other hand, blocking CCR2 alone could prevent the extravasation of pathogenic Th17 cells and potentially diminish tissue damage in Th17-cell driven disease.
Intrinsic Alteration of Differentiation and Glycolysis in T Lymphocytes Immediately After TCR Activation Through PSGL-1 Signaling
Authors: Hope Jennifer L , Otero Dennis , de Jong Petrus , Ma Jiadai , Henriquez Monique , Powis Garth , Bradley Linda
Published: January 2023

P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesion molecule expressed on the surface of naïve, effector, and memory CD4+ and CD8+ T cells. We identified PSGL-1 to be an immune checkpoint inhibitor as antibody-mediated ligation of PSGL-1 promotes T cell exhaustion and deletion of PSGL-1 prevents chronic viral infection and inhibits tumor. To investigate the role of PSGL-1 signaling in the development of T cell responses, we assessed the differentiation state, expansion capacity, and glycolytic profile of PSGL-1-deficient T cells. PSGL-1+/− OT-II CD4+ T cells demonstrated increased skewing towards IL-17A+ T cells compared to wild-type OT-II CD4+ T cells under TH17 conditions, and reduced IFNg+ cells under both TH0 and TH1 conditions. In a B16-OVA tumor model, we observed increased IL-13+ CD4+ T cells among the intratumoral T cell compartment in PSGL-1−/− mice. In vitro activation with a sub-optimal dose of αCD3 of PSGL-1−/− OT-II CD4+ T cells demonstrated increased expansion and expression of CD25 compared to wild-type OT-II CD4+ T cells. Further, adoptively transferred in vitro-activated PSGL-1−/− CD4+ T cells demonstrated greater expansion in vivo upon adoptive transfer into RAG−/− host mice. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. Further, in situ activation of PSGL-1−/− CD8+ T cells demonstrates that at both sub-optimal and optimal levels of αCD3 stimulation, PSGL-1−/− CD8+T cells have increased glycolysis and increased glycolytic capacity. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses.
IL7R regulates fetal tissue resident macrophage development
Authors: Leung Gabriel , McCann Taylor , Valencia Clint H. , Worthington Atesh , Forsberg Camilla , Beaudin Anna E
Published: January 2023

Tissue-resident macrophages (TRMs) play critical roles in tissue homeostasis and disease. Many populations of TRMs derive from fetal progenitors and independently self-maintain across the lifespan through in situ proliferation. Here, we have identified the interleukin-7 receptor (IL7R) as a novel regulator of TRM development. Using an IL7R-Cre lineage tracing model, we observed that adult TRMs in the brain, epidermis, liver, and lung were highly labeled by IL7R-cre, in the absence of IL7Ra mRNA or protein expression. To gain insight into developmental expression of IL7Ra, we profiled surface expression, mRNA expression, and IL7R-cre driven labeling across fetal development. Erythromyeloid progenitors, putative TRM precursors, were barely labeled by IL7Ra-cre, and IL7R deletion did not affect YS hematopoiesis. In contrast, we observed IL7Ra mRNA expression in fetal monocytes, and robust IL7Ra surface expression on developing TRMs during late gestation. Sorted Ly6chi fetal liver monocytes cultured ex vivo with M-CSF differentiated into macrophages expressing IL7R, suggesting a precursor-product relationship. Blockade of the IL7R with a monoclonal antibody during gestation impaired liver, lung, and epidermal TRM cellularity at birth, with a concomitant increase in cellularity of liver monocytes, suggesting that IL7Ra regulates TRM differentiation from fetal monocytes during fetal development. These data reveal dynamic regulation of IL7Ra expression in TRMs and TRM precursors during late gestation, and provide evidence that IL7R signaling regulates fetal TRM development. Ongoing work addresses downstream signaling and the specific developmental processes regulated by IL7R signaling during fetal TRM development.
Bone marrow resident Tregs maintain stromal cell function via IL-10
Authors: Camacho Virginia , Patel Sweta , Matkins Victoria , Turnquist Heth R , Welner Robert S
Published: January 2023

Regulatory T cells within the bone marrow (BM Tregs) represent a specialized population whose functions are incompletely understood. In this study, we provide a novel mechanism by which BM Tregs support the maintenance of stromal cells to direct hematopoiesis. When compared to Tregs found in spleen and lymph node, they express genes associated with activation as well as chemokine receptors. As another distinguishing characteristic, BM Tregs highly express both CD49b, a marker of circulating Tregs, and S1PR1, a receptor involved in trafficking. This was corroborated by parabiosis and transplant experiments in which BM Tregs were exchanged at a higher frequency than Tregs from other tissues. This indicates that they are highly migratory, but preferentially return to the marrow. Additionally, we found that BM Tregs are high producers of IL-10, an immunosuppressive cytokine that limits inflammation. Indeed, Tregs are the primary source of this cytokine in the marrow. Loss of IL-10 production by Tregs altered the BM microenvironment in several ways. Numbers of CD51+ stromal cells, and PDGFRα+ bone forming progenitors declined. There was a marked shift towards myeloid cell production, a decrease in B cell production, and perturbed differentiation of stem and progenitor cells in IL-10 deficient marrow. Remarkably, Tregs enhanced the supportive capacity of stromal cells through IL-10 by promoting the maintenance of hematopoietic stem cells. Collectively, our observations suggest that Tregs residing within BM are master regulators of hematopoiesis and that production of IL-10 accounts for at least some of their functions. This and other properties have the potential to be exploited in the context of transplantation or hematopoietic disease.
Combined immune and airway epithelial cell profiles define asthma severity phenotypes
Authors: Oriss Timothy B , Zhou Xiaoying , Camiolo Matthew , Scholl Kathryn L , Gorry Michael , Gauthier Marc , Ray Prabir , Wenzel Sally E , Nadeau Kari Christine , Ray Anuradha
Published: January 2023

Asthma constitutes a spectrum of conditions that affect a substantial proportion of the population in developed countries. A large segment of asthmatics are characterized by an immunologic type 2 allergic profile. A preponderance of conventional and biologically-based therapies are available to treat these subjects. However, a small minority of individuals clinically classified as severe asthmatics (SA), are highly refractory to treatment and account for a significantly disproportionate share of the clinical and economic impact of the asthma condition as a whole. In order to better define the immune response in SA with the ultimate goal of developing novel therapeutic modalities, we analyzed bronchoalveolar lavage (BAL) cells from a cohort of SA, mild to moderate asthmatics (M/M), and healthy controls by mass cytometry (CyTOF). Unbiased bioinformatic assessment revealed 34 clusters of BAL cells with assignment of subjects to one of four groupings by principal component analysis (PCA). The majority of SA (78.9%) fell into two of these groups and constituted 65.2% of their makeup, with M/M representing the remaining 34.8%. Similarly, analysis of airway bronchial epithelial cells (BECs) by RNA sequencing (RNASeq) revealed three subject groups with SA falling largely into one of these. Synthesis of CyTOF and RNASeq data in the context of relevant clinical parameters resulted in a combined profile for each subject, which was able to distinguish those with the most severe disease. Our approach demonstrates that an unbiased, multifactorial assessment of immune and epithelial profiles can predict asthma severity, but may also reveal mechanism-based clues for targeted, personalized therapeutic intervention in severe asthmatics.
The transcriptional repressor Blimp-1 promotes Th2 development and lung inflammatory disease in a house dust mite induced model of allergic asthma
Authors: Poholek Amanda C , Hettinga Angela , He Kun , Kale Sagar , Schmitz Elizabeth , Pandya Shivani , Xie Markus Ming , Dent Alexander , Oriss Timothy B , Ray Anuradha
Published: January 2023

Asthma is a T cell-mediated chronic inflammatory lung disease that is thought of as a type 2/Th2 disease, yet precisely how Th2 cell development in the lung is initiated is unknown. Blimp-1 is a transcriptional repressor that regulates effector T cell responses to constrain T cell-mediated autoimmunity. We describe Blimp-1 as an unexpected regulator of allergen-induced airway inflammation that is critical to promote Th2 cell development in the lung and subsequent airway inflammation. Using a house dust mite (HDM) antigen model to drive allergic lung disease, we show that the T cell specific absence of Blimp-1 leads to reduced lung inflammation, a significant decrease in eosinophil recruitment to the lung tissue, and a near absence of Th2 cells in the lung. Intriguingly, Th1 and Th17 responses are intact, suggesting Blimp-1 is specifically required for Th2 differentiation in this model. Furthermore, despite a lack of Th2 cells in the lung, IgE responses are intact, dissociating IL-4-mediated antibody driven responses from Th2 cells in the lung. Bcl6 expression is increased in both the draining lymph node and lung and using T cell specific Bcl6 and Blimp-1 double deficient mice, we find that Th2 cells are restored when Bcl6 is deleted in addition to Blimp-1. Bcl6 is a potent repressor of GATA3, suggesting Blimp-1 acts to indirectly promote GATA3 expression by suppression of Bcl6. Furthermore, we show that STAT3 signaling in T cells is responsible to upregulate Blimp-1 and subsequent Th2 development, implicating this pathway as a possible therapeutic target. Taken together, our data suggests Blimp-1 plays an unexpected and context-dependent role in allergic lung inflammation to promote Th2 cell differentiation and subsequent lung disease.
Inhibition of allergen-specific tissue resident memory Th2 cells through blockade of CD3-mediated signaling ameliorates allergic lung inflammation
Authors: Gracias Donald Tom , Gupta Rinkesh Kumar , Croft Michael
Published: January 2023

Allergic asthma remains a major public health concern, with more than 26 million Americans suffering from the disease. The secretion of cytokines such as IL-4, IL-5, and IL-13 and their ability to persist in the host long after the primary allergen encounter are a reason memory Th2 cells are a major driving force behind allergic asthma morbidity. The selective removal of pre-existing memory Th2 cells could be a key therapeutic venture to enhance tolerance. One such method could involve targeting CD3 through the use of inhibitory anti-CD3 antibodies which successfully induced tolerance in animal models of autoimmune diseases and also show promise in clinical trials. To determine if we could induce airway tolerance, we tested blocking CD3 complex signaling in a HDM model of allergic inflammation. Importantly, pre-sensitized C57BL/6J mice displayed strongly ablated lung inflammation and Th2 responses when treated at the time of memory T cell reactivation with F(ab′)2 anti-CD3 antibody. This treatment was found to inhibit HDM-specific CD4+ T cell proliferation and Th2 cytokine production. Interestingly, while mice treated with anti-CD3 also showed an increased frequency of Treg cells, protection to allergic inflammation was maintained even after Treg depletion. Anti-CD3 treatment was found to specifically inhibit eosinophilic lung inflammation driven by allergen-specific lung tissue resident memory (Trm) Th2 cells as determined by treatment of mice with fingolimod (FTY720), which prevents the recruitment and contribution of circulating memory T cells to the allergic response. These data suggest that blocking CD3 complex signaling could be employed to inhibit Trm recall responses to complex allergens thus promoting airway tolerance.
Endogenous UDP-Glc acts through the purinergic receptor P2RY14 to exacerbate eosinophilia and airway hyperresponsiveness in a protease model of allergic asthma
Authors: Karcz Tadeusz , Whitehead Gregory , Nakano Hideki , Jacobson Kenneth A , Cook Donald N
Published: January 2023

Human asthma represents a wide spectrum of pathologies that likely arise through distinct molecular pathways, and a major therapeutic goal is to target individual therapies to specific forms, or endotypes, of this disease. Here we studied the role of UDP-glucose (UDP-Glc), an alarmin which we found to be released into the airways of allergen-sensitized mice upon their subsequent challenge with allergen. UDP-Glc signals through the purinergic receptor, P2Y14R, and mice lacking this receptor display marked attenuation of both eosinophilia and airway hyperresponsiveness (AHR) in a protease model of allergic asthma. By contrast, these features of asthma were similar in wild type and P2ry14-deficient mice in a TLR-ligand model of asthma, indicating an endotype-specific role for UDP-Glc and P2Y14R. Importantly, administrationof PPTN, a small molecule antagonist of P2Y14R, also led to reduction of airway inflammation and AHR in the protease mediated asthma. Although its mechanism of action remains unclear, P2ry14 was most highly expressed in recruited eosinophils and neutrophils, suggesting UDP-Glc acts directly on these cells. Our current findings suggest that antagonists of P2Y14R might be selectively used to control asthma exacerbations in specific forms of human asthma.
Development of conventional, but not tissue resident NK cells requires pH-controlling vacuolar a2v-ATPase
Authors: Beaman Kenneth D , Schnederman Sylvia , Katara Gajendra , Dambaeva Svetlana
Published: January 2023

Innate lymphoid cells are abundant in uterine endometrium and their number dramatically increases during pregnancy. In addition to conventional NK (cNK) cells, unique uterine-specific NK cells populate the uterus. These, tissue-resident NK (trNK) cells are characterized by CD49a expression. A2v-ATPase is a proton pump molecule that acidifies intracellular vesicles and is critical in processing of Notch receptors. A knockout of a2v- ATPase in VAV-1 expressing cells (cells of hematopoietic origin) leads to dramatic reduction of NK cells in blood and spleen of the genetically modified animal (a2vKO mouse). Uterine tissues of a2VKO mice were analyzed for NK cell presence based on their reactivity with the lectin DBA. DBA-reactive cells were readily observed in uterine stroma of pregnant a2vKO mice. Although the number of DBA+ cells in a2vKO mice was lower than in wild type (WT) controls, the difference was not statistically significant. Flow cytometry analysis of uterine tissues from virgin a2vKO mice revealed a dramatic decrease of cNK cells (NK1.1+NKp46+CD11b−/lowDx5+CD49a−). However, the frequency of trNK cells, that possess phenotype NK1.1+NKp46+CD11b−Dx5−/+CD49a+, was comparable with what was found in WT. Similar findings were observed in pregnant a2vKO mice. Despite the absence of cNK cells that compose a significant proportion of lymphocytes in pregnant uterine tissue, mice fertility was not critically affected and this suggested a role for trNK cells in supporting pregnancy. In conclusion, the development of NK cells that takes place in endometrium appears to be different from NK cells originated in bone marrow, namely it is not dependent on a2vATPase; this can explain uniqueness of trNK cells.
Intrinsic factor Recognition Promotes Th17/Th1 Autoimmune Gastric Inflammation in Patients with Pernicious Anemia
Authors: Troilo Arianna , Grassi Alessia , D'Elios Mario Milco
Published: January 2023

The intrinsic factor (IF) is the major humoral autoantigen in pernicious anemia (PA)/autoimmune gastritis. Although many studies have examined the autoantibody response to intrinsic factor and H+,K+-ATPase, no information is available on possible pathogenic mechanisms mediated by IF-specific gastric T cells. The aim of this study was to investigate IF-specific T cells in the gastric mucosa of PA patients and define their functional properties. In vivo-activated T cells from the infiltrates of the gastric mucosa of PA patients were isolated and cloned. We investigated the ability of the gastric T-cell clones to proliferate and produce cytokines in response to IF, as well as their helper function for antibody production and their cytolytic potential. For the first time we provide evidence that gastric mucosa of PA patients harbour a high proportion (20%) of autoreactive activated CD4+ T-cell clones that specifically recognize IF. Most of these clones (94%) showed a Th17 or Th1 profile. Virtually all IF-specific clones produced TNFα, IL-21 and provided substantial help for B-cell immunoglobulin production. Most mucosa-derived IF-specific T-cell clones expressed perforin-mediated cytotoxicity and induced Fas-Fas ligand-mediated apoptosis in target cells. Altogether, our results suggest that chronic autoantigen-induced T cell– dependent B-cell activation is responsible for the local synthesis of IF autoantibodies found in the sera and indicate that activation of IF-specific Th17 and Th1 T cells in the gastric mucosa represent a key effector mechanism in PA suggesting that the Th17/Th1 pathway may represent a novel target for the prevention and treatment of the disease.
Differentiation and dynamics of circulating and tissue-resident memory CD8 T cells in the lung
Authors: Anthony Scott M , Harty John T
Published: January 2023

Memory CD8 T cells are capable of providing robust heterosubtypic immunity to Influenza. In contrast to other tissues, relatively little is known about the differentiation and localization of the memory CD8 T cell subsets within the lung. We utilized intravital 2-photon microscopy of live mice to examine the real-time dynamics of circulating and/or resident memory (Trms) CD8 T cells in the lung. LCMV-induced transgenic P14 CD8 memory T cells, which are exclusively circulating, were highly motile, while Influenza-experienced P14s, which are comprised of both circulating and resident memory CD8 T cells, exhibited heterogenous motility patterns including both highly motile cells and those exhibiting a slow to moderate motility pattern. Selective depletion of circulating memory CD8 T cells resulted in the loss of highly motile cells, suggesting that lung CD8 Trms exhibit slow to moderate motility. Intranasal administration of cognate peptide or recombinant Influenza virus infection induced the arrest of a large number of LCMV-experienced circulating P14s in the lung shortly after administration in an antigen-specific manner. Interestingly, within 12–48 hours post stimulation, the majority of lung-residing LCMV-experienced circulating P14s became un-arrested and began a moderate scanning motility phenotype that persisted in a substantial fraction of P14s and correlated with the generation of secondary memory CD103+ Trms in the lung. Overall, these data shed light into the differentiation and dynamics of circulating and resident CD8 memory T cells in the lung.
Small Intestinal Cytotoxic CD4 T Cells: Novel Mechanisms of Development and Unique Protective Capability
Authors: Chen Angeline , Iwaya Hitoshi , Thiault Nicolas , Cheroutre Hilde
Published: January 2023

Conventional CD4 T helper T cells convert to CD4 cytotoxic T lymphocytes (CTL) in the small intestinal epithelium during steady state, but the mechanism and antigens that drive their generation and purpose(s) at this barrier are largely unknown. The unique localization of mucosal CD4 CTL to the small intestinal epithelium suggests local non-pathogen antigens and organ-specific properties that direct CD4 CTL to develop from CD4 T helper (Th) cells.

Similar to traditional antigen presenting cells (APCs), intestinal epithelial cells (IECs) express MHC class II (MHCII), indicating a possible influence over nearby CD4 T cells. In addition, IECs are constantly processing luminal contents and therefore are likely to present non-pathogen-derived antigens (e.g. diet or commensal bacteria-derived) to CD4 T cells at its baso-lateral side. Taken together, we hypothesized and now show that the constant presentation of dietary antigens by IECs in a non-inflammatory environment leads to the development of CD4 CTL in the small intestinal epithelium.

These CD4 CTL accumulate over time and fortify the small intestine as an innate-like defensive arm specifically designed to contain infections while minimizing inflammation at this delicate interface. Many types of immune cells are involved in combating Salmonella infection, but CD4 CTL represent a distinct niche. Here, we demonstrate the protective capacity of CD4 CTL to rapidly and effectively kill infected epithelial cells thereby preventing systemic spread of pathogens as well as excessive immune pathology caused by infiltrating peripheral adaptive immune cells.
A fast and robust protocol for immunomagnetic isolation of human CD45+ leukocytes from samples with variable start frequencies
Authors: Posarac Vesna , Ewen Catherine L. , Jovanovic Vida K. , Hayer Elisha , Kokaji Andy I. , Woodside Steven M. , Eaves Allen C. , Thomas Terry E.
Published: January 2023

The study of immune cell function in non-lymphoid tissue and tumors has emerged as an exciting research area, with the promise to provide novel strategies for the treatment of immune disorders, infectious diseases and cancer. A major challenge in the field is the isolation of leukocytes from tissues due to the complexity of starting samples that can be highly variable in frequency and subset composition. We describe a new 28 minute protocol optimized for the isolation of particle-free human CD45+ leukocytes from varied sample sources. Using the EasySep™ Release Human CD45 Positive Selection Kit, leukocytes are labeled with antibody complexes linked to magnetic particles and separated using an EasySep™ magnet. The isolated cells are then released from particles by resuspension in EasySep™ Release Buffer and a final magnetic separation step. Performance was assessed using human leukocytes spiked into a single cell suspension of mouse thymocytes across a range of frequencies to simulate the variable leukocyte content in tissue and tumor samples. Human CD45+ purities were 95.4 ± 4.3% from start ranges of 0.5–2.0% (n=15) and 99.1 ± 0.7% from start frequencies of ≥10% (n=18). The isolated cells are compatible with further downstream isolation and are functional, as demonstrated by T cell proliferation and IFN-gamma production assays. We have also assessed our CD45+ isolation kit from tissues of humanized NSG mice, expanding the application of our protocol to highly relevant disease and cancer models. The EasySep™ Release Human CD45 Positive Selection Kit provides a robust method for high purity isolation from diverse tissues and starting frequencies, generating particle-free cells ready for downstream evaluation.
Identifying antibodies and T-cell receptors with known specificity in repertoire sequencing data
Authors: Mahajan Swapnil , Vita Randi , Yan Zhen , Nielsen Morten , Sette Alessandro , Peters Bjoern
Published: January 2023

The adaptive immune system in vertebrates has evolved to recognize an immense number of pathogens. B and T cells play important role in the antigen recognition by expressing a substantially diverse repertoire of antigen-specific receptors. The recent advances in the high-throughput immune repertoire sequencing has led to a tremendous increase in the antibody and T-cell receptor information. Identification of specific epitopes recognized by the receptors is vital in analyzing the repertoire sequencing data. Here, we present a tool that examines data from an immune receptor repertoire experiment, and identifies receptors (or highly related variants) that are present in the Immune Epitope Database (IEDB). The IEDB is a free resource which compiles experiments characterizing immune epitopes. IEDB also captures the epitope-specific immune receptor sequences and groups them based on their epitope specificity. IEDB currently has information on over 26,000 receptor groups. Users can provide receptor repertoire sequence data in multiple formats, and specify different CDR sequence identity cutoffs to restrict or broaden the receptor search. We expect that this tool will provide new insights into B and T cell responses captured in the receptor repertoire experiments by identifying specific epitopes, and facilitate development of new strategies for monitoring and treating of infectious, allergic and autoimmune diseases.
MHC clustering regulates selectivity and effectiveness of T-cell responses
Authors: Anikeeva Nadia , Fischer Nicholas O , Blanchette Craig D , Sykulev Yuri
Published: January 2023

Although formation of nano-scale MHC clusters on the cell membrane of antigen-presenting cells has been well documented, the essence of MHC clustering and the role in controlling antigen presentation and T-cell responses have been not systematically studied. This is due to the absence of available approaches to evaluate how the clusters’ size, density and composition of MHC clusters influence TCR engagement and triggering of TCR signaling. We exploited nano-scale discoidal membrane mimetics (nanolipoprotein particles, or NLPs) to capture and present pMHC ligands at various densities and examined the binding of these model membrane clusters to the surface of live human CD8+ T cells and the subsequent triggering of intracellular Ca2+ signaling. The data demonstrate that the proximity of pMHC ligands, high association rate of CD8-MHC interactions, and relatively long lifetime of cognate TCR-pMHC complexes emerge as essential parameters explaining the significance of MHC clustering. Rapid rebinding of CD8 to clustered MHC suggests a dual role of CD8 in facilitating the T cells’ hunt for a rare foreign pMHC ligand and the induction of rapid T-cell response. Thus, our findings provide a new understanding of how MHC clustering influences multivalent interactions of pMHC ligands with CD8 and TCR on live T cells that regulate antigen recognition, kinetics of intracellular signaling, and the selectivity and efficiency of T-cell responses.
Real-time visualization and quantification of neutrophil activation and function using live-cell analysis
Authors: Bevan Nicola , Lovell Gillan , O’Clair Belinda , Kelsey Lauren , Szybut Clare , Campwala Hinnah , Jackson Tim , Holtz Nevine , Endsley Eric , Dale Tim , Trezise Del
Published: January 2023

Polymorphonuclear neutrophils (PMNs) have multiple functions during the resolution of inflammation. At the inflammation site, increases in chemokines induce infiltration of PMNs via CXCR receptor activation, resulting in cell clearance via phagocytosis and NETosis. Here we describe characterization of the activation and function of PMNs using IncuCyte® live-cell analysis.

Changes in cell shape and CD marker expression are known indicators of PMN activation. Freshly isolated PMNs were seeded in 96-well plates, in the presence of FabFlour-488 labeled CD11b antibody for live-cell immunocytochemistry. Phase and fluorescence images were captured every 30 min for 6 h with IncuCyte S3. Cell-by-cell analysis enables individual cell segmentation and yields area, shape (eccentricity) and fluorescent intensity metrics. Subsets of PMNs could be analyzed by classifying on each of these parameters. CXCL8 induced a time- and concentration-dependent increase in eccentricity (EC50 0.63 nM) and concomitant increase in CD11b expression (EC50 0.22 nM). CXCL8-induced activation was attenuated by an anti-CXCL8 antibody. CXCL1 or CCL2 yielded little or no change in either parameter.

As a follow up, we assessed CXCL8-dependent chemotaxis using IncuCyte ClearView Chemotaxis plates, phagocytotic activity with IncuCyte pHrodo® E. coli Bioparticles® and PMA-induced NETosis (IncuCyte Cytotox Green). In all three cases robust, time-dependent signal changes were observed, consistent with known PMN function. We conclude that live-cell analysis is a flexible and powerful method for analyzing neutrophil activity, where morphological, protein and functional parameters can be readily quantified and integrated over time.
An advancement in single cell genomics allows for T cell population analysis at high resolution
Authors: Sukovich David J , Taylor Sarah E B , Pfeiffer Katherine A. , Stubbington Michael J T , Lee Josephine Y , Sapida Jerald , Roidan Daniel P. , Barrio Alvaro Martinez , Walter Dagmar , Brix Liselotte , Jacobsen Kivin , Yeung Bertrand , Zhao Xinfang , Mikkelsen Tarjei S
Published: January 2023

Progress in our understanding of immunology and cancer immunotherapies requires a comprehensive view of immune cell behavior and the interactions of these cells with their environment. Recent technological innovations have facilitated the combination of cell-surface protein, transcriptome, immune repertoire, and antigen specificity measurements from the same single cells, providing thorough and high-throughput lymphocyte characterization.

Using the 10x Genomics Single Cell Immune Profiling Solution with Feature Barcoding technology along with oligo-conjugated antibodies and peptide-MHC (pMHC) Dextramers®, we performed multi-omic characterization of PBMCs from cytomegalovirus (CMV) seronegative and seropositive patients. Next generation sequencing libraries were made following the 10x Genomics workflows, where transcriptome and immune repertoire libraries are generated alongside libraries from DNA barcodes conjugated to antibodies or pMHC.

Full length, paired TCRα/β sequences with specificity to known CMV antigens were identified in the seropositive donor, but not in the seronegative donor. Interestingly, a large Epstein Barr Virus (EBV) pMHC specific T cell expansion was identified in the CMV seronegative donor, suggesting an active EBV response. Moreover, the combination of transcriptomic and cell surface protein information resulted in an increase in resolution of cell type identification. This multi-omic workflow allowed the identification of enriched amino acid motifs within the TCR sequences that contained novel and known CDR3 amino acid sequences specific to CMV.

These technological advancements provide new biological insights that are critical for progress in the field.
Controlled temp for cell plating eliminates room temp cell settling in 96-well plates, reducing Edge Effect and improving cell conditions
Authors: Henn Alicia D. , Darou Shannon , Henn Stassa P.D. , Frank Allayna M. , Alm Kersti , Yerden Randy
Published: January 2023

The “edge effect” has always limited the use of edge wells of widely-used 96-well plates, reduced useable assay space by 37%, increased materials, time, and costs for these assays. Methods reported to reduce edge effect include leaving the plates at room temperature (RT) for extended cell settling times (30–120 min). We previously reported that controlling conditions to 37° C during cell plating prevents thermal currents and non-random distribution of cells seen during cell settling in edge wells. Here we report studies with human A549 lung carcinoma cells, used for in vitro viral assays, and human bone marrow mesenchymal stromal cells (MSC). Our null hypothesis is that there would be no difference in cell density or viability between cells plated/settled at constant 37° C and extended cell settling times at RT conditions. We plated A549 or MSC under controlled conditions in modular cell handling and incubation chambers in the Xvivo System. We pre-incubated plates for 30, 60, 90, and 120 min at RT and compared these with cells handled at constant 37 degrees. We used the HoloMonitor M4 (PHI, Inc) to monitor cell settling and assessed cell density at 24 and 48 hrs later using crystal violet. We found that cell settling at RT resulted in delayed cell attachment, delayed time to first division, and different cell densities as compared with cell handling at 37° C. Cell agitation and thermal variation was still seen between center and edge wells during cell settling with medium warmed to 37 degrees before traditional cell plating at RT. We concluded that full-time control of 37° C during cell plating is necessary to prevent cell density and cell viability edge effect that occurs during routine handling of cells under RT conditions.
The Immune Epitope Database enables and accelerates research
Authors: Salimi Nima , Vita Randi , Mahajan Swapnil , Overton James A , Dhanda Sandeep Kumar , Martini Sheridan , Cantrell Jason R , Wheeler Daniel K , Sette Alessandro , Peters Bjoern
Published: January 2023

The Immune Epitope Database and Analysis Project (IEDB) is a freely available resource funded by the National Institute of Allergy and Infectious Diseases, to advance immunology research. The database allows easy searching of all published experimental data characterizing antibody and T cell epitopes studied in humans, non-human primates, and other animal species. Epitopes involved in infectious disease, allergy, autoimmunity, and transplant are included. The data were derived from more than 19,000 publications. The IEDB also hosts tools that assist in the prediction and analysis of B cell and T cell epitopes. In recent years, the IEDB has updated its search interface and added highly sought after data, such and antibody and TCR sequence information. We will detail how the IEDB can be used to facilitate immunological research.
Intracellular pairing of multiple mRNA species at the single-cell level using DNA Origami
Authors: Ulrich Peaches Rebecca , Schoettle Louie , Dubois Courtney , Jones Kylee , Chaudhary Neelam , Anderson Karen S. , Blattman Joseph
Published: January 2023

Current research has demonstrated the importance of identification of antigen-specific T cells for use in adoptive cell therapy to treat cancer. By identifying rare T cells, we may be able to determine highly effective reagents for tumor regression. A major problem in identifying TCR for clinical applications has been development of high-throughput methods for obtaining CDR3 sequence information from TCRαβ pairs from individual cells. To this end, we have developed novel DNA origami nanostructures with “bowtie”-barcoded capture probes to simultaneously capture human TCRαβ mRNA from single cells. Origami nanostructures are delivered into T cells via electroporation, and TCRα and β mRNA are co-captured onto integral nanostructure probes. Individual TCRαβ sequences are then reverse transcribed and appended to unique origami-specific bowtie oligonucleotide barcodes that can be matched in downstream next-generation sequencing. We have validated this approach using a Jurkat T cell line, expressing known TCRαβ sequences. Upon the synthesis of DNA origami with TCRα and β capture probes, Jurkat T cells were transfected with the origami scaffold, resulting in the capture and amplification of TCRαβ sequences. Validation using Sanger sequencing confirmed the capture of barcoded TCRα and TCRβ CDR3 sequences. Using a custom “bowtie” dual-barcoding system, we have developed an approach for single cell analysis of multiple mRNA species. Although our current focus is on the TCR repertoire, the “bowtie”-barcoded probes may be modified to capture sequence information from virtually any two genes of interest.
Innovative drug discovery for autoimmune diseases aiming at BAFF signaling pathway
Authors: Yoshimoto Keiko , Suzuki Katsuya , Seki Noriyasu , Sugahara Kunio , Chiba Kenji , Takeuchi Tsutomu
Published: January 2023

Background and Purpose

It is well known that BAFF (B cell activating factor) signaling is involved in the pathogenesis of autoimmune diseases. To search for specific inhibitors against BAFF signaling, we have established our original high throughput screening system and discovered a pyrroloprymidine derivative, BIK-13, which inhibits BAFF binding to its receptor, BR3. In this study, we explored the possibility of BIK-13 as a drug to treat autoimmune diseases.

Methods

Human PBMC were stimulated in vitro with a mixture of BAFF, IL-21, and anti-IgM and anti-CD40 antibodies in the presence of BIK-13. Differentiation of B cells and the expression level of Activation-induced cytidine deaminase (AID) in the cells were analyzed by FACS and qPCR, respectively. The amounts of IL-6, IL-10 and IgG in the culture supernatants were measured by ELISA. Serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in BIK-13-treated autoimmune model mice, MRL/lpr, were measured by ELISA. The proportion of B cells among splenocytes and infiltration of lymphocytes into organs of the mice were analyzed by FACS and immunohistochemistry, respectively.

Results

In vitro experiments revealed that BIK-13 suppressed 1) differentiation of B cells, 2) AID expression and 3) IL-6, IL-10 and IgG production in a dose dependent manner. BIK-13 decreased the serum levels of an anti-dsDNA antibody, IL-6 and IL-10 in MRL/lpr mice. Notably, infiltration of B cells into lacrimal glands and the proportion of B cells among splenocytes were remarkably suppressed in BIK-13-treated mice.

Conclusion

Our data collectively suggest that BIK-13, a low molecular weight BAFF-signaling inhibitor, is a promising drug candidate for autoimmune diseases by suppressing B cell activation.
SINGLE-TARGET PAN-LIVER AUTOIMMUNE DISEASE-SUPPRESSING NANOMEDICINES
Authors: Umeshappa Channakeshava Sokke , Singha Santiswarup , Blanco Jesus , Shao Kun , Nanjundappa Roopa Hebbandi , Yamanouchi Jun , Pares Albert , Serra Pau , Yang Yang , Santamaria Pere
Published: January 2023

Peptide-major histocompatibility complex class II (pMHCII)-based nanomedicines displaying tissue-specific autoantigenic epitopes can blunt specific autoimmune conditions by re-programming cognate antigen-experienced CD4+ T-cells into disease-suppressing T-regulatory type 1 cells. Here, we show that single pMHCII-based nanomedicines displaying epitopes from primary biliary cholangitis (PBC)- or autoimmune hepatitis (AIH)- relevant mitochondrial, endoplasmic reticulum or cytoplasmic antigens can broadly blunt PBC, AIH and Primary Sclerosing Cholangitis in various murine models in a non-disease-specific manner without suppressing general or local immunity against infection or metastatic allogeneic tumors. Thus, CD4+ T-cell autoreactivity against liver-enriched autoantigens in liver autoimmune inflammation is not disease-specific and can be harnessed to treat various liver autoimmune diseases broadly.
Angiotensin-converting enzyme inhibitor suppresses Type I interferon responses, decreases neuroinflammation and improves depression-like behavior in MRL/lpr lupusprone mice
Authors: Sriram Uma , Nocito Cassandra , Hand Michelle , Lubinsky Cody , Seliga Alecia , Zuluaga-Ramirez Viviana , Winfield Malika , Persidsky Yuri
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ damage. Interferon- α (IFN-α) is a central mediator in disease pathogenesis. Neuropsychiatric lupus (NPSLE) is one of the most common manifestations of human SLE often causing depression. Administration of IFN-α in patients with chronic viral infections or cancers causes depressive symptoms. Angiotensin-converting enzyme (ACE) is part of the kallikrein-kinin/renin-angiotensin (KKS/RAS) system that regulates many physiological processes, including inflammation and brain functions. It is known that ACE degrades bradykinin into inactive peptides. We have previously shown in an in vitro model of mouse bone-marrow-derived dendritic cells that the effects of captopril (an ACE inhibitor) on IFN responsive gene inhibition were reversed when bradykinin receptors were blocked, suggesting that ACE, in part, acts via kinin receptors to bring about the suppressive effects. We used the MRL/lpr lupus-prone mouse model, an established model to study NPSLE, to check the in vivo effects of captopril on IFN-induced immune responses, neuroinflammation, and behavior. Exposing MRL/lpr mice to IFN-α increased depressive-like behavior and enhanced ACE expression in the brain. Administering captopril in MRL/lpr mice decreased expression of IFN responsive genes in brain and kidney, decreased microglial activation (IBA1 expression) and reduced depressive-like behavior as assessed by the forced swim test. Thus, ACE inhibitors may have potential for therapeutic use for systemic and neurolupus.