bluuebunny/crossref_metadata_2025 huggingface dataset:
NADPH-derived superoxide influences CD4 T cell activation by oxidizing membrane thiols
Authors: Morgan Nadine N , Tse Hubert Ming
Published: January 2023

Reactive oxygen species play a key role in mediating T cell autoreactivity in the development of autoimmune diseases including Type 1 diabetes (T1D). It was recently shown that redox regulation of cell surface thiol levels can influence T cell activation, and the development of autoimmunity. Enhanced susceptibility in developing autoimmune arthritis has been attributed to an increase in the number of reduced cell surface thiols on arthritogenic CD4 T cells. We hypothesize that NAPDH (NOX)-derived superoxide can modulate the oxidation status of cell surface thiols on diabetogenic CD4 T cells, and thereby, influence T cell effector responses. We utilized the NOD.BDC-6.9.Ncf1m1J TCR transgenic mouse that possesses superoxide-deficient CD4 T cells that recognize the putative autoantigen, islet amyloid polypeptide. BDC-6.9.Ncf1m1J CD4 T cells stimulated with their cognate BDC-6.9 hybrid peptide displayed a 3.5- and 1.5-fold increase in IFN-γ and TNF-alpha production, respectively, in comparison to NOX-sufficient BDC-6.9 CD4 T cells. Hybrid peptide-stimulated BDC-6.9.Ncf1m1J CD4 T cells also displayed a 1.8-fold increase in the percentage of CD25 and a 1.5-fold increase in the percentage of CD69 in comparison to BDC-6.9 T cells. We examined the number of reduced cell surface thiols on CD4 T cells using Alexa Fluor 647 coupled with maleimide. Interestingly, hybrid peptide-stimulated BDC-6.9.Ncf1m1J CD4 T cells exhibited a 2.3-fold increase in the percentage of reduced cell surface thiols in comparison to stimulated BDC-6.9 CD4 T cells. Our data indicates that NOX-derived superoxide mediates the oxidation status of T cell surface thiols to influence diabetogenic CD4 T cell effector responses.
The tetraspanin CD151 is elevated on activated human CD4+ T cells with enhanced cytotoxic potential
Authors: Seu Lillian , Sabbaj Steffanie , Duverger Alexandra Emilie , Wagner Frederic Houston , Goepfert Paul Andres , Kutsch Olaf
Published: January 2023

Circulating cytotoxic CD4+ T cells are absent in healthy individuals, but have been reported in autoimmune and inflammatory states. Cytotoxic CD4+ T cells arise largely as a result of chronic viral infection, such as CMV. These cells are characterized by the loss of CD28/CD27, their memory differentiation status (TEM/EMRA), and a phenotype primed towards the targeting of viral antigens. While cytotoxic CD4+ T cells are speculated to complement cytotoxic CD8+ T cell activity, they may also contribute to MHC class II-directed immunopathogenic states (e.g., vascular complications resulting from endothelia damage). The molecular mechanism of how cytotoxic CD4+ T cells are propagated remains elusive. Recently, tetraspanins have been shown to influence the cytotoxic potential of immune cells- CD63 aids in mast cell degranulation whereas CD53 modulates NK cell cytotoxicity. Here, we report that the tetraspanin CD151 is elevated on circulating CD4+ T cells with enhanced cytotoxic potential. We observed that CD151+ frequencies were most elevated on CD28neg and TEM/TEMRA CD4+ T cells. Importantly, CD151+ frequencies were robustly enriched on CD4+ T cells containing preformed granzyme B (GzmB) (51 ± 18% vs. 14 ± 6%)- cell populations reported as elevated after primary CMV infection. Within CMV-seropositive donors, CD151+ frequencies were robustly enriched on IFN-γ+GzmB+ CD4+ T cells responding to overlapping peptides from CMV pp65 (60 ± 21% vs. 17 ± 8% baseline). In summary, the tetraspanin CD151 is a cell surface molecule that demarcates circulating CD4+ T cells with enhanced cytotoxic potential, particularly against CMV antigen. Tetraspanins may therefore be a class of molecules that tunes the effector cell response to persistent antigens.
ROCK is essential for directionally persistent migration of CD8+ effector T cells within inflamed lung
Authors: Mrass Paulus , Oruganti Sreenivasa R , Byrum Janie R , Moses Melanie E , Cannon Judy L
Published: January 2023

Recruitment of effector T lymphocytes into peripheral tissues, such as inflamed lung, contributes to an effective protection against infection and cancer. The rate of pathogen detection might be further influenced by the motility of recruited immune cells within three-dimensional tissues. Indeed, recent two-photon studies have shown that effector T cells navigate actively through inflamed lung tissue, but the molecular mechanisms that regulate this process are poorly characterized. Here, we used two-photon imaging of a murine lung treated with endotoxin to quantitatively analyze tissue navigation of lung-infiltrating CD8+ effector T cells. Tracking of individual T cells within inflamed lung tissue for several hours revealed that T cell movement transitions between periods of high directional persistence and confinement. Some T cells also moved in alignment with the vasculature. Treatment with pertussis toxin to inhibit chemokine receptor-dependent Gi-type G protein signaling led to a moderate reduction of the speed of lung-infiltrating T cells. Strikingly, pharmacological inhibition of the molecule ROCK, which promotes cytoskeleton-dependent squeezing through dense environments, led to a pronounced reduction of speed and almost completely abolished directional persistence of lung-infiltrating effector T cells. Together, these results show that migration of lung-infiltrating T cells is fine-tuned by environmental signals and dependent on the cell-intrinsic ROCK-signaling pathway.
Unique pattern of PMN-MDSC migration in cancer
Authors: Patel Sima , Condamine Thomas , Dominguez George , Partlova Simona , Languino Lucia R , Vonderheide Robert H , Gabrilovich Dmitry
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are pathologically activated immature myeloid cells that contribute to an immunosuppressive tumor microenvironment. In order to exert their effects at either the primary tumor site or in metastases, PMN-like MDSC (PMN-MDSC) must first migrate to these sites. There is some evidence to suggest that chemokine gradients can mediate their recruitment. We have observed that human PMN-MDSC accumulation in colon and lung tumors strongly correlates with the expression of S100A9, CXCL1 and CXCL8 in those tumors. Moreover, we have found that, in comparison to PMN, patient PMN-MDSC have an increased chemotactic response to CXCL8 in spite of decreased cell surface CXCR2 expression. Similarly, in murine transgenic models of melanoma, pancreatic and prostate cancer, we have found that PMN-MDSC migrate more than PMN when stimulated with CXCL1, in spite of similar levels of cell surface CXCR2 expression. Interestingly, murine PMN-MDSC are more migratory even without stimulation, and have greater speed, mean squared displacement and random motility coefficient than PMN. It is possible that in addition to specific factors secreted in the primary and metastatic tumor sites, PMN-MDSC recruitment to these sites is regulated by an altered migratory behavior of the cells themselves.
Alpha-beta T cell positive selection is enhanced by CD3-delta via a quantitative developmental signaling mechanism
Authors: Wilton Katelynn Marie , Huseby April M , Ferrer Alejandro , Neier Steven C , Hoffmann Michele , Wettstein Peter , Carmona Eva M , Kappes Dietmar , Pages Diana Gil , Johnson Aaron J , Limper Andrew H , Schrum Adam G
Published: January 2023

CD3-delta has a critical role in alpha-beta T cell development; its absence greatly diminishes thymocyte positive selection. Despite this critical role, mice lacking CD3-delta have a low, but reproducible numbers of peripheral alpha-beta T cells, indicating that some T cell development occurs independently of CD3-delta. These T cells lacking CD3-delta developed via positive selection as evidenced by genetic Major Histocompatibility Complex-dependence, progression to single positive thymocytes in fetal thymic organ culture and the presence of functional immune responses in vivo. Given this data, CD3-delta must have either a qualitative or quantitative impact on positive selection. Specifically, a qualitative impact would mean that CD3-delta is required for a subset of T cell receptors (TCRs) to be selected, resulting in a starkly skewed TCR repertoire with corresponding absolute deficiencies in certain immune responses, or an altered immunodominance to specific infection. In contrast, a quantitative role in positive selection would show a conserved, diverse peripheral TCR repertoire with a decreased quantity of each TCR clone paired with present, though decreased, immune responses. The TCR repertoire complexity of T cells lacking CD3-delta was complex for both alpha and beta transcripts, likely indicating a quantitative role. Similarly, in vivo, mice lacking CD3-delta responded effectively to three independent immune challenges. These findings indicate that the CD3-delta protein quantitatively increases the efficiency of positive selection, likely through enhanced signal amplification, allowing the alpha-beta T cell pool to be more efficiently generated.
Multiparameter mass cytometry of T cell development and selection
Authors: Reich-Zeliger Shlomit , Setty Manu , Tadmor Michelle , Salame Tomer Meir , Pe’er Dana , Friedman Nir
Published: January 2023

T cell development and selection in the thymus play key roles in shaping the adaptive immune system and maintaining self-tolerance. Previous studies characterizing thymic development have largely relied on genetic perturbations and subsequent cell sorting that invariably perturb specific developmental compartments. Using mass-cytometry, allow us to capture the entire developmental progression of T cells, from the double-negative (DN) stages, through double-positive (DP) and the CD4 and CD8 branches, without perturbation. With 42 channels simultaneously measured, we collected a mass cytometry dataset profiling the mouse thymus with T-cell surface markers and transcription factors, chosen based on their broad functionality in T cell development. Using the newly developed “Wishbone” algorithm, we recovered the known stages in T cell development with high accuracy and developmental resolution. This allowed us to place DN, DP, CD4+ and CD8+ cells from a single thymus along a unified bifurcating trajectory. Our data allows for precise ordering of multiple events along the trajectory using un-sorted cells from mice that were not perturbed genetically. To characterize the selection process along the development trajectory we used Nur77-GFP mice in which GFP is induced by TCR signaling that occurs during positive and negative selection. By following GFP strength together with cell trajectory we were able to recognize and characterize the behavior of proteins that participate in selection and that are involved in the transition from the DP to SP stage. This approach enables us for the first time to place and follow coordinated changes in multiple markers that take part in T cell development and selection in the thymus using single snapshots
Development of regulatory T cells in the thymus - two paths to the same goal?
Authors: Farrar Michael A , Owen David L , Mahmud Shawn A.
Published: January 2023

Mature thymic regulatory T cells (Tregs) are defined by dual expression of Foxp3 and CD25 on CD4SP thymocytes. However, the cellular pathways by which CD4 thymocytes differentiate into Tregs remain incompletely understood. Previous reports demonstrated that Treg development occurred via a two-step process. In step one, strong TCR signaling induces expression of CD25 leading to a CD25+ Foxp3− Treg progenitor (TRP). In step two, exposure of this TRP to IL2 results in conversion to a CD25+Foxp3+ Treg. Recently, work by Singer and colleagues have defined a CD25−Foxp3lo TRP that can also be converted by IL2 into CD25+Foxp3+ Tregs. A key question is why two distinct cellular pathways involving distinct TRP populations are needed to reach the same endpoint. Using a combination of maturation marker analysis and RAG2-GFP reporter mice we observed that CD25+Foxp3− TRP arise more rapidly than the CD25−Foxp3lo TRP following positive selection. Analysis of Nur77-GFP reporter mice revealed that these two progenitor populations also display distinct affinity spectrums for self-peptide. Further, TCR sequencing has provided evidence that these 2 progenitor populations contain largely distinct TCR repertoires, suggesting these different TRP populations may be required to provide tolerance to different antigens. Indeed, our preliminary data suggests that these TRPs home to different locations upon adoptive transfer. Our results suggests a model whereby cells are driven by high affinity or high avidity interactions with self-peptide through two discrete pathways to form a comprehensive Treg repertoire capable of protecting against autoimmunity and reactivity to commensal organisms.
Thymic epithelial cells control let-7 up-regulation in thymic B progenitors during the neonatal to adult transition
Authors: Xiao Shiyun , Zhang Wen , Manley Nancy
Published: January 2023

The postnatal thymus is an efficient microenvironment for T cell specification and differentiation. B cells are also present in the thymus, and have been recently implicated in the central tolerance. In Foxn1lacZmutant mice, which undergo premature thymic involution beginning 1 week after birth, T committed progenitors were progressively reduced, however, thymic B cells started to increase at 1 week and transiently formed a peak at 3–4 weeks. These increased B cells were developed from neonatal derived BM progenitors possessing a high B potential, were originally generated in the thymus. Most of them showed CD19loB220loCD24hiCD43+/−IgM−progenitor phenotype with increased expression of Ly51 but decrease of CD25 accumulating at pre-B-II stage. These B progenitors showed a delayed down-regulation of Lin28b, an impaired up-regulation of Let-7 with an increased expression of Arid3a. However the treatment of these progenitor B cells with Vitamin D3 might up-regulate Let-7 and down-regulate Arid3 in vivo coculture. These changes were associated with a transient increase in IL7, decreased CCL25, DL4 and Cyp27b1, IL15 in thymic epithelial cells (TECs), which also reflected the changes of progenitor switch from fetal to adult type during neonatal. Our data show that signaling provided by TECs control the development of thymic B cells by regulating Let-7 and Arid3a expressions in the thymic progenitor B cells during the neonatal to adult transition.
The role of Pdia3-dependent 1α,25-dihydroxy vitamin D3 signaling on IgE-mediated mast cell activation
Authors: Baker Bianca , Barnstein Brian , Clark Nicholas , Boyan Barbara , Schwartz Zvi , Ryan John J
Published: January 2023

Active 1,25(OH)2 Vitamin D3 (1,25D3) is most commonly known for its role in the regulation of bone metabolism. However, research has now indicated it is a potent immune regulator beyond its calcemic effects. Vitamin D deficiency has been implicated in the pathogenesis of allergic diseases including asthma and atopic dermatitis. 1,25D3 is known to signal through the canonical Vitamin D receptor (VDR) facilitating its anti-inflammatory properties and regulating over 100 genes. However, protein disulfide isomerase family A, member 3 (Pdia3), was recently shown to mediate rapid-membrane responses to 1,25D3. In this study, we show that upon IgE-mediated activation, VDR knockout mast cells display enhanced cytokine production. Additionally, 1,25D3 suppresses cytokine production independently of VDR supporting the possibility of an alternative receptor facilitating the suppressive properties of Vitamin D on mast cells. Pdia3 protein and mRNA levels were upregulated in response to 1,25D3 in both WT and VDR KO mast cells. Upon blocking Pdia3 signaling, VDR KO mast cells lost their suppressive response to Vitamin D. Prostaglandin E2, which is released during Pdia3-dependent 1,25D3 signaling, mimicked Vitamin D effects on WT and VDR KO mast cells. Suppressor of Cytokine Signaling 3 (SOCS3), which is known to be upregulated by PGE2, was induced by 1,25D3 in both WT and VDR KO mast cells. Interestingly, VDR KO mast cells showed an enhanced induction of SOCS3 in comparison to WT. Collectively, our data depict a novel signaling mechanism utilized by mast cells to respond to 1,25D3 that could be explored as a potential therapeutic target for the treatment of allergic and inflammatory diseases.
Suppression of established food allergy by a combination of anti-TSLP, anti-IL-33, and anti-IL-25 monoclonal antibodies.
Authors: Khodoun Marat , Krishnamurthy Durga , Strait Richard T , Tocker Joel , Potter Crystal , Finkelman Fred D
Published: January 2023

Food ingestion generally induces antigen (Ag)-specific tolerance rather than immunity. In contrast, we recently reported (JACI, 131:442–50, 2013) that Ag-specific, IgE-mediated food allergy (FA) can be induced when a protein Ag is repeatedly ingested with a widely used nutrient, medium chain triglycerides (MCT), without any additional sensitization. Mice that have been repeatedly inoculated orally with MCT plus egg white (EW) develop a Th2 cytokine and an IgE response as well as shock (hypothermia) following oral EW + MCT challenge. Because MCT ingestion induces increased intestinal epithelial cell expression of the pro-Th2 cytokines TSLP, IL-25 and IL-33, we investigated the importance of these cytokines in establishing and maintaining FA. We found that treatment with neutralizing mAbs to any of the pro-Th2 cytokines could suppress MCT + EW-induced FA development, with aTSLP mAb causing near-total suppression and aIL-25 and aIL-33 each causing considerable suppression. In contrast, treatment with aTSLP mAb, by itself, had little effect on established FA to EW. Treatment of established FA with combinations of mAbs to any two pro-Th2 cytokines had a partial suppressive effect, with the combination of aTSLP + aIL-33 being most suppressive. Simultaneous treatment with mAbs to all three pro-Th2 cytokines caused complete loss of the hypothermia response to EW + MCT challenge and considerably reduced IL-4, IL-13, IgE, and mouse mast cell protease 1 responses. Thus, all three pro-Th2 cytokines are essential for FA induction, but these cytokines are somewhat redundant for FA maintenance in our model. These results provide a better understanding of the FA mechanisms and lead to the development of novel therapies.
High-molecular-weight kininogen and its cleavage product bradykinin are critical in the pathogenesis of autoantibody-induced arthritis
Authors: Xie Zhanli , Dai Jihong , Wu Yi
Published: January 2023

High-molecular-weight kininogen (HK) is a major component of plasma kallikrein-kinin system (KKS), the activation of which mediates HK cleavage to release bradykinin (BK). BK is a nonapeptide with potent proinflammatory and vasoactive activities. Although patients with rheumatoid arthritis (RA) exhibits the activation of the KKS and the cleavage of HK in plasma, the role of HK in arthritis pathogenesis has never been characterized. In this study we evaluated the phenotype of our newly-generated HK-deficient (Kng1−/−) mice in arthritis. To induce autoantibody-induced arthritis, two groups of Kng1+/+ and Kng1−/− mice were injected intraperitoneally with anti-type II collagen antibody cocktail (4 mg) on day 0 and lipopolysaccharide (LPS, 50 μg) on day 3. Although wild-type (WT) mice developed severe arthritis, the Kng1−/− mice exhibited significantly attenuated disease severity as evaluated by joint diameter change and clinical scores (p<0.0001). Compared with WT mice, Kng1−/− mice displayed reduced synovial hyperplasia and cartilage erosions, and decreased protein and mRNA levels of IL-1b and IL-6 in joint tissue (p<0.001). BK level and kallikrein activity in Kng1−/− plasma were significantly lower than that of WT mice. Incubation with collagen-anti-collagen antibody complex mediated BK production in human plasma. Consistent with the phenotype of Kng1−/− mice, the mice lacking BK receptors also displayed a reduced arthritis severity, as well as a decrease in IL-1b and IL-6 production in joint tissue. Our study provides the first genetic evidence indicating an important role of HK in the pathogenesis of arthritis, and the blockade of HK cleavage to prevent BK production might be a novel therapeutic strategy for RA treatment.
Characterization of CD4+ T cells expressing an elevated interferon-stimulated gene signature in SLE patients
Authors: Murray Sara , Speake Cate , Mason Michael J , Whalen Elizabeth , Buckner Jane H , Linsley Peter S
Published: January 2023

The type I interferon (IFN) signaling pathway plays a key role in Systemic Lupus Erythematosus (SLE) pathology, and a majority of patients display elevated expression levels of interferon-stimulated genes (ISGs) in the peripheral blood. However, it is unclear which immune cells are responding to IFN to drive disease pathology. We compared the ISG signatures of cell types sorted from peripheral blood of IFNβ-treated relapsing-remitting multiple sclerosis (RRMS) patients with those of SLE patients. While systemic IFNβ treatment stimulated ISG expression in the monocytes and neutrophils of RRMS patients, the ISG signature in SLE patients was primarily detected in CD4+ T and B cells. This suggests these cells may be exposed to higher levels of IFN in microenvironments of inflammation within SLE patients, such as upon interaction with APC presenting SLE antigens. We hypothesize that a subset of primarily antigen-specific CD4+ T cells expand and express ISGs during SLE disease activity, and that these cells and their signature pathways can serve as disease biomarkers and therapeutic targets. To examine ISG expression by CD4+ T cells in SLE, we compared the expression of several ISG-correlated surface markers between high- and low-IFN score patients and healthy controls. Subsets of CD4+ T cells in high-IFN score patients expressed elevated levels of PD1, CD66a, and OX40, while the majority of cells expressed a similar level as cells in healthy controls. This suggests that only a small subset of CD4+ T cells is activated by localized IFN sources and is involved in SLE pathology. We have further characterized PD1+, OX40+ CD66a+, and SLE-specific memory CD4+ T cells by RNAseq and flow cytometry.
A novel mechanism for enhanced neutrophil recruitment, retention, and podocyte damage in immune complex-mediated glomerular disease.
Authors: Powell David W , McLeish Kenneth R , Tan Min , Sheehan Ryan M , Zheng Shirong , Caster Dawn J , Korte Erik A
Published: January 2023

Glomerulonephritis (GN) is characterized by immune complex deposition and ensuing inflammation within the glomerulus of the kidney. Frank J. Dixon, a pioneer in immunologic kidney disease research, reported in 1965 that injection of nephrotoxic antibodies in rats and rabbits resulted in proteinuria and glomerular accumulation of neutrophils, and that depletion of circulating neutrophils reduced this immune-mediated glomerular damage and proteinuria. In the decades since, studies have confirmed a role for glomerular neutrophil accumulation in human GN, but the mechanisms for neutrophil recruitment and neutrophil-directed injury have not been resolved. We previously reported genetic variants for a NF-κB inhibitor ABIN1 as risks for GN. Others also report that glomerular ABIN1 gene expression is altered in GN in mice and humans. Here we present that transgenic disruption of ABIN1 function results in exacerbated podocyte injury, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in a mouse model of acute antibody-mediated GN. Moreover, a novel inhibitor of neutrophil granule release (SNAP23 peptide) also attenuates podocyte injury in this model. These in vivo findings are supported by in vitro experiments showing that secretome from homologous ABIN1 mutant podocytes activates neutrophil chemotaxis and granule release and neutrophil granule contents specifically disrupts cytoskeletal organization of ABIN1 mutant podocytes. These studies unfold a role for ABIN1 dysfunction in a novel neutrophil-mediated mechanism of podocyte injury in GN and presents inhibition of neutrophil granule release as a promising novel therapeutic direction for kidney inflammation.
Targeting the HVEM-BTLA-CD160-LIGHT network in Psoriasis
Authors: Sedy John R , Veny Marisol , Nguyen Jennifer , Balmert M Olivia , Niemela Nichole , Norris Paula S , Ware Carl F
Published: January 2023

Genetic studies have identified heritable linkages to psoriasis. However, complete identification of psoriasis susceptibility loci remains elusive. We have identified the HVEM-BTLA-CD160-LIGHT pathway as a critical regulator of innate and adaptive cell function in several autoimmune diseases, and recently in a mouse model of experimental psoriasis. Specifically, we have shown that the inhibitory receptor BTLA regulates the activity of innate γδ T cells, correlating with worsened dermatitis in BTLA-deficient animals, and attenuated disease in animals treated with agonistic BTLA antibodies. Additionally, our data indicates that the TNF receptor protein HVEM activates CD160 receptors in innate cells, promoting inflammatory signaling. More recently we have observed that skin inflammation is also worsened in animals lacking the TNF ligand for HVEM, LIGHT. In order to promote anti-inflammatory activities of HVEM, we sought to develop ligand specific biologics that could selectively engage inhibitory pathways through BTLA. Our initial studies showed that wild-type HVEM-Fc worsens disease. Additionally, we find that LIGHT-specific proteins that block HVEM-LIGHT interactions also worsen disease, consistent with our results in LIGHT-deficient animals, and identifying a unique regulatory role for LIGHT in skin inflammation. Finally, animals treated with proteins specific for BTLA and CD160 show reduced pathology compared to animals treated with wild-type HVEM-Fc. Through our efforts to synthesize ligand specific HVEM biologics, we have shown how select ablation of HVEM ligands can result in both pro- and anti-inflammatory signaling that can be targeted for the development of therapeutics for inflammatory diseases.
The nature and role of autoimmunity in cystic fibrosis lung immunopathology
Authors: Skopelja Sladjana , Hamilton JoNell , Jones Jonathan D. , Ashare Alix , Gifford Alex H. , Rigby William F.C.
Published: January 2023

Cystic Fibrosis (CF) arises due to the altered activity of the CF transmembrane regulator (CFTR) protein. While the lung disease in CF is associated with chronic infection by Pseudomonas aeruginosa (PA), identical CFTR mutations exhibit considerable clinical variability. To elucidate this, we evaluated the autoantibody response to bactericidal permeability-increasing protein (BPI) and anti-citrullinated proteins (ACPA) in CF patients (n=38) and compared it with the autoantibody profile in sera from Rheumatoid Arthritis (RA) patients (n=50), given the association of airway inflammation with ACPA induction in RA. The presence of ACPA was restricted to RA patients; no antibodies to BPI were seen. In contrast, 42% of CF patients had anti-BPI IgG, but no ACPA. Contrary to ACPA, the autoantibodies to BPI did not bind to post-translationally modified epitopes but mapped to the protein C-terminus (CT-BPI). Patients with high levels of anti-BPI IgG exhibited worse lung function (lower FEV1), the presence of anti-Cif antibodies (CFTR inhibitory factor), and the absence of the homozygous F508del CFTR mutation. Neutrophil incubation with PA led to the cleavage of BPI into a ~32kDa fragment corresponding to CT-BPI, in an elastase dependent process. In addition, anti-BPI IgG levels were higher in patients harboring the more pathogenic (mucoid) PA strain infection. These findings prompt the model that autoantibody responses reflect greater impairment of PA clearance, which is the hallmark of this chronic inflammatory disorder. Alternatively, given that no clear association between bacterial colonization and CF disease severity exists, the model that autoantibodies independently exacerbate CF lung pathology merits consideration.
Identifying the human B cell population that produces atheroprotective natural antibodies
Authors: Buchta Claire M , McSkimming Chantel C , Taylor Angela M , McNamara Coleen A
Published: January 2023

B cells act in a subset-dependent fashion in the development of murine atherosclerosis: conventional B-2 cells are atherogenic, while B-1 B cells have atheroprotective functions, mainly through production of IgM natural antibodies. Many of these antibodies are specific for oxidation-specific epitopes (OSE) such as malondialdehyde (MDA) on LDL and block macrophage uptake of oxidized lipids, preventing foam cell formation. In human subjects, high levels of plasma IgM to MDA-LDL are associated with reduced coronary artery disease and cardiovascular events; however the human B cell subset that produces these antibodies has not yet been identified. Recently, a putative human B-1 cell (CD20+CD27+CD43+) was defined. In PBMC from a cohort of human subjects presenting with cardiovascular disease, we show that expression of the chemokine receptor CXCR4 on human B-1 cells (n=54) positively correlated with plasma levels of IgM to MDA-LDL (p=0.004) and is inversely associated with coronary artery plaque volume as measured by intravascular ultrasound (IVUS) (p=0.0001). Using CyTOF mass cytometry technology, we demonstrate that CXCR4 expression was localized to a specific subset of human B-1 cells, suggesting that these cells may have distinct effector functions. Additionally, we developed biotinylated MDA-LDL and demonstrate that MDA-LDL showed enriched binding to a subset of IgM+ human B-1 cells, suggesting that these cells are responsible for producing IgM to MDA-LDL. Ongoing experiments will examine antibody production from these cells. Identification of the human cell type that produces IgM to MDA-LDL has the potential to lead to the development of strategies to enhance production of this atheroprotective antibody.
Uterine NK cells of preeclamptic patients express reduced Fas
Authors: Loewendorf-Snead Andrea I , Nguyen Tina A , Kahn Daniel A
Published: January 2023

Preeclampsia, a disease of pregnancy, despite extensive investigation has escaped a defining mechanism, though risk factors suggest underlying immunopathology. A lymphocytic infiltrate of the decidua includes the specialized uterine NK cells. Uterine NK cells have distinct phenotype and function and are essential for healthy pregnancy.

After UCLA IRB approval (#11-002962), patients with normal and preeclamptic third trimester pregnancies undergoing cesarean section were recruited for sampling of maternal peripheral blood, and endometrium at the time of delivery. Lymphocyte populations and functionality were characterized using flow cytometry and in-vitro re-stimulation assays.

As expected, we found increased proportions of CD56hi CD16- uterine NK cells in the endometrial decidua compared to the peripheral blood of the same patient. Only uterine NK cells exclusively displayed an activated (CD69+) phenotype. In preeclamptic patients, the percentage of peripheral NK cells expressing Fas was significantly increased whereas Fas expression was decreased on uterine NK cells (CD56+CD16−). Interestingly, intracellular cytokine production of uterine NK cells is similar in healthy and preeclamptic patients (Perforin A, Granzyme A, IFNγ). While proportions of γδ T cells isolated from the decidua are similar in healthy and preeclamptic patients, γδ T cells from the decidua of preeclamptic patients displayed reduced expression of CD127 and CD27. This pattern along with reduced Granzyme A-expression suggests reduced functionality similar to the exhaustion observed in αβ T cells in the context of persistent inflammatory stimulation.
Using dimensionality reduction techniques on cross project phospho-flow data to cluster patients based on signaling patterns
Authors: Couse Michael , Wang Weiqi , Holmes Tyson , Maecker Holden T
Published: January 2023

Phospho-flow is an assay that measures the phosphorylation level of various cellular proteins on a per cell basis. This approach allows researchers to apply numerous stimulations and measure the phosphorylation level in a variety of cell types for each patient, thus creating a patient specific signaling “fingerprint”. This technique has been used to identify perturbations underlying a single disease, however little work has been done to compare these “fingerprints” across diseases. In this study, we compile phospho-flow data performed at the Stanford Human Immune Monitoring Center (HIMC) and use dimensionality reduction techniques to cluster patient’s “fingerprints” from a variety of diseases.

The HIMC provides immune monitoring assays and catalogs these assays on the Stanford Data Miner (SDM), a data repository with mining tools that allows for the upload and integration of clinical and demographic data as well as data from HIMC assays. Through this, we were able to sort on only phospho-flow studies and subsequently extract the data from 164 patients comprising 11 different conditions. Each sample received 7 different stimulations; 3 STAT proteins were measured in 8 cell types totaling over 27,000 data points.

Through principal component analysis we are able to see distinct clustering of patients by disease when graphing the 1st 3 components. This clustering picture is validated when looking at the clustering that occurs through analysis by tSNE and unsupervised hierarchical clustering. Next, we isolated the top loadings from the first three principal components to identify the signaling pathways driving the clustering. Specific signaling pathways were identified to explain differences seen between diseases and within diseases.
Intravenous rasburicase-induced immune tolerance is associated with depletion of specific lymphocytes and up-regulation of regulatory T cells
Authors: Xu Hui , Mills Frederick , Max Edward E
Published: January 2023

Therapeutic proteins can be potent agents for treating serious diseases, but in many patients these proteins provoke antibody responses that blunt therapeutic efficacy. Intravenous administration of high doses of some proteins induces immune tolerance, but the mechanisms behind this effect are poorly understood. As a model to study tolerance induction in mice, we used Rasburicase, a commercial recombinant uricase used for treatment of hyperuricemia. Intraperitoneal (i.p.) injection of Rasburicase without or with alum provoked a clear anti-Rasburicase antibody response, but intravenous (i.v.) injection did not. The lack of response to i.v. Rasburicase was apparently due to active immune suppression, since i.v.-treated mice showed blunted antibody and reduced T cell responses to subsequent i.p. injections of Rasburicase. This blunted response was associated with a decrease in Rasburicase-specific B cells and T cell proliferation, and an increase in proportion of CD4+ FoxP3+ regulatory T cells (Treg). We examined the number of lymphocytes in peripheral blood after Rasburicase i.v. injection. Intravenous injection of rasburicase temporary reduced numbers of B cells and T cells, but robustly depleted Rasburicase-specific B cells in blood. Further results showed that rasburicase i.v. injection decreased the number of lymphocytes by inducing apoptosis of B cells and activated T cells. Thus, our data suggests that Rasburicase i.v. injection-induced apoptosis and depletion of lymphocytes and up-regulation of Treg cells play important roles in the intravenous therapeutic protein-induced immune tolerance.
Chemokine production by regulatory T cells is required for therapeutic attenuation of autoimmunity and allograft rejection
Authors: Pesenacker Anne M , Patterson Scott J , Wang Adele Y , Mojibian Majid , Morishita Kim , Tan Rusung , Kieffer Timothy J , Verchere C Bruce , Panagiotopoulos Constadina , Levings Megan K
Published: January 2023

Regulatory T cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and non-harmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require Tregs to be near their targets of suppression, but how Treg-to-target proximity is controlled is unknown. We found that Tregs produce chemokines to attract CD4+ and CD8+ T cells close to their proximity in vitro and in vivo. Mouse and human lineage committed Tregs, as well as murine in vitro induced Tregs, produced CCL3 and CCL4 at message and protein level. Chemokine producing Tregs had an activated Treg phenotype with high expression of multiple proteins associated with suppressive function. Furthermore the human CCL3 and CCL4 promoter could be transactivated by FOXP3. In vitro and in vivo migration experiments indicated that CCR5 was the major receptor required by target cells. CCL3 and CCL4 deficient Tregs were impaired in their ability to prevent experimental autoimmune encephalomyelitis or islet allograft rejection, but suppressive in a standard in vitro assay. Moreover, Tregs from subjects with established type 1 were impaired in their ability to produce CCL3 and CCL4. These results demonstrate a previously unknown facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their in vivo function and therapeutic effect in autoimmunity and transplantation.
Functional angiotensin and bradykinin receptors are expressed on primary human mast cells
Authors: Fukuoka Yoshihiro , Hite Michelle R , Schwartz Lawrence B
Published: January 2023

Purpose

The role of mast cells (MCs) has traditionally been recognized as an effector cell for IgE-mediated allergic diseases, involving ~20% of USA citizens. However, everyone has MCs, indicating their involvement in biologic and pathobiologic conditions beyond allergy. Angiotensin converting enzyme inhibitors (ACEI) and angiotensin II type 1 receptor (AT1R) blockers are used to treat cardiovascular disorders such as hypertension, while a bradykinin type 2 receptor (B2R) inhibitor is used to treat attacks of hereditary angioedema. We examined the expression and biological function of AT1R, B2R and B1R on primary human MCs.

Methods

MCs were dispersed from fresh surgical human skin obtained from CHTN, and placed into culture with SCF (100 ng/ml) for 4–8 weeks. FACS analysis, western blotting, qRT-PCR, degranulation (β-hexosaminidase release), cytokine/chemokine secretion of IL-6, IL-8 and CCL5, nitric oxide (NO) production (DAF-AM diacetate) were measured.

Results

FACS analysis and western blotting showed the AT1R and B2R are expressed on MCs. Angiotensin II (0.1–10 μM) and bradykinin (BK, 0.1–2 μM) stimulated MCs to release cytokines (1–4 ng/ml) which was inhibited by specific inhibitors. BK also stimulated the production of NO. Both the B2R and B1R are up-regulated by TNFα (10 ng/ml for 3 days), leading to enhanced activation of MCs by BK (1 μM) and desArg-LysBK (1 μM). Exposure of MCs to angiotensin II (2 μM), angiotensin (1–7)(10 μM) or ACEI (ramipril 1 μM) also enhanced cytokine secretion triggered by low concentration of BK (0.03–0.25 μM).

Conclusion

These findings suggest functional interactions of AT1R, B2R, ACE and Mas on human MCs, providing novel roles for MCs in cardiovascular, inflammatory and allergic disorders.
The effect of LPS on CD103 expression on dendritic cells
Authors: Brassard Julyanne , Bernatchez Emilie , Langlois Anick , Blanchet Marie-Renée
Published: January 2023

Control of the intensity and duration of the pulmonary immune response is crucial in homeostasis maintenance in the lung. Dendritic cells (DCs), the main antigen presentation population in the lung, play a crucial role in these mechanisms. Notably, CD103+ DCs play a regulatory role while CD11b+ DCs are considered pro-inflammatory cells. Therefore, the balance between these two important DC populations is crucial in homeostasis maintenance and host-defense. However, the mechanisms regulating this tight balance remain poorly understood. Preliminary data obtained in our laboratory showed that Saccharopolyspora rectivirgula exposure downregulates CD103+ DCs levels in vivo as early as 18h following antigen exposure, impacting the pro/anti-inflammatory DC balance. However, the mechanisms involved in this response are still undefined. As TNF is an important pro-inflammatory cytokine secreted rapidly in response to antigens (such as LPS), lung- and spleen-isolated DCs were stimulated in vitro for 18h with increasing concentrations of TNF and LPS. Flow cytometry and qRT-PCR analysis demonstrated that CD103 protein and mRNA expression are downregulated following stimulation. The percentage of CD103+ DCs was also decreased in vivo 18h after mouse LPS exposure (mouse model). Furthermore, exposure of Cd103−/− mice to LPS led to an impaired resolution of airway inflammation and return to homeostasis, supporting the importance of CD103 expression for homeostasis maintenance in the lung in response to antigens. Our results suggest that modulation of CD103 expression in response to antigens is a crucial step in host defense and homeostasis maintenance in the lung.
NLRX1 attenuates damage following traumatic brain injury through negatively regulating NF-κB signaling
Authors: Allen Irving C , Meza Armand , Brickler Thomas , Coutermarsh-Ott Sheryl , Ives Angela , Bertke Andrea , Theus Michelle
Published: January 2023

Mechanical trauma to the CNS results in the disruption of the cellular microenvironment leading to massive necrotic and apoptotic loss of neuronal and glia populations. The progressive cascade of secondary events, including ischemia, inflammation, excitotoxicity and free radial release contribute to neural tissue damage. Members of the NLR family of pattern recognition receptors are essential mediators of the host immune response. Recently, our lab and others identified a novel sub-group of NLRs that function as negative regulators of inflammation. One of the members of this sub-group, NLRX1, is a potent regulator of interferon, NF-κB, ROS production and autophagy. Thus, we hypothesized that NLRX1 attenuates Traumatic Brain Injury (TBI) through the negative regulation of overzealous innate immune system signaling. To evaluate this hypothesis, we utilized Nlrx1−/− mice in a controlled cortical impact (CCI) injury model. The Nlrx1−/− mice exhibited significantly larger brain lesions and increased motor deficits following CCI injury. We also observed significant proliferation of microglia within the Nlrx1−/− lesions compared to wild type animals. Mechanistically, our data indicates that NLRX1 attenuates TBI progression through the microglia compartment via negative regulation of NF-κB signaling and IL-6 production. Together, our data extends the function of NLRX1 beyond its currently characterized role in host-pathogen defense and identifies this highly novel NLR as a significant modulator of TBI progression.
BTLA+ dendritic cells govern induction of extrathymic regulatory T cells and tolerance.
Authors: Jones Andrew , Bourque Jessica , Opejin Adeleye , Gross Cindy , Hawiger Daniel
Published: January 2023

Dendritic cells (DCs) initiate immunity and also antigen-specific tolerance mediated by extrathymic regulatory T (Treg) cells that prevent subsequent T cell priming. It remains unclear how DCs regulate induction of such tolerance. Here we report that T cell activation by CD11c+ DCs in steady state had no lasting impact on subsequent immune responses and resulted in neither induction of Treg cells, nor priming of autoimmune T cells. Our studies further revealed that efficient induction of Treg cells required BTLA expressed specifically in DEC205+ CD8+ CD11c+ DCs. Ligation of HVEM, a receptor of BTLA, in Foxp3neg T cells promoted Foxp3 expression through upregulation of CD5. In contrast, T cells activated in the absence of BTLA and HVEM-mediated functions, remained CD5lo and therefore failed to resist the inhibition of Foxp3 expression in response to effector-differentiating cytokines. Thus only T cells specifically stimulated by BTLA+ DCs efficiently converted into Treg cells that mediated antigen-specific tolerance.
Anti-inflammatory role of cannabinoid receptor type 1 antagonist AM251 in high fat diet-induced obesity— a focus on microRNAs.
Authors: Miranda Kathryn , Mehrpouya-Bahrami Pegah , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Obesity is a major health problem in the U.S. that continues to increase in prevalence. Excessive calorie consumption leads to expansion of adipose tissue through both hypertrophy and hyperplasia. As a result, adipocytes die and macrophages infiltrate into the adipose tissue to repair damage. Activation and infiltration of immune cells contribute to the chronic low-grade inflammation present in obesity. Immune cells express cannabinoid receptors that regulate inflammation. In this study, a mouse model of high fat diet-induced obesity was used to determine the effect the CB1 receptor antagonist AM251 has on adipose tissue inflammation. C57BL6 mice were fed a high fat diet (HFD) for twelve weeks to induce obesity and then given AM251 (10mg/kg/day) oral gavage for four weeks while continuing HFD. Treatment improved metabolic parameters while reducing fat mass and pro-inflammatory cytokine production. To further investigate the anti-inflammatory effects of AM251, infiltrating F4/80+ cells were isolated from epididymal fat and used for microRNA microarray. Nine miRNAs were differentially expressed as a result of AM251 treatment. These miRNAs were analyzed using Ingenuity Pathway Analysis software (Qiagen) and were found to significantly affect the “Inflammatory Disease and Inflammatory Response” pathways. Upregulated miRNAs such as miR-466i-3p were found to target macrophage polarization genes including Pik3r1. The current study suggests that CB1 antagonists may mediate anti-inflammatory effects and amelioration of obesity through regulation of miRNA expression.

(Supported in parts by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641, and Veterans Affairs Merit Award BX001357.)
microRNAs profile expression in a mouse model of lupus triggered by lipids associated in non-bilayer phospholipid arrangements
Authors: Zarate Luz Angela , Wong Carlos , Bustos Israel , Resendiz Albany , Landa Carla , Pedroza Abraham , Wong Carlos , Perez Carlos , Baeza Isabel
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic multifactorial autoimmune disease; its pathology is mainly attributed to auto-antibodies that cause inflammation and tissue damage. Our group proposed that lipids associated in non-bilayer phospholipid arrangements (NPA) are involved in the development of SLE. BALB/c mice developed a disease very similar to human SLE when they received Mn2+ or drugs chlorpromazine, procainamide (which stabilize NPA in the plasma membrane), or liposomes bearing NPA induced by these drugs or by Mn2+. Recently, we found that the gene expression profile of mice with lupus was different to that found in healthy mice and was similar to patients with SLE: in mice with lupus genes involved in presentation of exogenous antigens, antibody production and TLR4 and NOD2 signaling and some genes encoded for complement proteins were over-expressed, while genes for NK cell recognition and apoptosis were under-expressed. In the present study, we used the mouse immunopathology miScriptTM miRNA PCR Array in order to compare mature microRNA profile between mice with lupus and healthy mice. The results indicated 9 microRNAs deregulated, 4 microRNAs were underexpressed (miR-155, miR-146b, miR-23, miR-let-7d) which can regulate positively transcription of RNAs associated with IL-6, IL-1 and 5 microRNAs were overexpressed (miR-205, miR-200a, miR-18a, miR-207 and miR-574) which can regulate negatively transcription of RNAs associated with apoptosis and T cell response. This results were validated used TaqMan microRNA Assays and were similar to those found in human LES. Then this study highlight the importance of microRNAs in regulate immune process that has implications in the pathogenesis of LES an inflammatory disease.