bluuebunny/crossref_metadata_2025 huggingface dataset:
PU.1 and IRF8 modulate the activation of NLRP3 inflammasome via regulating the monocyte-specific expression of the NLRP3 and its component genes
Authors: Yashiro Takuya , Yamamoto Machiko , Araumi Sanae , Nishiyama Chiharu
Published: January 2023

NLRP3 inflammasome plays crucial roles in the host defense against pathogens by activating Caspase-1, which catalyzes the processing of immature IL-1β and IL-18 into a biologically active form. NLRP3 expression is restricted in monocytic lineages such as monocytes, macrophages, and dendritic cells, thus its expression must be strictly regulated. However, the regulatory mechanism of lineage-specific transcription of the NLRP3 gene remains largely unknown. In this study, we investigated the molecular mechanisms of cell-type specific NLRP3 expression.

RT-PCR revealed that the NLRP3 gene was dominantly driven from the distal promoter in a human monocytic cell line, THP-1. By reporter assay, we identified several cis-acting elements including an Ets/IRF composite element (EICE), which is frequently associated with monocytic lineage-specific gene regulation, in the distal promoter of the human NLRP3 gene. EMSA and ChIP assay demonstrated that transcription factors, PU.1 and IRF8, both of which are known to play essential roles in the development of monocytic lineage, cooperatively bind to the EICE. The expression of NLRP3 was significantly decreased by the siRNA-mediated knockdown of PU.1 and/or IRF8. In addition, the knockdown of PU.1 and/or IRF8 down-regulated the expression of ASC and Caspase-1, which form inflammasome complex with NLRP3. Furthermore, we found that the silencing of PU.1 and IRF8 dramatically abrogated the processing of pro-IL-1β.

These results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific NLRP3 expression by binding to the EICE site within the distal promoter.
Targeting intracellular check points to unleash natural killer cell cytotoxicity
Authors: Barda-Saad Mira , Ben-Shmuel Aviad , Biber Guy , Raiff Anat , Sabag Batel , Matalon Omri
Published: January 2023

Natural killer (NK) cells are a potent weapon of the immune system against viral infections and tumor growth. The actomyosin network generates forces through the activity of actin filaments and myosin motors. This machinery is responsible for the conversion of mechanical forces into biochemical signals in a process termed mechanotransduction. However, the mechanism by which mechanotransduction controls the immune response, and specifically lymphocyte activity, is poorly understood. Here, we demonstrate that actomyosin retrograde flow (ARF) controls NK cell response through a novel interaction between beta-actin with the SH2-domain containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, thereby regulating NK cell cytotoxicity. Actin dynamics govern SHP-1 conformational structure dictating its catalytic activity. Indeed, blocking actin dynamics results in reduced SHP-1 activity, by confining SHP-1 to its inactivated “closed” conformation. This reduced enzymatic activity of SHP-1 leads to increased phosphorylation of SHP-1 substrates, an elevation of intracellular calcium flux, and NK cell cytotoxicity. Using multidisciplinary approaches including functional and advanced molecular imaging, we followed the signaling events in individual NK cells from the moment of target cell encounter until the final outcome – target cell killing or NK cell inhibition. Our data suggest that SHP-1 plays a major role as a sensor of ARF-generated forces in the process of mechanotransduction, and reveal a novel mechanism by which regulation of SHP-1 by ARF dictates NK cell killing decisions. Our data identify ARF as a master regulator of the lymphocyte response.
Phosphorylation of MAVS/VISA by Nemo-like kinase (NLK) for degradation regulates the antiviral innate immune response
Authors: Zhong Bo
Published: January 2023

MAVS is essential for antiviral immunity, but the molecular mechanisms responsible for its tight regulation remain poorly understood. Here, we show that NLK inhibits the antiviral immune response during viral infection by targeting MAVS for degradation. NLK depletion promotes virus-induced antiviral cytokine production and decreases viral replication, which is potently rescued by the reintroduction of NLK. Moreover, the depletion of NLK promotes antiviral effects and increases the survival times of mice after infection with VSV. NLK interacts with and phosphorylates MAVS at multiple sites on mitochondria or peroxisomes, thereby inducing the degradation of MAVS and subsequent inactivation of IRF3. Most importantly, a peptide derived from MAVS promotes viral-induced IFN-β production and antagonizes viral replication in vitro and in vivo. These findings provide direct insights into the molecular mechanisms by which phosphorylation of MAVS regulates its degradation and influences its activation and identify an important peptide target for propagating antiviral responses.
Glycan Remodelling Controls Germinal Centre B cell Responses
Authors: De La Pena Enterina Jhon Ralph , Sarkar Susmita , Macauley Matthew
Published: January 2023

Germinal centre (GC) B cells are routinely identified through distinct changes on their surface carbohydrates, called glycans; yet, whether these modifications play an essential role in GC reaction remain unknown. One striking change relates to the monosaccharide sialic acid. In mice, this change is mediated through downregulation of an enzyme called Cmah. As a consequence, GC B cells lose the preferred ligand for CD22, a member of the sialic acid-binding immunoglobulin-type lectins (Siglecs) and a co-inhibitory receptor of the B cell antigen receptor. Since CD22 regulates B cell function, we hypothesize that this specific glycan remodelling on B cells control the GC reaction by modulating the activity of CD22.

To test our hypothesis, we developed a mouse model that constitutively expresses Cmah in B cells. Following immunization, we find that sustained expression of Cmah in B cells results in impaired generation of antigen-specific GC B cells. More significantly, this observed defect is dependent on CD22, highlighting that coordinated loss of preferred ligands for CD22 on B cells plays a critical function in the GC. Furthermore, antibody affinity maturation is significantly impaired in immunized mice harbouring B cells which lack either CD22 or its cis glycan ligands, further supporting the role of CD22 and its glycan ligands in the GC. Ongoing efforts are focused on defining the GC-specific nature of these observations and examining potential mechanism related to BCR signalling, GC B cell survival and exit to plasma cells, and B-T cell interactions.
Role of peptide-MHCII complexes in germinal center B cell selection and plasma cell differentiation
Authors: Jing Zhixin , McCarron Mark , Fooksman David R
Published: January 2023

Germinal center (GC) selection is critical for generating affinity-matured antibodies in T-cell-dependent immune responses. Despite the essential help follicular T cells provide to GC B cells, it is unclear how GC B cell fate decisions are made. Although cognate peptide-MHCII (pMHCII) is critical for T cell help, how pMHCII density directly contributes to B cell selection and cell fate remain unclear. The a-DEC205-OVA (dec) model system has been widely used to interrogate GC B selection, by enhancing selection of WT (Ly75+/+) B cells over untargeted Ly75−/− (KO) B cells. However, under these conditions, it is unclear what the fate of B cells presenting intermediate levels of pMHCII is and whether T cell help is indeed limited.

To investigate these questions, we used Ly75+/− (DEC-het) B cells, which expressed intermediate levels (~50%) of surface DEC205 protein compared to WT B cells, and presented proportional amount of OVA peptide after dec treatment. Using competitive transfers, we found that WT B cells expanded two-fold more than DEC-het B cells. This 2-fold difference in expansion was maintained at a wide range of dec administration, indicating that in vivo, T cells can distinguish different densities of pMHCII on multiple B cells in the same GC and respond proportionally. Surprisingly, we found that upon dec stimulation, both WT and DEC-het GC B cells were equally capable of differentiating into plasma cells, suggesting that pMHCII density does not control B cell fate directly. Blocking of CD40 signaling removed the two-fold advantage of WT over DEC-het B cells during selection after dec treatment, suggesting CD40 signaling is proportional to pMHCII density. These results provide new interpretations of how GC selection occurs in vivo.
In vivo mechanisms by which Irf5 regulates BCR-, TCR- and TLR-induced plasma cell generation and antibody secretion
Authors: Brune Zarina , Rice Matthew , Matta Bharati , Lu Ailing , Barnes Betsy J
Published: January 2023

Generation of antibody secreting cells (ASCs) requires multiple B cell activation pathways, including B cell receptor (BCR), T cell receptor (TCR), and Toll-like receptors (TLRs). In human naïve B cells, we reported that knockdown of interferon regulatory factor 5 (IRF5) resulted in IgD retention, reduced proliferation and plasma cell differentiation, and reduced IgG secretion. Defects were due to early impairments in B cell activation and clonal expansion. Conversely, murine Irf5 was reported to regulate antibody production through direct control of class switch recombination (CSR) at the γ2a locus. To further elucidate distinct and overlapping roles between human and murine IRF5 in the regulation of plasma cell differentiation and antibody production, we performed in vivo analysis of B and T cell differentiation and function in wild-type (wt) and Irf5 knockout (ko) littermate mice after immunization. B and T cell subsets were analyzed by multi-color flow cytometry. Naïve B cells were in vitro differentiated to ASCs with CpG-B, anti-IgM, anti-CD40 and IL-21. Kinetics of B cell activation, AID expression and IgG production were determined. BCR and TCR signaling were examined by phospho-flow. A combination of T-dependent/independent and TLR-dependent/independent immunizations were used to study ASC differentiation. RAG−/− mice were used to determine cell type-specific effects. Results indicate distinct roles for human and murine IRF5 at the early stages of BCR signaling and redundant roles at the later stages of CSR, plasma cell differentiation and antibody secretion via regulation of AID.
Antigen valency dictates the affinity of B cells recruited to germinal centers and the magnitude of extrafollicular B cell responses
Authors: Kato Yu , Groschel Bettina , Abbott Robert K. , Menis Sergey , Irvine Darrell J. , Schief William R. , Crotty Shane
Published: January 2023

While it is widely agreed that antigen valency is a factor in immunogenicity, how antigen valency contributes to B cell selection during antigen-specific responses is poorly understood. Here, using HIV immunogens with a defined valency ranging from 1 to 60, we investigated the role of antigen valency during different phases of the antigen-specific B cell response. We show that highly multimerized immunogens rapidly activated cognate B cells in vivo with little affinity discrimination, initially. This subsequently led to strong extrafollicular and germinal center responses where B cells within a broad affinity range were efficiently recruited. Low-valency (1–8 copy) antigens induced less diverse effector B cell responses, with limited recruitment of low-affinity B cells. The data reveal roles of antigen valency in dictating affinity thresholds required for recruitment of B cells to germinal centers or rapid extrafollicular plasma cell responses, with important implications for vaccine design.
Liver-resident CD8 T cells do not need Levy walks to be efficient killers
Authors: Ganusov Vitaly V , Rajakaruna Harshana , O’Connor James , Cockburn Ian A
Published: January 2023

Movement strategies by which antigen-specific CD8 T cells search for sites of infection are not fully understood. It was recently suggested that activated CD8 T cells, specific for Toxoplasma gondii, perform generalized Levy walks in murine brains; that is cells perform a local search and then displace randomly to distant locations. Several theoretical studies found that Levy walkers are an order of magnitude more efficient at finding rare targets than Brownian walkers. We investigated movement patterns of Plasmodium-specific liver-resident CD8 T cells using intravital microscopy. Based on the distribution of movement lengths Trm cells performed Brownian walks and yet displayed superdiffusive (Levy-like) behavior, i.e., on average they displaced further from the initial position than that predicted by the standard diffusion theory. Mapping out structure of liver sinusoids revealed that superdiffiusive nature of T cell walks in the liver is likely due to small branching angles of the sinusoids, instructing T cells to move directly. By simulating T cells moving as Brownian or Levy walkers in free space or physiologically constrained liver sinusoids we showed that structure may override the intrinsic pattern of cell movement. Interestingly, while Levy walkers indeed required less time than Brownian walkers to find rare targets when search was done in space with no constrains, on average, this advantage disappeared when search was done in the liver. Our results, thus, suggest that observed patterns of movement of T cells in the liver are likely to arise as a combination of cell-intrinsic search patterns and environmental constrains, and that evolutionary benefits of Levy walks may be limited in biologically-relevant environments.
Mechanistic mathematical representation of CD8+ T cell mediated drug-induced liver injury (DILI) Part 1: initial development using experimental antigen, ovalbumin (OVA)
Authors: Battista Christina , Kenz Zackary , Shoda Lisl
Published: January 2023

Idiosyncratic DILI (iDILI), rare liver injury not dependent on drug dose, is poorly understood and costly to patients and drug developers. Some iDILI events appear immune-mediated. This work builds on the established hepatocyte life cycle and intrinsic injury models in DILIsym, utilizing quantitative systems toxicology methods to incorporate direct activation of liver CD8+ T cells by OVA-expressing hepatocytes.

Simulated hepatocyte OVA expression leads to activation of naïve OVA-specific CD8+ T cells. Activated T cells differentiate to effector, memory, or exhausted T cells. T cell cytotoxicity induces apoptosis in antigen-presenting hepatocytes, with resultant ALT release. By design, the model reproduces a differential CD8+ T cell response to low vs. high levels of hepatocyte OVA expression consistent with data 1,2. Mechanistically, the differential response is driven by effector T cell mediated clearance of low antigen vs. persistent high antigen and differentiation of exhausted T cells3. Reducing T cell functional avidity by 70x, as described for OVA altered peptide ligands4, reduced IFN-γ production and T cell cytotoxicity consistent with data5.

By reproducing these results, DILIsym establishes proof-of-concept in the quantitative relationships among antigen-expressing hepatocytes, antigen-specific T cell number, cytotoxicity, and ALT release. These quantitative relationships set the stage for translation to drug-mediated CD8+ T cell cytotoxicity6.
AR blockade promotes immunosuppression and pro-tumorigenic activity of myeloid cells
Authors: Udartseva Olga , Consiglio Camila Rosat , Ramsey Kimberley , Gollnick Sandra O.
Published: January 2023

It has been recently recognized that sex hormones can regulate hematopoiesis and modulate immune phenotypes that underlie sex disparities in immune response. Androgen receptor (AR) antagonism is known to compromise T cell response; however, the role of AR signaling in myeloid cell biology is poorly understood. To address this question, we analyzed the effect of AR blockage on functional activity of myeloid cells in MARKO (Myeloid AR KnockOut) mice and after pharmacological inhibition of AR. We demonstrated that AR antagonism promoted VEGFa, Arg1 and PD-L1 expression in MDSCs, and enhanced their immunosuppressive activity. AR blockage induced metabolic switch in myeloid cells by suppression of mitochondrial respiration and upregulation of glycolysis. AR-mediated metabolic reprogramming was regulated in a MPC/AMPK-dependent manner. To understand the role of AR antagonism in anti-tumor immunity, we utilized the AR-independent models of MC-38 colon and TRAMP C2 prostate carcinomas. Neither AR inhibition nor AR deletion in myeloid cells affected tumor infiltration with TAMs, MDSCs or T cells. However, in both models AR blockade and genetic deletion promoted tumor growth in immunocompetent but not immunodeficient mice, suggesting an important role of AR-mediated regulation of immunosuppression in anti-tumor immunity. Taken together, our findings reveal that AR signaling represents an important regulator of myeloid cell functional activity, and suggest a novel tumor-extrinsic mechanism of prostate cancer relapse.
Endoplasmic reticulum-targeting delivery of curcumin alters reactive oxygen species production and phenotypes in activated macrophages
Authors: Yen Chia Liang , Chen Ru-Fen , Shieh Dar-Bin , Jiang Si-Tse , Shieh Chi-Chang K
Published: January 2023

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that organelle-targeted delivery of sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitor curcumin with the endoplasmic reticulum (ER)-targeting peptide decorated PLGA nanoparticles (NPs) rescues mutant H338Y-gp91phox protein maturation of a particular type of CGD with a CybbC1024T mutation, leading to ER retention of the mutant protein. However, how the rescuing of mutant H338Y-gp91 phox protein with ER-targeting curcumin-PLGA NPs on macrophages functions and differentiation are still unknown. In this study, we analyzed gp91phox maturation and macrophage M1/M2 polarization in bone marrow-derived macrophages (BMDMs) from wild-type (WT), Cybb−/−, and CybbC1024T transgenic Cybb−/−mice. First, we found that the levels of CD86 and CD206 expression with lipopolysaccharide+interferon-γ (LPS+IFN-γ) stimulation were similar in WT and CybbC1024T transgenic Cybb−/−BMDMs and CD206 expression was lowered in Cybb−/−mice when compared with WT and CybbC1024T transgenic Cybb−/−BMDMs. Second, we found that CD206 levels after LPS+IFN-γ stimulation were enhanced by free curcumin, curcumin-PLGA NPs and ER-targeting curcumin-PLGA NPs treatment in CybbC1024T transgenic Cybb−/−BMDMs, but not in WT and Cybb−/− BMDMs. Meanwhile, we found that ER-targeting curcumin-PLGA NPs enhanced gp91phox glycosylation in CybbC1024T transgenic Cybb−/−BMDMs. These findings may indicate that both the expression and maturation of gp91phox are crucial for macrophage M1/M2 polarization. Gp91phox maturation may be important for M2 phenotypes in activated macrophages.
Metabolic characteristics of Th2 and Th17 cells drive airway inflammation
Authors: Contreras Diana C , Cephus Jacqueline Y , Dahunsi Debolanle O , Newcomb Dawn C , Rathmell Jeffrey C
Published: January 2023

Asthma is an airway inflammatory disease that is mediated by T effector (Teff) cells, specifically Th2 and Th17 cells. Increases in disease severity leading to neutrophilic asthma is characterized by a shift towards a higher Th17 response. Typical treatment of asthmatic patients include the use of glucocoriticoid (GC) steroids. These drugs are effective at controlling inflammation in mild cases but as disease severity increases towards neutrophilic disease there is resistance by Th17 cells to the effects of these drugs. A key therapeutic objective is to identify either alternative treatments for asthma or targets that make Th17 cells more susceptible to GCs. Studies have shown that cells that have increased glycolysis and oxidative phosphorylation are more resistant to the effects of GCs. We hypothesized that the intrinsic metabolic phenotype of Th17 cells allow them to be more resistant to GCs. In this study we show, that in the murine model of airway inflammation there are differences in the expression of metabolic proteins between Th2 and Th17 cells that are present in the lung. Additionally, we show that metabolic inhibitors alter the functionality of the Teff cells and can be a target to restore sensitivity to GCs. We have also performed CyTOF analysis of asthmatic patients’ PBMCs, these data demonstrate a much higher metabolic phenotype of asthmatic patients when compared to healthy controls. Overall our studies suggest that metabolic inhibition may be a viable therapeautic in conjuction with GCs in order to dampen the function of Teffs in the airway.
Maternal immunometabolic adaptation in pregnancy
Authors: Rees April , Jones Nick , Jenkins Ben , Richards Oliver , Cronin James , Thornton Cathy
Published: January 2023

Pregnant women undergo dynamic metabolic and immunologic changes to ensure provision of nutrients to, and prevent rejection of, the fetus. The mother increases glucose production, glucose intolerance and insulin resistance to support fetal-placental development and transitions from lipid storage to lipolysis to meet her own energy demands. Maternal immunoregulation prevents rejection of the fetal semi-allograft whilst maintaining protection against pathogens.

Hypothesis

Immunometabolic adaptation underpins maternal immune function changes in pregnancy.

Aim

To determine whether monocytes undergo metabolic adaptation in pregnancy and at which gestational stage this might occur.

Results

Peripheral blood monocytes of term pregnant (>37 weeks) and not pregnant women were compared using flow cytometry. Pregnant women had significantly more non-classical CD16+ monocytes, and CD16+ and CD16− subsets had more CD36 (fatty acid transporter; p = 0.0025) and less CD98 (amino acid transporter; p = 0.0043).

Bioenergetic analysis of monocytes showed oxidative phosphorylation was significantly reduced in pregnant women; glycolysis was unchanged. Also, oxidative phosphorylation was reduced significantly in the mononuclear cells of women with gestational diabetes at 28 weeks compared to healthy pregnant women at the same gestational stage.

Discussion

Ongoing work includes analysis of mitochondrial health and measuring transcriptomics, biosynthetic lipids, cytokines, and enzyme responses. Results are vital to understanding immunological adaptation during pregnancy. This could provide insight into adverse pregnancy outcomes and women’s long-term health in relation to immune-mediated diseases.
Metformin effect on human macrophage inflammatory response and phagocytosis of Mycobacterium tuberculosis
Authors: Cervantes Jorge L , Sanca Angel , Barragan Jose
Published: January 2023

Metformin (MTF) has a well-documented ability to cfontrol hyperglycemia, which has been shown to have effects on macrophage and lymphocyte functions that are key to controlling tuberculosis (TB) infection.

Here, we aimed to better understand the effects of MTF on the phagocytosis of Mycobacterium tuberculosis (Mtb) by human macrophages. PMA-differentiated THP-1 cells with two reporters for nuclear factor-κB (NF-κB), and interferon-regulatory factors (IRFs) were treated with 2mM of MTF for 4 hours, and then inoculated with Mtb from various lineages. Since MTF can also directly inhibit key metabolic processes of Mtb, we controlled this variable by using of gamma-irradiated mycobacteria. Phagocytosis was assessed by immunofluorescent assay.

Phagocytosis of Mtb increased in MTF-treated macrophages. NF-κB activation after Mtb stimulation was lower in MTF-treated macrophages. The effect on IRF activation was minimal.

Our results indicate that MTF improves phagocytosis of Mtb by macrophages, while it at the same time modulating their inflammatory response. Downregulation of type I IFN pathways, associated with active TB infection, could allow for improved activation of macrophages in the presence of TB infection. These results support the effects of MTF in keys steps TB infection control, and support its use as an additional treatment for TB.
Modeling Immune Effector Responses in Alveolar Epithelial Cell Driven Lung Injury and Fibrosis
Authors: Venosa Alessandro , Tomer Yaniv , Jamil Sarita , Beers Michael Francis
Published: January 2023

Mutations in the Surfactant Protein-C gene [SP-C] have been linked to pulmonary fibrosis (PF), with the missense isoleucine to threonine substitution at position 73 [I73T] as the most common. Clinically, PF is characterized by unrelenting tissue scarring and respiratory failure, intermitted by episodic exacerbations, bouts of inflammatory cell influx of unknown cause, which drastically reduce patient prognosis. Current murine models of PF rely on exposure to toxic agents or overexpression of pro-fibrotic signaling molecules in order to generate fibrotic endpoints. This reduces the overall relevance to human disease. To address these shortcomings, we developed a murine model of fibrosis designed to expressed mutant SP-CI73T. The resulting phenotype is accompanied by elevations in bronchoalveolar lavage cell counts, diffuse alveolar damage, and mortality. RNA and protein analysis demonstrate SP-CI73T epithelial cells initiate immune cell recruitment via CCL-2, IL-5, CCL-11, and CCL-17. Flow cytometric analysis highlighted temporally definite waves of inflammatory cells: an initial Ly6C+ inflammatory monocytes influx (3d), transient neutrophilic influx, and subsequent alveolar eosinophilia (2wk). RNA-seq analysis of sorted Ly6C+ monocytes revealed both inflammatory (inos, Il-6) and pro-fibrotic (col1a1, col1a2) phenotype. Pharmacologic and genetic monocyte ablation reduced inflammatory burden and improved survival of SP-CI73T mice. Taken together, we show that our mutant SP-CI73T model recapitulates the clinical features of PF. These findings support the concept that epithelial dysfunction is a key component of fibrogenesis and that inflammatory monocytes is responsible for disease exacerbation.
Transient immunosuppression blocks iBALT formation in the lung induced by helper-dependent adenoviral vector during gene delivery
Authors: Cao Huibi , Zhou Peter , Duan Cathleen , Hu Jim
Published: January 2023

Lung is an immune sensitive organ with presence of all immune cells at the frontline of immunity. Under chronic infection conditions, inducible Bronchus-associated lymphoid tissue (iBALT) can be formed to defend foreign infectious organisms. We wonder whether viral vectors for gene therapy could induce iBALT formation and whether this process can be controlled through immune modulation. To test these ideas, we repeatedly administered a helper-dependent adenoviral (HD-Ad) vector to mouse lungs and examined iBALT formation. HD-Ad vectors are based on adenovirus but with all viral coding sequences deleted and is one option for large gene delivery. In our previous studies, HD-Ad vectors have exhibited high in vivo efficiency in transduction of mouse, rabbit and pig airway epithelial cells, including stem/progenitor cells such as basal cells. HD-Ad vectors expressing therapeutic gene encoding cystic fibrosis transmenbrane conductance regulator (CFTR) rescue CFTR function in CF patient primary cells. In our mouse studies, we found that HD-Ad vectors induced iBALT formation and antibody production. We used cyclophosphamide or HD-Ad expressing IL-10 to modulate the host immune responses and to examine the effects on iBALT formation. Comparing with control group (HD-Ad vectors only), the mice treated with cyclophosphamide or IL-10 expressing HD-Ad vector showed a significant reduction of iBALT formation and infiltration of inflammatory cells, including CD4+ and B220+ as well as production of anti-Ad antibodies and neutralizing antibodies. Our results indicate that transient modulation significantly mitigated immunopathology and extended transgene expression in mouse airways following readministration of HD-Ad vectors.
Natural Killer cell immune synapse formation and cytotoxicity is controlled by mechanical tension of the target
Authors: Simmonds P , Friedman Daniel , Hale Alexander , Hodson Nigel W , White Michael RH , Davis Daniel M
Published: January 2023

Natural Killer (NK) cells can kill infected or transformed cells directly via the formation of a lytic synapse at the cell-cell interface. Diseased cells often present with altered mechanical properties, and cell stiffness is a feature of tumour cell migratory potential. However, little is known about how target cell stiffness impacts the assembly of the NK cell synapse or the subsequent functional response.

To study this, we set out to create surrogate targets with specific ligands presented on cell-sized beads of different stiffness. Sodium alginate cell-sized microbeads were synthesised where chemical composition was altered to produce beads of low (9kPa), medium (34kPa), or high (254kPa) stiffness. These were coated with antibodies against the NK cell activating receptor NKp30 and the integrin LFA-1 to model the 3D interaction between NK cells and target cells.

Imaging revealed that later steps in NK-cell activation differed significantly, including the degree of cytoskeletal arrangement, F-actin accumulation, microtubule organising centre polarisation and perforin accumulation. Stiffer beads induced polarisation of the MTOC in 59% of conjugates analysed compared to only 7% with low stiffness targets. Functionally, NK cell degranulation was higher when forming conjugates with stiffer targets. The metabolic profiles were similar between NK cells stimulated overnight by antibody-coated beads, regardless of their stiffness. Altogether, these data imply that stiffer targets are more potent in triggering the initial activating signals in NK cells. Thus, immune synapse mechanosensitivity represents a novel form of NK cell regulation.
Phenotypically Friendly Allospecific NK Cells Differentially Suppress Allospecific T cell Responses in Prenatal Hematopoietic Chimeras
Authors: Kang Hee Kap , Ott Katherine , Turner Lucas , Redden Catherine , Alhajjat Amir , Shaaban Aimen
Published: January 2023

Prenatal allospecific tolerance hinges on the developmental selection of NK cells expressing a friendly phenotype that is not cytotoxic to allogeneic donor cells. We wondered if prenatally educated friendly NK cells influenced allospecific T cell responses as an additional mechanism supporting prenatal tolerance. Therefore, we hypothesized that prenatally educated friendly NK cells exert suppressive effects on the induction of allospecific T cell responses.

To challenge this hypothesis, we examined the suppression potential of prenatally educated friendly NK cells through gain and loss of function experiments in stable prenatal Balb/c into B6.Thy1.2 chimeras (engrafter mice). We first depleted allospecific NK cells in host engrafter mice and then adoptively transferred Thy1.1+ naïve responder T cells along with CFSE-labeled allogenic or syngeneic target cells into the engrafter hosts. We found that the depletion of allospecific Ly49D+ NK cells in engrafter mice increased Thy1.1+ T cell alloimmunity in vivo. Conversely, adoptive transfer of friendly NK cells suppressed the induction of allospecific T cell responses in host mice. Furthermore, cultured friendly NK cells from engrafter mice showed stronger suppression on the induction of T cell alloimmunity in vitro than that of naïve mice. Lasltly, NK cell-mediated suppression of allospecific T cells required cell contact.

From these results, we conclude that prenatally educated friendly NK cell exert enhanced suppressive effects on T cell alloimmunity in vivo and in vitro. Depending on the context of their prenatal education, NK cells can differentially influence T cell responses in prenatal chimeras.
Changes in the gut microbiota caused by ASBTi suppress intrahepatic steatosis and inflammation in a mouse model of non-alcoholic fatty liver disease (NAFLD)
Authors: Matsui Masahiro , Asai Akira , Okamoto Norio , Nishikawa Tomohiro , Ohama Hideko , Tsuchimoto Yusuke , Fukunishi Shinya , Higuchi Kazuhide
Published: January 2023

Although the estimated prevalence of NAFLD is approximately 25%, an effective treatment has not been established. Recently, gut microbiota has been shown to affect the pathogenesis of NAFLD. In this study, the protective effect of Apical Sodium-dependent Bile acid Transporter inhibitor (ASBTi) against NAFLD was investigated in mice fed a high-fat diet for 12 weeks (HFD mice) via changing their gut microbiota. ASBTi mice were mice treated with ASBTi (2.5 μmol/kg/day) during HFD feeding. For fecal microbiota transplantation (FMT), stools were collected from HFD mice and ASBTi mice. Fecal materials obtained from HFD mice and ASBTi mice were transplanted to normal mice, fed HFD for 12 weeks, and designated as HFD-FMT mice and ASBTi-FMT mice, respectively. Microbiota in these groups of mice were analyzed by 16S rRNA gene sequencing. The degrees of hepatic steatosis and inflammation in these mice were determined pathologically. The shannon index of the microbiota from ASBTi mice (3.31±0.19) and ASBTi-FMT mice (3.29±0.18) was significantly high as compared to that from HFD mice (2.90±0.19) and HFD-FMT mice (2.89±0.16). Steatosis and inflammation in the liver of HFD mice and HFD-FMT mice were shown to be pathologically severe and NAFLD Activity Score (NAS) was 7.67±0.58 in HFD mice and 5.67±2.08 in HFD-FMT mice. However, those in the liver of ASBTi mice and ASBTi-FMT mice were improved and NAS was reduced in ASBTi mice (1.67±0.58) and in ASBTi-FMT mice (2.67±0.58). These results suggest that changes in the gut microbiota caused by ASBTi suppress intrahepatic steatosis and inflammation in NAFLD mice. Elobixibat can be expected to prevent fatty liver.
Malaria-driven atypical memory B cells are blinded to soluble but not membrane-associated antigens
Authors: Sohn Haewon , Ambegaonkar Abhijit A. , Kwak Kihyuck , Manzella-Lapeira Javier , Brzostowski Joseph , Pierce Susan K.
Published: January 2023

A common feature of many chronic human infectious diseases, including malaria, includes the large expansion of a population termed atypical memory B cells (MBCs) in peripheral blood, which are phenotypically and transcriptionally distinct from naïve B cells and classical MBCs. One shared distinguished phenotype among these is their high expression of a variety of inhibitory receptors including FcγRIIB. Despite the potential importance of atypical MBCs, if or how they function in chronic infectious diseases has yet to be clearly known. We reported earlier that in malaria atypical MBCs, in contrast to naïve B cells and classical MBCs, are refractory to activation by soluble antigen. Thus, atypical MBCs have been described as hypo-responsive to antigen-driven B cell activation and believed to be ‘exhausted’ and to contribute to the poor acquisition of immunity in chronic infectious diseases. However, we show the evidence here that atypical MBCs respond robustly to membrane-associated antigen by signaling through the B cell antigen receptor, gathering and internalizing antigen for presentation and initiating differentiation toward plasma cells. We discovered that on antigen-containing membranes the immune synapses of atypical MBCs actively exclude the inhibitory receptor FcγRIIB thus allowing BCR signaling. The high expression of inhibitory receptors by atypical MBCs may serve to restrict B cell responses to antigens presented on cell surfaces, avoiding the potential deleterious effects of chronic soluble antigen exposure.
IL12RB1 allelic expression imbalance in polarized human TH cells
Authors: Mejia Oscar Rosas , Williams Amanda , Robinson Richard
Published: January 2023

Allelic Expression Imbalance (AEI) occurs when two alleles of a given gene are expressed at different levels, either due to epigenetic inactivation and/or genetic variation in regulatory regions. Human autosomal genes are widely presumed to be equally expressed from both alleles (biallelic expression); this is not always the case, however, as AEI affects expression of odorant receptor and adhesin genes in neurons, as well as immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes. Ig and TCR gene AEI is an important regulator of immune development and disease resistance; therefore, it is reasonable to believe AEI of other immune receptors may similarly impact immune development and disease resistance. Here we demonstrate that the human cytokine receptor gene Interleukin 12 Receptor Beta 1 (IL12RB1) exhibits AEI in differentiated T cells, as measured by a significant deviation of the allelic mRNA expression ratios from that of the allelic gDNA ratios. Specifically, naïve human CD4+ T cells were purified from healthy adult donors and incubated with polarizing cytokines to drive their differentiation into specific T helper subsets (e.g. TH0 cocktail: IL-2. TH1 cocktail: IL-2, anti-IL-4, IL-12. TH17 cocktail: IL-2, IL-1b, TGF-b1, IL-23, anti-IFNg, anti-IL-4). The extent of IL12RB1 AEI was then quantified and related to T cell expression of IFNg (TH1) and IL17 (TH17). Collectively, our data suggest that IL12RB1 AEI associates with the expression of anti-mycobacterial effector genes in T cells. Future experiments will determine the epigenetic or molecular mechanism behind IL12RB1 AEI.
REV-ERB-dependent heme signaling regulates Th17 cell activity
Authors: Mosure Sarah A , Solt Laura A
Published: January 2023

T helper 17 (Th17) cells are important for protective immunity at mucosal surfaces, but their dysregulation is implicated in the pathogenesis of autoimmune and chronic inflammatory diseases. A better understanding of the signaling pathways governing Th17 cell-mediated immunity and pathogenicity would aid in the development of novel therapeutics to treat Th17 cell-mediated diseases. We recently showed that the REV-ERB nuclear receptors (REV-ERBα and REV-ERBβ) are critical regulators of Th17 cell development and pathogenicity. As ligand-regulated transcription factors, the REV-ERBs can bind their natural ligand, heme, which modulates REV-ERB function. To investigate heme’s role as a REV-ERB-dependent signaling molecule in Th17 cells, we differentiated naive CD4+ T cells under Th17-polarizing conditions and found heme treatment repressed differentiation. Repression was partially ablated in cells lacking the REV-ERBs, indicating heme can specifically activate the REV-ERBs in Th17 cells. We further found that enzymes involved in heme biosynthesis and transport were upregulated during Th17 cell differentiation, indicating heme is actively produced in Th17 cells. To investigate the role of endogenous heme, we overexpressed REV-ERB mutants incapable of binding heme, which relieved repression of Th17 cell differentiation relative to over expression of wild type (WT) REV-ERBs. RNA-seq of mutant- vs WT-overexpressed cells revealed that heme modulates a specific subset of REV-ERB target genes. Together, our findings suggest that conditions affecting heme levels in Th17 cells can alter the transcriptional phenotype of the cell via heme modulation of REV-ERB activity.
Identifying key transcriptional regulators that establish T cell memory during viral infection
Authors: Tsuda Shanel M , Diao Huitian , Getzler Adam J , Milner Justin J , Goldrath Ananda , Crotty Shane , Pipkin Matthew E.
Published: January 2023

During infections, naive CD8 T cells differentiate into terminal effector cells (TE) that are relatively short-lived, and memory precursor (MP) cells that give rise to long-lived memory CD8 T cells, but the transcriptional control of this process is still unclear. In naïve CD8 T cells, cis-regulatory regions that become accessible in chromatin during the first 24 hours of TCR stimulation and that remain accessible in mature memory T cell subsets are highly enriched with motifs encoding binding sites for the ETS- and bZIP-families of transcription factors (TFs), many of which also overlap Runx-TF binding sites. The ETS and bZIP TF families are encoded by 88 genes, and are differentially expressed between naïve, early effector, and memory CD8 T cell subsets. To interrogate their requirements functionally, we applied an in vivo pooled RNA interference screen using short hairpin RNAs in microRNA contexts (shRNAmirs) to suppress individually all TFs from both families in CD8 T cells responding to lymphocytic choriomeningitis virus (LCMV) infection. In addition, we focused on Ets1, the most highly expressed ETS TF in naïve, effector and memory cell subsets. Ets1 suppression with shRNAmirs impaired overall effector CD8 T cell numbers in vivo, and increased the frequency of TE-like cells at early times after LCMV infection, which resulted in reduced long-lived effector cells and an increased fraction of altered central memory-like cells at later time points. Enforced Runx3 expression in Ets1-suppressed cells partially restored normal MP cell formation at early times. These results suggest that Ets1 and Runx3 are each necessary for the normal differentiation of MP cells, and could function cooperatively to program their differentiation.
Establishment And Application of An in Vitro Cd8+ T Cell Exhaustion System
Authors: Zhao Manzhi , Kiernan Caoimhe H. , Leon Leticia G. , van Meurs Marjan , Brouwers-Haspels Inge , Mueller Yvonne M , Katsikis Peter D
Published: January 2023

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL). In vivo models of CTL exhaustion yield very few exhausted CTL, limiting the analysis can be done on these cells. Furthermore, the milieu in these models can obscure the phenotype of exhausted CTL. An in vitro system could therefore greatly facilitate the study of this cell state.

Purified OT-I CTL were cultured and repeatedly stimulated with OVA(257–264) SIINFEKL peptide to induce exhaustion. On day 5, inhibitory receptors (IRs) and cytokine production were assayed. Sorted live CTL were transferred into WSN-OVA influenza infected mice and on day10 post infection the number of the donor CTL were measured. Gene expression changes were determined by RNAseq and DNA methylation sequence.

In vitro exhausted CTL exhibited upregulation of multiple IRs as well as impaired polyfunctionality. They expanded less than controls in vivo. In vitro exhausted CTL exhibited all the transcriptomic characteristics of in vivo exhausted CTL.

Repeated stimulation of CTL with their specific peptide, induced all the functional, phenotypic and transcriptomic characteristics of exhausted CTL. This in vitro system can be used to identify genes and signalling pathways involved in exhaustion and facilitate the screening of reagents that prevent/reverse CTL exhaustion.
C C Chemokine Receptor 7 Differentially Regulates Trafficking of T Cells to the Bone Marrow During the Adaptive Immune Response
Authors: Vines Charlotte M , Cervantes Jaisel , Vazquez Aaron , Bill Colin A
Published: January 2023

It has become increasingly evident that the bone marrow is an important niche for memory B cells and memory T cells that can be activated to become effector T cells during a secondary immune response. However, signaling events that promote bone marrow persistence of memory cells remain largely unknown. We have found that the C-C chemokine receptor 7 (CCR7) signaling plays an important role in regulating the presence of memory cell populations of the bone marrow. In mice homozygously deleted for CCR7 (CCR7−/−) we find that the memory immune cell repertoire is redistributed following the secondary immune response. Specifically, instead of homing to the lymph nodes or spleen, the CCR7−/− effector memory T cells, which respond quickly to antigen, localize to the bone marrow. Additionally, using a CCR7 conditional knockout mouse, we found that when CCR7 is deleted only in T cells, numbers of activated B cells and antibody titers increase, while the regulatory T cells that would normally suppress the immune response, have reduced numbers within the bone marrow. Moreover, we found that in our CCR7f/f-CD4-Cre mice in the absence of CCR7 in T cells, bone marrow activated B cells increased in numbers on day 30. This was not due to differences in the immune response to B-cell cytokine IL-4, which was not significantly increased in any of the mice tested. Instead, we observed a difference in the overall percentage of activated B cells in CCR7f/f-CD4-Cre mice compared to CD4-Cre controls. From these data we infer that CCR7 may promote differential migration of effector memory T cells into the bone marrow that have a direct effect on the composition of the memory B-cell compartment.
The LncRNA Snhg1 is Essential for Memory CD8 T Cell Establishment
Authors: Zhang Yanyan , Bai Qiang , Ni Ting , Yang Liuqing , Jiang Lubin , Ye Lilin , Wu Yuzhang
Published: January 2023

The efficient induction and long-term persistence of pathogen-specific memory CD8 T cells are pivotal to rapidly curb the reinfection. Recent evidence indicates that lncRNAs possess higher lineage and stage specificity than mRNAs in T cell development, differentiation and function. However, the pivotal lncRNAs regulating CD8 T cell differentiation remain largely unclear. Through profiling of CD8 T cell lineages in this study, the lncRNA Snhg1 with the conserved on-off-on expression pattern in naïve-effector-memory CD8 T cells from mouse to human, is found to be required for memory CD8 T cell generation. Further, Snhg1 is found interacting with the highly conserved vesicle trafficking protein Vps13D to facilitate IL-7Rα membrane location specifically. IL-7 has been reported to sustain memory survival through STAT5-BCL2 axis, but whether IL-7 can directly promote memory differentiation remain cloudy. In this research, Snhg1/Vps13D depletion impair memory formation through IL-7-STAT3-TCF1-Blimp1 axis. Whether TCF-1 is induced by canonical Wnt-β-Catenin signaling in memory CD8 generation remain controversial. This work finds gene Tcf7 is regulated by direct binding of p-STAT3 in CD8 stimulated with IL-7. Furthermore, TCF-1 is found promoting memory with blocking effector mainly through impeding Prdm1 expression directly to balance Blimp1-BCL6 and Blimp1-ID3 axis. Thus, this study proposes a model for lncRNA as critical factor that couples membrane trafficking of cytokine receptor with transcriptional induction to promote memory CD8 T cell establishment.
Siglec 5 - a novel checkpoint receptor in T cells
Authors: Vuchkovska Aleksandra , Iwashima Makio
Published: January 2023

Immune checkpoint molecules have become a major target for designing immunotherapies. Across fourteen malignancies, the FDA has approved six drugs targeting three different inhibitory checkpoint receptors. Even though TCR stimulation through MHC-peptide and CD28 co-stimulation are the major requirements for T cell activation, it is the engagement of checkpoint receptors that dictate the functional outcome of a particular T cell. Here, we describe a new candidate checkpoint receptor, Siglec 5, expressed on activated T cells. To date, Siglec 5 is defined as a self-associated molecular pattern receptor that exhibits an inhibitory response upon interaction with sialic acids, subsequently dampening immune system activation against self. Current literature shows that Siglec 5 is expressed mainly by myeloid cell, such as neutrophils and monocytes, and that T cells do not express any Siglecs, including Siglec 5. However, we found that Siglec 5 is a unique, late-activation marker of T cells, upregulated at 48 hours, with peak expression at 72 hours post-TCR stimulation. Similar to PD-1, Siglec 5 has an ITIM and a SWITCH domain, both well described for their inhibitory function. In this study, we sought to determine the function of Siglec 5 on T cell activation. We found that overexpression of Siglec 5 in T cells results in a significant inhibition of T cell activation measured by reporter NFAT and AP-1 activity. Mutagenesis studies show that both of the intracellular domains, ITIM and SWITCH, of Siglec 5 are required for mediating T cell inhibition. Together, these data demonstrate that Siglec 5 is a previously unknown immune checkpoint molecule found on T cells.