bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a humanized mouse model that recapitulates the fibrotic response to biomaterial implants
Authors: Doloff Joshua C , Sadraei Atieh , Farah Shady , Bortell Rita , Brehm Michael A , Greiner Dale L , Anderson Daniel , Langer Robert
Published: January 2023

Implanted biomedical devices reside within tens of millions of patients in the United States alone, and are involved in millions of new surgeries every year. They comprise a major component of modern medicine and are essential for many clinical applications ranging from hip/knee replacement, tissue repair/reconstruction, controlled release, sensing, and cell transplantation. Limiting therapeutic performance and lifespan, immune-mediated foreign body rejection results in sequestration behind dense layers of fibrotic scar tissue. Current approaches for management of long-term biomedical device implantation often involve broad-spectrum anti-inflammatories. However, these agents are not specific to individual immune populations, and have side effects in vivo. To avoid such issues, improved biomaterials need to be identified with reduced immunogenicity. Deeper understanding of immune-mediated foreign body responses was leveraged to generate a variant of the humanized mouse model, previously unreported in the field of host rejection of biomaterial implants, that is capable of recapitulating fibrosis. Here, we present this model in comparison to wildtype systems, including rodents and non-human primates. Cellular and cytokine responses were determined with numerous biomaterials—hydrogel alginate, ceramic glass, and polymer polystyrene, across multiple implant sites. This model allows us to investigate human immune cells interacting directly with implantables, without putting patients at risk. It is our hope this model can be used as a pre-clinical diagnostic tool for screening implantable synthetic as well as natural materials for immunogenicity.
Invariant NKT cells recruit CD103+ dendritic cells via XCL1-XCR1 axis, thereby promoting allergic airway resistance
Authors: Woo Yeon Duk Chris , Kim Hye Young , Chung Doo Hyun
Published: January 2023

Among immune cells, dendritic cells (DC) and invariant (i) NKT cells are critically implicated in allergic asthma. However, the interaction between two subsets remains elusive in asthma. To address this, we investigated how these two subsets interact and exert regulatory effects on asthma. Upon intranasal inoculation of WT mice with synthetic ligand, a-galactosyl ceramide (a-GalCer), CD103+ DCs were significantly recruited into the lungs in WT but not CD1d KO mice, suggesting that iNKT cells contribute to recruiting CD103+ DCs in the respiratory system. Moreover, microarray assay revealed that iNKT cells highly expressed XCL1, a chemokine for CD103+ DCs expressing XCR1. Consistently, a-GalCer treatment increased production of XCL1 by iNKT cells in vitro and in vivo, and XCL1 KO mice showed a deficiency in iNKT cell-mediated recruitment of CD103+ DCs into the lungs. In the OVA-induced allergic asthma model, XCL1 KO mice attenuated airway resistance, inflammation, CD103+ DC number, and Th2 type immune responses in the lungs. Furthermore, adoptive transfer of XCL1-deficient iNKT cells did not alter airway resistance and recruit CD103+ DCs in CD1d KO mice, whereas WT iNKT cells or CD103+ DCs restored allergic asthma. Taken together, this study demonstrates that iNKT cells crosstalk CD103+ DCs via XCL1-XCR1 axis, thereby promoting allergic asthma.
Dynamic monitoring of pathological and immunological changes and treatment effect of killer artificial antigen presenting cells in a murine experimental autoimmune encephalomyelitis model
Authors: wan xin
Published: January 2023

Multiple sclerosis (MS) is a chronic, autoimmune disease of the central nervous system. Experimental autoimmune encephalomyelitis (EAE) are widely used to study the pathological features and mechanisms underlying MS. Here, a murine EAE model was induced. The disease degree was daily assessed; The local inflammatory infiltration was observed and the expressions of MBP and GFAP were detected. The frequencies and reactivity of MOG antigen-specific CD4+ T cell and CD8+ T cells were concurrently investigated. As a result, the dynamic changes of inflammation, myelin loss and astrocyte proliferation in CNS were highly consistent with clinical manifestations observed in EAE course. Data also emphasize the pathogenic roles of both CD4+ T cells and CD8+T cells in the development of EAE, while their reactivity in response to the antigen was different from their dynamic changes of frequencies.

Killer artificial antigen presenting cells (KaAPCs) are thought to be a powerful tool to inhibit autoimmune effect and kill autoreactive T cells avoiding systemic immune suppression. Here, a KaAPC was generated by covalently co-coupling targeted binding molecules (H-2Db/MOG40-54 dimmer, H-2I-Ab/MOG35-55 multimer), negative regulation molecules (anti-mouse Fas mAbs, PD-L1-Ig, TGF-β1) and antiphagecytic molecules (CD47-Ig) onto poly lactic-co-glycolic acid (PLGA) microspheres. We found decreased clinical score of EAE mice after several times treatment of KaAPCs.

Collectively, this study presented a clear histopathologic and immunologic pattern of mouse EAE model, which may contribute to better understanding of the EAE process. Also, KaAPCs are feasible strategy for treating EAE, which lead to a new approach for MS therapies.
NKG2D signaling between human NK cells enhances TACE-mediated TNF-α release
Authors: Markiewicz Mary A , Sharma Neekun
Published: January 2023

Natural killer group 2 member D (NKG2D) is a strong NK cell activating receptor, with engagement by ligands triggering granule release and cytokine production from NK cells. The function of NKG2D signaling in NK cells has largely been studied in the context of engagement of the receptor by ligands expressed on the surface of target cells, such as tumor cells. Here we report that upon activation with IL-12, IL-15 and IL-18, human NK cells expressed NKG2D ligands of the UL16 binding protein (ULBP) family on the cell surface. NKG2D-ULBP engagement during homotypic interaction between cytokine-stimulated NK cells increased the activity of the metalloprotease TNF-α-converting enzyme (TACE). This enhanced TACE activity significantly increased the release of TNF-α and ULBP proteins from the surface of the NK cells. These results demonstrate that NKG2D signaling is critical for maximal TNF-α release by NK cells. Further, they are the first to demonstrate a role for NKG2D-ligand interaction via homotypic NK cell contact in NK cell effector function.
Neutrophil Cathepsin G inhibits hapten-presenting dendritic cell production of IL-12 and skewing of hapten-reactive CD4 T cell development to IFN-γ- and IL-17-producing effector cells in allergic contact sensitivity
Authors: Kish Danielle , Min Susie , Fairchild Robert L
Published: January 2023

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive CD8 T cells that produce IFN-γ and IL-17 and mediate CHS and hapten-reactive CD4 T cells producing IL-4 that cannot mediate CHS. Although CXCR2-dependent neutrophil recruitment into the hapten challenged skin is required to direct effector CD8 T cell infiltration into the site to elicit CHS in WT mice, CHS was elicited following challenge of hapten sensitized CXCR2−/− mice. Sensitization of CXCR2−/− mice or antibody-mediated depletion of neutrophils during sensitization of WT mice generated hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 and either T cell population could mediate CHS independently of the other. The hapten-reactive CD4 cells producing IFN-γ and IL-17 and their CHS responses were not generated during hapten sensitization of IL-12−/− mice depleted of neutrophils or WT mice depleted of neutrophils and treated with anti-IL-12 mAb. Neutrophil depletion during hapten sensitization of WT mice markedly increased numbers of IL-12-producing dendritic cells in the skin draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to challenge. Induction of CHS effector CD4 T cells in neutrophil-depleted WT mice was eliminated by subcutaneous injection of recombinant cathepsin G during sensitization. Thus, hapten skin sensitization induces neutrophil production of cathepsin G that regulates hapten-presenting dendritic cell production of IL-12 and the development of CHS effector CD4 T cells.
Skin-resident effector memory CD8+CD28− T cells exhibit a pro-fibrotic phenotype in patients with systemic sclerosis
Authors: Fuschiotti Patrizia , Larregina Adriana T , Domsic Robyn T , Stolz Donna B , Medsger Thomas A , Lafyatis Robert A
Published: January 2023

Loss of CD28 expression by CD8+ T cells occurs with age and during chronic inflammatory conditions. CD8+CD28− T cells are a heterogeneous cell subpopulation whose function ranges from immunosuppressive to effector. Here we analyzed the role of CD8+CD28− T cells in the pathogenesis of systemic sclerosis (SSc), a connective tissue disorder characterized by autoimmunity, vasculopathy and extensive cutaneous and visceral fibrosis. We show that the frequency of CD8+CD28− T cells is increased in the blood and affected skin of SSc patients, independent of patient age, and correlates with the extent of skin fibrosis. We found that the majority of skin-tropic CD8+CD28− T cells are resident in the skin lesions of patients in the early stage of the disease, exhibit an effector memory phenotype and present a strong cytolytic activity ex vivo. Skin-resident and circulating SSc CD8+CD28− T cells produce high levels of the pro-fibrotic cytokine IL-13, which induces collagen production by normal and SSc dermal fibroblasts. Thus, our findings indicate that CD8+CD28− T cells represent a pathogenic T-cell subset in SSc and likely play a critical role in the early stage of SSc skin disease.
Group 2 Innate Lymphoid Cells Recruit and Activate Airway Macrophages to facilitate Pulmonary Epithelial Regeneration
Authors: Copenhaver Alan M , Dagher Rania , Berlin Aaron A , Besnard Valerie , Maret Marielle , Hamidi Fatima , Aubier Michel , Kolbeck Roland , Pretolani Marina , Humbles Alison A
Published: January 2023

Group 2 innate lymphoid cells (ILC2s) are a subset of ILCs that are resident in many tissues including the lung. These cells respond to epithelial cytokines and in turn potently produce many type 2 associated cytokines, such as IL-4, IL-5, and IL-13. Beyond cytokine production, ILC2s are thought to aid tissue repair after damage by producing growth factors such as amphiregulin or by expressing the enzyme arginase-1. Alternatively activated macrophages and epithelial progenitor cells also play important roles in directing successful repair after damage, but it is not known whether ILC2s work in concert with either of these cells to mediate effective repair. Using a model of sterile epithelial damage, we investigated the role of ILC2s during the course of epithelial repair and regeneration. Lung ILC2s were significantly activated 4–6 days after epithelial damage and produced increased levels of GM-CSF, IL-5 and IL-13. Interestingly, mice lacking ILCs (Rag2−/−Il2rg−/−) had reduced numbers of macrophages in the lung upon injury and subsequently decreased levels of macrophage-derived growth factors, such as IGF-1 and BRP-39. Reconstitution of ILC2s into Rag2−/−Il2rg−/− mice restored macrophage numbers and growth factor expression within the lung. These data suggest that ILC2s are part of a larger, multicellular circuit required for both recruitment and stimulation of alternatively activated macrophages which initiate the epithelial repair process. Thus, ILC2s can mediate both inflammation and repair at barrier sites by coordinating the activation state of specific effector cells.
IL-33/ST2 macrophage crosstalk promotes lung epithelial repair
Authors: Dagher Rania , Copenhaver Alan M , Besnard Valerie , Maret Marielle , Hamidi Fatima , Berlin Aaron A , Aubier Michel , Kolbeck Roland , Humbles Alison A , Pretolani Marina
Published: January 2023

Increasing evidence points to a central role for macrophages in tissue regeneration, however, the molecular mechanisms underlying this function remain unknown. Here, using depletion and adoptive transfer approaches, we demonstrate a major role of alveolar macrophages in facilitating bronchial re-epithelialization. In addition, we identify a novel, and paradoxical, protective role for the IL-33/ST2 axis in epithelial repair following naphthalene (NA)-induced lung injury. We defined two subsets of ST2 expressing myeloid cells, namely recruited monocyte-derived cells and resident alternatively activated macrophages (AAMs), which infiltrate the airways during the regeneration of the bronchial epithelium. Further, ST2-deficient mice exhibited an incomplete epithelial repair which was associated with a dysfunctional AAM phenotype and a reduction in the proliferation of a self-renewing subset of Clara progenitor stem cells. Notably, reconstitution of ST2+ AAMs, post NA, completely restored the epithelium in ST2-deficient animals. In contrast, the monocyte/macrophage-dependent CCL2/CCR2 axis was redundant for repair, however, anti ST2-antibody treatment to CCR2−/− mice resulted in ineffective epithelial regeneration and confirmed the observations seen with ST2-deficiency after NA-induced injury. Thus, the IL-33/ST2 pathway regulates effective Clara cell regeneration and bronchial re-epithelialization via AAM polarization and may lead to new therapeutic insights in acute respiratory diseases.
Novel inhibitory receptor signatures revealed by 18 parameter flow cytometry and mapped via a combination of automated analysis algorithms
Authors: Belkina Anna C , Fleury Michelle , Olson Alex , Dooms Hans , Kukuruzinska Maria , Lin Nina , Snyder-Cappione Jennifer
Published: January 2023

Inhibitory Receptors (IRs) such as PD-1, TIM-3, CD160, and TIGIT are upregulated during activated, exhausted, and pathological immune cell states. How the combinational expression of multiple IRs on individual cells tracks with functional status and/or fate is largely unknown. Due to paucity of human samples, large panels for immune cell characterization are of a high value, and combining them with automated platforms of multi-dimensional single cell analysis would allow objective and comprehensive population characterization. We report an 18-parameter (16-color) fluorescent panel that allows IR signature analysis as well as cell sorting for functional studies, and adopt algorithmic methods previously used for mass cytometry into a novel platform to analyze fluorescent datasets in an unbiased analytical workflow. PBMC from diseased and healthy control subjects as well as tumor specimens were stained and analyzed on a FACSARIA cell sorter. For the automated analysis, the datasets were processed with t-SNE algorithm into a 2D map of phenotypic space that was subsequently clustered using SPADE. From the samples, a 32-plex combinational IR signature was determined for the CD4+ T, CD8+ T, NK, iNKT, and γδ T cell lineages. The results indicate novel combinational IR ‘hits’ for particular immune subsets and some immune subset IR signatures correlated with disease status. In conclusion, 18-parameter flow cytometry with automated analysis provides robust results from human blood and solid tissue samples. In combination with functional and other readouts, measurement of single immune cell IR signatures could provide new insight into activation, exhaustion, and pathogenic processes and potentially reveal novel therapeutic targets.
Recombination signal sequences shape antigen receptor repertoires and enforce allelic exclusion
Authors: Wu Glendon S , Yang-Iott Katherine S , Bassing Craig H
Published: January 2023

Antigen receptor loci are arrays of variable (V), diversity (D), and joining (J) gene segments that the RAG endonuclease assembles into complete AgR genes. RAG binds to and cleaves at recombination signal sequences (RSSs) that flank gene segments. V(D)J recombination of Tcrb, Igh, and Igk loci is regulated such that functional AgR genes are assembled on and expressed from one allele (allelic exclusion) in most cells. It is assumed that allelic exclusion is achieved by mono-allelic initiation and feedback inhibition of the V-to-(D)J recombination step, and intrinsic features of RSSs have been proposed to mediate mono-allelic V-to-DJ recombination of Tcrb and Igh loci. We established and analyzed mice with the Trbv2 or Trbv31 RSS replaced by an RSS that possesses greater intrinsic recombination activity. Each of these RSS substitutions causes a profound increase in the development of T cells expressing either Trbv2+ or Trbv31+ TCRβ chains, reflecting that the RSSs flanking Trbv segments are major determinants of Trbv recombination frequency. Each RSS replacement also promotes a modest increase in the fraction of cells expressing Trbv2+ or Trbv31+ TCRβ chains along with another type of TCRβ chain (allelic inclusion). Strikingly, mice with Trbv2 and Trbv31 RSS replacements on opposite alleles have a 30-fold higher than normal level of allelic inclusion for Trbv2+ and Trbv31+ TCRβ chains, demonstrating that RSSs flanking Trbv segments enforce TCRβ allelic exclusion. We conclude that the infrequency of Trbv recombination as determined by inherent inefficiencies of Trbv RSSs limits bi-allelic assembly of Tcrb genes within the time window before feedback inhibition permanently cements allelic exclusion.
Rhbdf1 is a novel target of TRAF3 inactivation in B lymphocytes
Authors: Zhu Sining , Luo Chang , Lalani Almin , Hart Ronald , Xie Ping
Published: January 2023

TRAF3 is a new tumor suppressor gene in B lymphocytes. Deletions and mutations of TRAF3 were detected in human non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). We previously reported that specific deletion of TRAF3 in B lymphocytes causes prolonged survival of mature B cells, which eventually leads to B lymphoma development in mice. Here we identified Rhbdf1, a 7-transmembrane domain protein implicated in the trafficking of ER proteins, as a novel target of TRAF3 inactivation in B cells. Rhbdf1 expression was strikingly up-regulated in TRAF3-deficient premalignant B cells, B lymphomas and human MM at both the mRNA and protein levels. Normal physiological stimuli such as BAFF, CD40 or BCR could not induce the expression of Rhbdf1 in B cells regardless of the presence of TRAF3, suggesting that Rhbdf1 is specifically and aberrantly induced by TRAF3 inactivation in B cells. Biochemical fractionation studies revealed that Rhbdf1 was mainly localized at the ER in mouse splenic B cells and human MM. Interestingly, lentiviral shRNA vector-mediated knockdown of Rhbdf1 induced apoptosis and inhibited proliferation in human MM cells with TRAF3 deletions or mutations. Furthermore, bortezomib, an effective clinical drug for MM, drastically decreased Rhbdf1 protein levels in both TRAF3-deficient mouse B lymphoma and human MM. Taken together, our findings suggest that Rhbdf1 is a novel target of TRAF3 inactivation in B cells and may contribute to oncogenic B cell survival.
IL-7-dependent STAT1 activation limits homeostatic CD4 T cell expansion
Authors: Catalfamo Marta , Le Saout Cecile , Luckey Megan A. , Villarino Alejandro , Smith Mindy , Hasley Rebecca B. , Myers Timothy G. , Imamichi Hiromi , Park Jung-Hyun , O’Shea John J , Lane H. Clifford
Published: January 2023

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that under steady state conditions, IL-7 signaling is principally mediated by activation of Signal Transducers and Activators of Transcription 5 (STAT5). In contrast, under lymphopenic conditions there is a modulation of STAT1 expression resulting in an IL-7-dependent STAT1 and STAT5 activation. Consequently, the IL-7-induced transcriptome is altered with enrichment of interferon (IFN) stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4 T cells undergoing Lymphopenia-Induced Proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, “switching on” an alternate IL-7-dependent program. This mechanism could be a physiological process to “sense space” regulating the size of the CD4 T cell pool. During HIV infection the virus could exploit this pathway leading to the homeostatic dysregulation of the T cell pools observed in these patients.
Knockout validation of antibodies to Ki67: a marker for cellular proliferation
Authors: Dreja Hanna S , Ayton Julie , Bruce David , Lochead Julia , Renshaw Simon , Parton Laura , Hamilton Bruce J , Beer Samantha , Munro Melanie , Solache Alejandra
Published: January 2023

At Abcam, providing a high level of validation has always been a priority and our strategies are constantly evolving as new technologies are developed. The recent knockout (KO) validation initiative, made possible through a partnership with Horizon Discovery and the use of their CRISPR/Cas9 KO cell lines, addresses both antibody specificity and reproducibility on a large scale.

Ki67 is a proliferation marker and determining its expression levels has prognostic and predictive values in cancer (1–5). Hence, it is critical that antibodies used to target Ki67 are highly specific. This study demonstrates the evolving strategies used in our laboratory to validate antibodies to Ki67. The use of a human haploid cell lines where Ki67 expression has been knocked out has unequivocally identified antibodies that bind specifically to Ki67.

Utilising this technology has enabled KO validation of antibodies and, at the time of abstract submission, there are over 650 KO validated antibodies on our catalogue. It is our hope that by using this technique we can help to raise industry standards and contribute to the generation of reproducible research.
Immunology Antibody Validation: PTEN a target case in the use of human KO cell lines for antibody validation
Authors: Ayton Julie S , Dreja Hanna S , Bruce David , Lochead Julia , Koch Sofia , Parton Laura , Hamilton Bruce J , Munro Melanie , Solache Alejandra
Published: January 2023

At Abcam we are constantly improving our manufacturing processes and adopting more robust antibody validation technologies as they are developed. Recently we have implemented the use of human knockout (KO) cell lines to interrogate the specificity of our antibodies on a large scale. This has been possible through a partnership with Horizon Discovery and the use of their KO cell lines generated using CRISPR/Cas9 technology.

PTEN, is a well-known tumour suppressor that plays a critical role in the host immune response. Recent research has highlighted the importance of this protein with regards to a) lineage stability and homeostasis in Treg cells (1) and b) negative regulation of the NK cell immune response (2, 3). In both cases PTEN acts by regulation of the PI3-kinase signalling pathway.

We have identified several monoclonal antibodies to both PTEN and PI3K and confirmed their specificity by KO validation. We performed this analysis in either denatured (western blot) or native (immunocytochemistry) conditions. Our KO models represent true negative controls and provide confidence that these antibodies are binding to their intended target. This technology has allowed us to develop a growing range of over 650 KO-validated antibodies, including a number that are applicable to immunology/oncology research.
Role of innate immunity in neonates during a viral CNS infection
Authors: Ganesan Priya , Kuhn Alison M , Dunphy Abigail A , O’Donnell Lauren A
Published: January 2023

Viral infections in the central nervous system (CNS) are associated with devastating neurological consequences in newborns. This may be due, in part, to deficiencies in the immature innate and adaptive immune response. To study viral CNS infections in neonates, we use a transgenic mouse model of neuronally-restricted measles virus (MV) infection (NSE-CD46 mice), where the human isoform of CD46, a MV receptor, is expressed under the control of the neuron-specific enolase promoter. Adult CD46+ mice survive infection and control MV in an interferon gamma (IFNg)- and T cell-dependent manner. In contrast, neonatal CD46+ mice succumb to the infection despite T cell infiltration into the brain parenchyma. CD46+/RAG2-KO neonates, which lack T- and B-cells, experience less severe disease, prolonged survival, and lower viral load compared to CD46+ neonates. CD46+/RAG2-KO neonates also show greater infiltration of neutrophils and natural killer cells and higher expression of the pathogen recognition receptors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIGI) during infection. Both CD46+ and CD46+/RAG2-KO neonates upregulate IFNg but CD46+/RAG2-KO neonates show higher expression of IFN-beta. CXCL10, a IFNg inducible chemokine, is also upregulated in CD46+ neonates (84-fold) and CD46+/RAG2-KO neonates (771-fold). Current studies aim to explore the role of IFN-beta in early viral control in the neonates and explore the contribution of CXCL10 in the infiltration of innate immune cells in the CNS.

Supported by 1R15NS087606-01A1.
Mechanisms of T follicular helper (Tfh) impairment in chronic HIV infection
Authors: Chakhtoura Marita , Cubas Rafael , Talla Aarthi , TARDIF Virginie , Haddad Elias
Published: January 2023

Human immunodeficiency virus (HIV) infects millions of people worldwide. In secondary lymphoid organs, site where the anti-HIV immune response is mounted, the virus has been shown to induce a predominant dysregulation and disturbance of the microvenvironment. Previous work in our laboratory has identified the lack of protective anti-HIV antibodies in patients to be due, at least in part, to an inadequate T follicular helper (Tfh) cell help to germinal center (GC) B cells in draining lymph nodes through impaired PD-1/PD-L1 interactions. Since the mechanism of this impairment is not yet fully elucidated, we have used our co-culture assay followed by a unique gene array analysis to screen for key molecular pathways that could be accountable for the difference between helper programs in Tfh cells of HIV positive and negative individuals. The major driver of gene profile clustering was observed within Tfh proliferation programs as well as infection status. Gene set enrichment analysis included repair mechanisms, cell cycle regulation, metabolism, signal transduction and co-stimulation in the top 10 enriched pathways. Careful examination of gene fold changes in proliferating vs non-proliferating Tfh cells between healthy and HIV individuals showed a decrease with HIV, in immune regulatory gene expression such as the transcription factor c-maf and its mediators, responsible for Tfh differentiation, development, survival and performance. Investigating this pathway would contribute to a better understanding of the HIV-mediated dysregulation.
AXL Promotes Zika Virus Infection in Astrocytes by Antagonizing Type I Interferon Signaling Pathway
Authors: Xu Jianqing , Zhang Xiaoyan , Liu Jia
Published: January 2023

Zika virus (ZIKV) is known to be associated with neonate microcephaly and Guillain-Barré syndrome in human adults. Despite the progress on understanding the causal link between ZIKV infection and microcephaly, the life cycle and pathogenesis of ZIKV remains elusive. In particular, recent studies have revealed a controversial role of AXL, a TAM family kinase receptor previously deemed as the entry receptor of ZIKV. In this study, we show that though genetic ablation of AXL protects primary human astrocytes and U-251MG astrocytoma cells from ZIKV infection, AXL knockout (KO) does not block the entry of ZIKV, instead it facilitates the up-regulation of type I interferon (IFN) signaling genes, including interferon regulatory factor 7 (IRF7), a key interferon transcriptional factor, and a number of type I interferon and interferon stimulating genes (ISGs). Knockout of type I IFN receptor a chain (IFNAR1) rescues the vulnerability of AXL KO astrocytes to ZIKV infection. In addition, AXL KO in astrocytes results in the down-regulation of SOCS1 in primary astrocytes and the failure to retain the homeostasis of SOCS1 in astrocyte cell lines, a known inhibitor of type I IFN signaling pathway, thus revealing important roles of AXL in maintaining the homeostasis of SOCS1 and thereby in containing type I interferon signaling pathway. Taken together, our study demonstrates that AXL does not function as the sole, if at all, entry receptor of ZIKV, but instead promotes ZIKV infection in astrocytes by SOCS1-mediated antagonism of type I interferon (IFN) signaling.
Dissemination of HIV-1 from latently infected cells to resting CD4+ T cells requires CD4 and actin remodeling.
Authors: Lan Jie , Li Wei , Shepherd Nicole , Xing Yanyan , Yu Andy
Published: January 2023

The major barrier to HIV-1 eradication is the replication-competent proviruses in long-lived latently infected CD4+ T cells. Current efforts to purge latently HIV-1-infected cells have focused on developing “shock and kill” approaches to force reactivation of the proviruses in these cells to be killed after reactivation of virus gene expression by viral cytopathic effects, host immune responses, or both. Cell-free virus infection and cell-to-cell transmission via the virological synapses (VS) are the two modes of HIV-1 dissemination between CD4+ T cells. Several studies have demonstrated that cell-to-cell spread, in which HIV-1 disseminates through direct contact of infected cells with surrounding uninfected cells, is a far more efficient process than that of cell-free virus infection. In this work, we studied the mechanism of cell-to-cell transmission between primary CD4+ T cells and the HIV-1 latently infected cell line ACH-2. When primary resting CD4+ T cells were co-cultured with ACH-2 cells under latency reversing agent Romidepsin stimulation, about 20.7% cells had been infected without becoming activated. Additionally, blocking with anti-CD4 or anti-gp120 neutralizing antibodies (VRC01) decreased de novo infection, and neither anti-CXCR4 antibodies nor chemokine inhibitors demonstrated the ability to inhibit cell-to-cell transmission. We also found that co-cultured resting CD4+ T cells carried higher levels of active cofilin, the actin-depolymerizing factor, suggesting actin plays a role in VS formation and viral pathogenesis. These results suggest HIV-1 Env gp120 binds with the CD4 receptor, not chemokine coreceptor to form VS during cell-to-cell transmission.
Zika and Dengue Virus Specific and Cross-reactive Memory B Cell Responses
Authors: Andrade Paulina E , Coloma Josefina , Michlmayr Daniela , Balmaseda Angel Lazaro , Harris Eva
Published: January 2023

Zika virus (ZIKV) belongs to the Flaviviridae family and is transmitted to humans by Aedes mosquitoes. Since 2015, ZIKV has spread rapidly to 70 countries, most notably in the Americas, where it co-circulates with other flaviviruses that also cause human disease, such as dengue virus (DENV). DENV is comprised of four serotypes that are conserved 65–70% in amino acid sequence of the envelope protein among the DENV serotypes and conserved 54–59% with ZIKV. The antigenic similarity of ZIKV and DENV has raised important biological concerns regarding the effect of immunological cross-reactivity on protection and potential enhancement of ZIKV and DENV infections and vaccines. To date, the cross-reactivity of ZIKV and DENV B cell responses is not fully understood in the context of natural human infections. Here, we use a novel ELISPOT-based assay designated Quad-Color Fluorospot that allows investigation of the DENV serotype specificity vs. cross-reactivity of the memory B cell (MBC) population at a single-cell level, adding a fifth color to include ZIKV. We analyzed a unique set of peripheral blood mononuclear cells from the Nicaraguan Pediatric Dengue Cohort Study, a prospective study of dengue, chikungunya, and Zika now in its 13th year. Samples were collected ~2 weeks and several months after RT-PCR-confirmed ZIKV infection from children who were previously DENV-immune or DENV-naïve. We also included a set of dengue patients who were ZIKV-naïve. Preliminary results show that despite the antigenic similarity between DENV and ZIKV, MBCs from ZIKV-infected subjects were highly specific to ZIKV, with lesser cross-reactivity to DENV. These studies provide much-needed information on how the human immune response reacts to ZIKV and DENV over time
Global Phosphoproteomic and Structural Analyses of Ebola Virions
Authors: Parry Christian , Ivanov Andrey , Ramanathan Palaniappan , Ilinykh Philipp A. , Lin Xionghao , Ammosova Tatiana , Bukreyev Alexander , Nekhai Sergei
Published: January 2023

Ebola virus causes severe hemorrhagic fevers with high mortality characterized by unchecked viral growth and inflammation with marked suppression of host innate and adaptive immune responses. Ebola virus infects macrophages and dendritic cells preferentially, repressing the development of MHC and antigen presentation. Patients who succumb to Ebola virus disease show little evidence of a humoral immune response. VP35 inhibits critical immune signaling early in infection. We have carried out global phosphoproteomic analysis of Ebola virions produced in monkey and bat cells using high resolution MS/MS. We detected all viral proteins with high coverage and identified novel phosphorylation sites in all seven proteins of each virion type. We compared phosphorylation patterns and validated the results by testing the role of the phosphorylation sites of interferon-modulatory VP35 using mutagenesis, in vivo phosphorylation and minigenome assay. We carried out biophysical analyses, structural modeling and simulations of wildtype versus phosphorylated proteins. The phosphorylation pattern differs between virions grown in monkey cells and those grown in bat cells. Nearly all phosphorylation sites were found to be solvent accessible with the majority in flexible regions. VP30, VP35 and NP were characterized by inherent disorder. Mutagenesis, phosphorylation analysis and minigenome experiments showed that VP35 Ser-210 undergoes phosphorylation and is important for transcription. Ser-210 lies in a linker region connecting the coiled coil oligomerization domain and the interferon inhibitory domain. Our findings point to an important role of VP35 phosphorylation and a means of developing inhibitors as drugs against Ebola virus.
Glucocorticoids Drive Circadian Changes in T-Cell Homing and Response via IL-7 Receptor
Authors: Shimba Akihiro , Ikuta Koichi
Published: January 2023

The immune system is deeply affected by steroid hormones. Glucocorticoids are a group of steroid hormones with strong immunosuppressive effects and widely used in clinical practice. However, whether glucocorticoids physiologically control homeostasis and response of the immune system remains unclear. We found that glucocorticoid receptor up-regulates IL-7 receptor (IL-7R) of T cells by binding to an enhancer of the IL-7R locus, and the induction followed circadian rhythm consistent with circadian change of glucocorticoid production. The circadian regulation of IL-7R supported T-cell survival and redistribution between spleen, peyer’s patch and blood by controlling the expression of a chemokine receptor, CXCR4. CXCR4 upregulation induced T cell migration to CXCL12-positive area in spleen and peyer’s patch. Consequently, T cell accumulation in spleen promoted immune responses against systemic bacterial infection. Furthermore, GR enhanced IL-4 production in Th2 condition and helped B cell activation and class-switch to IgG1. Our results reveal the beneficial role of glucocorticoids in enhancing adaptive immunity, and provide insights how the immune function is influenced by circadian rhythm.
T cell up-regulation of CD127 is associated with reductions in the homeostatic set point of the peripheral T cell pool during malnourishment
Authors: Bupp Melanie R Gubbels , Murphy Sarah , Patrick Kristin , Thoner Timothy , Edwards R. Whitney , Litvin Seth
Published: January 2023

The following study was undertaken to better understand the impact of short-term malnutrition on T cell homeostasis, with particular attention focused on the CD127/IL-7 signaling dynamic of peripheral T cells in mice. We report that the total number of peripheral naïve and memory CD4+ and CD8+ T cells notably declined after one week of mild malnourishment--a time period too short to be entirely due to malnutrition-induced thymic involution. Peripheral malnourished T cells expressed higher levels of the IL-7 receptor component, CD127, and were less sensitive to death-by-neglect as compared to control T cells. Adoptive transfer studies revealed that CD127 expression did not correlate with increased survival in vivo and that all naïve CD8+ T cells upregulated CD127, regardless of initial expression levels. Corticosterone levels were elevated in malnourished mice and this correlated in time with peripheral T cell up-regulation of CD127 and the diminishment of the peripheral T cell pool. Overall, these data suggest a model in which CD127 levels are up-regulated quickly during malnourishment, thereby increasing the scavenge rate of IL-7, and providing a mechanism to rapidly adjust the total number of T cells during malnutrition.
Regulatory T cells exhibit distinct lymph node homing and trafficking kinetics as compared to conventional CD4+ T cells
Authors: Tong Alexander , Forestell Benjamin , Murphy Daniel V. , Nair Aditya , Allen Frederick , Mandl Judith N. , Huang Alex Yee-Chen
Published: January 2023

Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. We have previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal significant differences in Treg versus Tconv homing and transit through peripheral and mesenteric LNs. We find that Tregs with an effector phenotype are preferentially retained in LNs, and complementary transwell assays demonstrate that Tregs have blunted response to CCL21 compared to Tconvs. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs, taking advantage of a novel gut SLO stabilization device allowing up to 10 hours of continuous imaging. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in LNs.
Development and function of skin-homing invariant natural killer T cells
Authors: Wang WeiBei , Xiong Na
Published: January 2023

Invariant natural killer T (iNKT) cells are innate-like T cells that express restricted T cell receptors (TCR) to recognize self and foreign lipid antigens. Distinct iNKT subsets can quickly produce numerous cytokines such as interferon (IFN)-g (iNKT1), interleukin (IL)-4 (iNKT2), and IL-17 (iNKT17) to regulate immune responses in microbial infection, allergic disease, autoimmune disease and cancer. Different iNKT cell subsets have unique transcription factor profiles such as T-bet, PLZF and RORgt that determine their cytokine-producing abilities. However, mechanisms directing the tissue localization preference of different iNKT cell subsets are not well understood. Using a novel strain of CCR10-knockout (KO)/EGFP-knockin (KI) reporter mice, we found that the skin-homing chemokine receptor CCR10 is highly upregulated in iNKT cells during their thymic development stages. Analysis of cytokine production in stimulated thymic iNKT cells demonstrated that CCR10+ iNKT cells are predominantly iNKT2 cells producing IL-4, implying that iNKT2 cells are preferentially programmed in the thymus to migrate into the skin. Confirming this, CCR10-expressing iNKT2 cells are enriched in the skin at the steady state. Taken together, these findings suggest that intrathymic programming orchestrate generation of skin-homing innate-like iNKT cells with specific functional potentials to provide immediate protection of the border tissue.
Supplementing dams with both arachidonic acid and docosahexaenoic acid has beneficial effects on growth and immune development beyond single fatty acid supplementation
Authors: Hadley Kevin , Guimont-Desrochers Fanny , Bailey-Hall Eileen , Field Catherine J , Yurko-Mauro Karin , Salem Norman
Published: January 2023

Omega-3 long-chain polyunsaturated fatty acids (LCPUFAS) modulate immune cells in vitro and in vivo. This study investigated the effects of enriching the maternal diet with the n-6 and -3 LCPUFAs, arachidonic (20:4n-6, 0.6%wt ARA) and docosahexaenoic acid (22:6n-3, 0.32%wt DHA), or a 2:1 ratio (2ARA:1DHA) on total lipids in milk, total lipids, and immunophenotypes in plasma, lymph nodes, and spleen from isolated immune cells from 28d old pups. From day 15 of gestation to post-partum day (ppd)3, Sprague-Dawley dams were fed a commercial chow. On ppd3 litters were culled and pups (4 males and 2 females) randomly cross-fostered to dams who were randomized to one of the 3 experimental diets resulting in 20 male and 10 female pups/diet group. Dams fed ARA or ARA:DHA had 28–36% more 20:4n-6 in milk and feeding DHA or 2:1 ARA:DHA doubled 22:6n-3 in milk lipids (P<0.05). Feeding 2ARA:1DHA or ARA alone resulted in greater pup weight at weaning (P<0.05). Feeding ARA (-DHA) resulted in lower DHA in plasma and spleen while feeding DHA (-ARA) resulted in higher DHA in plasma (P<0.05). Compared to the 2ARA:1DHA pups, there was general trend for ARA fed pups to have higher and DHA fed pups to have lower total, T helper, T cytotoxic, and B lymphocytes in blood, and spleen. In summary, perinatal supplementation with 2ARA:1DHA, compared to ARA or DHA alone, resulted in a higher content of ARA and DHA in breast milk and tissues and had positive effects on growth. DHA without ARA increased 22:6n-3 in milk, plasma, spleen, and reduced 20:4n-6 in milk. Manipulation of dietary LCPUFAs was reflected by lipid content in tissues, and accompanied by evidence of effects on immunophenotype. These changes suggest functional effects but this remains to be confirmed by future investigations.
Embryonic fate mapping uncovers the critic role of miRNAs in skin-resident γδ T cell ontogeny
Authors: Yao Yi , Liu Queping , Martin Carly Elizabeth , Zhou Li , Mi Qing-Sheng
Published: January 2023

Epidermis-resident γδ T cells (termed dendritic epidermal T cells, DETCs) are the first T cells developing in embryonic thymus. Given lack of a fate mapping tool, DETC ontogeny remains poorly understood. Recently, colony stimulating factor 1 receptor (CSF1R) Cre reporter was used to fate map tissue resident macrophages. To study the role of miR-155 in embryonic resident macrophages, we generated Csf1rCremiR-155GFP mice in which both miR-155 and GFP expressions are induced by Csf1rCre. As expected, the majority of skin macrophages highly expressed GFP. Surprisingly, the epidermal Vγ3+DETCs that emerged at E16.5 were all GFP+ cells, and nearly all fetal thymic γδ T cells were also GFP+, although overexpression of miR-155 did not disturb embryonic macrophages and γδ T cells. This observation was confirmed in Csf1rCreRosa26EGFP mice, suggesting CSF1R promoter driving both embryonic myeloid and lymphoid cell lineages. To further study the role of miRNAs in γδ T cell ontogeny, we next generated Csf1rCreDicerKO mice in which miRNAs are deficient. DETCs were almost completely eliminated in Dicer. KO epidermis at E18.5. The number of thymic Vγ3+ T cells were severely reduced (P < 0.0001) in KO embryos, and the frequency of annexin V+ apoptotic Vγ3+ T cells was raised (P < 0.001). Moreover, CD122+ or CD45RB+ mature Vγ3+ T cells were decreased (P < 0.01) in KO embryos, while immature CD24+Vγ3+ T cells were increased (P < 0.01). Thus, loss of miRNAs contributes to defects on survival and maturation of fetal thymic Vγ3+T cells, leading to DETC deficiency. Overall, this is the first study to demonstrate that Csf1rCre serves as a new tool for studying resident γδ T cell embryonic development and miRNAs regulate DETC ontogeny.
Kruppel-like factor 10 regulates innate-like IL-17+CD27−Vγ4+ γδ T cell development
Authors: Kim Girak , Gu MinJeong , Kim Soo Ji , Han Seung Hyun , Cho Jae-Ho , Yun Cheol-Heui
Published: January 2023

γδ T cells, an important source of innate IL-17 in mouse and human, are known to act critical contributions to host immune responses. However, certain factors that control their development and homeostasis are poorly elucidated. In the present study, we examined the role of zinc finger transcriptional factor, Kruppel-like factor 10 (KLF10) in the development and homeostasis of IL-17-committed CD27− γδ T (γδ27−-17) cells. We found a selective augmentation of Vγ4+ γδ27− cells, but not of other IL-17-producing αβ T cells, with higher IL-17 production in KLF10-deficient mice. Although either cytokine (IL-7 or IL-1β plus IL-23) or T cell receptor (TCR) stimuli could induce Klf10 on γδ T cells, KLF10 inhibited γδ27− cell responsiveness to IL-7 or IL-1β plus IL-23 but not TCR stimuli. Surprisingly, KLF10-deficient CD127hiVγ4+ γδ27−-17 cells expressed CD5, a stable indicator of TCR strength, higher than their wild-type counterparts. This was also observed in mature (CD24loCD44hi) but not in immature (CD24hiCD44lo) Vγ4+ γδ27− cells from the neonatal thymus of KLF10-deficient mice, with enhanced maturation of these subsets, indicating that KLF10 deficiency promoted high-affinity TCR bearing Vγ4+ γδ27− cell maturation. Finally, mixed bone marrow chimera study indicated that intrinsic KLF10 signaling is mandatory to limit Vγ4+ γδ27−-17 cells. Collectively, these results suggest that the development and homeostasis of innate-like Vγ4+ γδ27−-17 cell are dependent on a transcriptional control by KLF10 which is differently involved in cytokine or TCR signaling.