bluuebunny/crossref_metadata_2025 huggingface dataset:
STAT1 -/- mice are protected against exacerbation of MRSA pneumonia during influenza co-infection (VIR2P.1021)
Authors: Lee Benjamin , Robinson Keven , McHugh Kevin , Mandalapu Sivanarayana , Manni Michelle , Alcorn John
Published: January 2023

In mice, bacterial lung defense requires Th17 activation, which is attenuated by preceding influenza, resulting in exacerbation of pneumonia. Influenza induced-type I IFN requires STAT1 signaling and inhibits dendritic cell production of Th17-activating IL-23. We explored the need for STAT1 signaling in influenza-associated Th17 suppression in a mouse model of influenza and MRSA co-infection using STAT1 -/- mice. WT and STAT1 -/- mice were infected with influenza A PR/8/34/H1N1 or PBS. Mice were challenged with PBS or MRSA on day 6 and sacrificed on day 7; BAL and lungs were harvested for cell count, flow cytometry, bacterial burden, cytokine levels, and gene expression. Compared to WT, influenza-infected STAT1 -/- mice had increased day 6 weight loss (82.3% vs 88.5% of baseline) and higher BAL neutrophil count (2.85 x104 vs 4.15 x105). Co-infected STAT1 -/- mice had higher total cell count (1.04 x106 vs 1.61 x106 cells), but did not show increased bacterial burden compared to WT mice. IL-17 and IL-22 producing CD4+ and γδ T cells were reduced in co-infected WT mice versus MRSA alone but were increased overall in STAT1 -/- mice. No differences in Th17 cytokines (IL-17, IL-22, IL-23) or in IFN-β gene expression were detected in whole lung. These data suggest that STAT1 -/- mice are protected from exacerbation of MRSA pneumonia during influenza co-infection despite increased influenza severity and lung inflammation, but the mechanism of these findings remains unclear.
Viral neuraminidase and the biphasic immune regulation by host cytokine interleukin-10 in influenza (VIR2P.1030)
Authors: Dutta Avijit , Chen Tse-Ching , Lin Chun-Yen , Chiu Cheng-Hsun , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Ching-Tai
Published: January 2023

Influenza virus invades the host while the host immune response defends to clear the virus. These responses may result in excessive inflammation with tissue injury, which is as detrimental as the invasiveness of the virus. Here we demonstrate a biphasic immune regulation by the host T cell cytokine interleukin-10. During the early phase of infection, viral neuraminidase increases with multiplication of the virus. The abundance of neuraminidase mediates a robust release of the suppressive cytokine TGF-β. Host interleukin-10 interacts with neuraminidase, counteracts the release of TGF-β, and supports the antiviral immunity. Neuraminidase activity wanes with eradication of the virus. In the late phase of the infection, interleukin-10 preferentially binds to up-regulated interleukin-10 receptors and attenuates the immune responses to avoid immune-mediated damage to the host.
A role for MHC-mediated signalling in the functional and phenotypic heterogeneity of naive T cells (IRC8P.454)
Authors: Rath Satyajit , Rane Sanket , Das Rituparna , Gund Rupali , Balyan Renu , Das Arundhoti , Khalsa Jasneet , Ranganathan Vidya , Prabhu Savit , Mattoo Hamid , Durdik Jeannine , George Anna , Bal Vineeta
Published: January 2023

Variability in cellular activation in the immune system can have diverse consequences, resulting in heterogeneity in effector programming and function as well as in cell survival. While examining relationships between lymphocyte heterogeneity and functionality, we find that apparently unimodally distributed coreceptor heterogeneity among naive T cells, simplistically expected to be due to intrinsic noise, is not only correlated with major functional variation but is modulated by extrinsic microenvironmental cues in vivo. Thus, when naive T cells from young mice are separated into coreceptor-hi and coreceptor-lo subsets, we find that coreceptor-lo cells are smaller, have higher CD5 levels, respond poorly, are more susceptible to death, and show altered effector differentiation. Human coreceptor-lo and coreceptor-hi naive T cells show similar differences. Coreceptor-lo and coreceptor-hi subsets of 'single-positive' (SP) thymocytes do not show these differences, whereas peripheral coreceptor-lo and coreceptor-hi subsets of naive T cells from TCR-transgenic mice do. Adoptive transfer-mediated parking in vivo in MHC-sufficient but not in MHC-deficient mice causes reduction in coreceptor levels, increase in CD5 levels and hyporesponsiveness in naive T cells. Naive T cells from aged mice show similar phenotypic and functional differences. Thus, our data show complex roles for tonic signaling in the functional heterogeneity of naive T cell populations.
LRBA deficiency in in Mexican patients with common variable immunodeficiency (IRM10P.616)
Authors: Moreno Corona Nidia , Berron Ruiz Laura , Mogica Martinez Dolores , Nakashimada Yamazaki , Segura Mendez Nora , Santos-Argumedo Leopoldo , López-Herrera Gabriela
Published: January 2023

CVID is one of the most common primary immunodeficiency diseases and is a diagnosis of exclusion. CVID is characterized by hypogammaglobulinemia and recurrent bacterial infections. About two third of CVID subjects have an autoimmune condition. The etiology of about 80% of CVID remains unknown. Genetic linkage studies in such families have found distinct homozygous mutations in the gene encoding LRBA. Since then, all 11 LRBA deficient CVID subjects identified to date have autoimmune diseases, all patients, except one presented hypogammaglobulinemia. All mutations reported caused the loss or LRBA expression in PBMCs. In this work, we explore the frequency of LRBA deficiency in a cohort of 40 patients with clinical diagnosis of CVID diagnosis, most of which, consanguinity is unknown. Two patients with LRBA deficiency were identified in this cohort. All of the forty patients showed hypogammaglobulinemia. We also correlate the deficiency of LRBA with Freiburg classification from which patients with mutations in LRBA were from group Ia an Ib.
Ikaros tumor suppressor function in pre-B ALL: potential role of Ikaros target gene Ctnnd1 (IRM10P.621)
Authors: Schjerven Hilde , Eggesbo Linn , Lindeman Ida , Muschen Markus
Published: January 2023

Ikaros is a zinc finger transcription factor required for B-cell development and proper hematopoiesis, and an important tumor suppressor in developing lymphocytes. To understand the mechanism of Ikaros tumor suppressor function and potentially develop new and improved targeted therapies, it is important to elucidate the downstream target genes involved. With this aim, we combine mouse models of pre-B ALL and in vitro culture of human patient-derived pre-B ALL cells. A mouse model with targeted deletion of the fourth DNA-binding zinc finger of Ikaros resulted in loss of tumor suppressor function, with a limited set of deregulated genes useful to narrow down the list of putative relevant Ikaros target genes (Schjerven et al., 2013). To specifically address the role of Ikaros as a tumor suppressor in human pre-B ALL, we developed TET-regulated Ikaros expression in human pre-B ALL cells. This enables us to test specific Ikaros target genes from the mouse model, and allows for genome-wide expression analysis by RNA-seq to elucidate all genes downstream of Ikaros in human pre-B ALL cells. To help distinguish direct from indirect target genes, we have mapped the genome-wide binding sites of Ikaros in these human pre-B ALL cells by ChIP-Seq. This approach has identified the Ikaros target gene Ctnnd1. Ongoing experiments explore the potential role of Ctnnd1 and test the current hypothesis that Ikaros-mediated repression of Ctnnd1 limits CyclinD levels and leukemic growth in pre-B ALL.
Analysis of germinal centers in mice with Chagas’ disease treated with NIPOx-B
Authors: Mora Albany Reséndiz , Baeza Carlos Wong , Marin Daniela Cruz , Flores Elizabeth Celso , Ramírez Isabel Baeza , Ramírez Carlos Wong
Published: January 2023

Chagas’ disease is caused by the parasite Trypanosome cruzi; it affects 10 million people in Latin America and it remains incurable: the only drugs available for its treatment, Benznidazole (Bz) and Nifurtimox (Nx), are inefficient to cure patients. The chronic phase of the disease is characterized by megacolon and megaesophagus; this phase is established by parasite evasion of the host adaptive immunity, a process that is facilitated by the free radicals generated by Bz and Nx, which affect lymphocytes and other immune cells. We designed and synthesized the benzyl ester of N-isopropyl oxamic acid (NIPOx-B) and determined that it is an effective inhibitor of alpha-hydroxy acid dehydrogenase II enzyme, a key enzyme in the parasite metabolism. NIPOx-B is more efficient than Bz and Nx in decreasing mice parasitemia. In this work, we studied by flow cytometry the cell populations in germinal centers in mice with Chagas’ disease, untreated or treated with Bz or with NIPOx-B. The percentage and the activation of B cells, macrophages, CD4 lymphocytes, dendritic and follicular dendritic cells in germinal centers were higher in mice treated with NIPOx-B than in mice treated with Bz. The IgG and IgE antibodies titers were also higher in mice treated with NIPOx-B. Untreated mice had the lowest cell percentages and antibody titers. Mice treated with NIPOx-B did not develop megasyndromes, while untreated mice or mice treated with Bz developed them. Because NIPOx-B has trypanomicidal activity and did not affect the percentage of cells involved in the immune response it can be proposed as a drug for the treatment of Chagas’ disease.
Immune triage: prioritization of host immune responses.
Authors: Jamieson Amanda Mercedes , Crane Meredith , Xu Yun , Lee Kayla
Published: January 2023

The immune response is important in many functions, including host defense against pathogens, wound healing, development, response to cancer, and maintenance of homeostatic physiological responses. We are interested in the concept of immune triage, in that a given host must be able to deal effectively with multiple insults, and at times prioritize immune responses. It is important for the overall health status of the host that the immune system responds effectively to protect essential organs. We have developed several mouse models, focusing on the lung immune response, that allow us to examine different aspects of immune triage. The lung is an essential and delicate organ and thus pulmonary immune responses must be tightly regulated. We have determined that lung infection with influenza A virus (IAV) alters the response to bacterial lung infections. Depending on the bacterial infection, previous infection with IAV can suppress or augment the immune response to bacteria. We have also determined that pulmonary infection with IAV alters many aspects of the systemic immune response. There is a global suppression to systemic bacterial infection, and a decrease in the wound healing response. Our data indicate that the immune system prioritizes lung infections over many other responses. This is most likely due to the importance of the lung in host survival. We have established several regulatory mechanisms by which this immune triage occurs. By understanding how the immune system responds to multiple insults we can improve our understanding of the immune network on a global level.
Investigating mechanisms of sex bias in lung fibrosis using mice with sex chromosome complement
Authors: Valera Isela C , Kafaja Suzanne , Pham Brandon , Fishbein Michael , Singh Ram R
Published: January 2023

Men with systemic sclerosis (SSc) have more end organ damage, an accelerated course, and lower survival than women. Mechanism underlying such sexual dimorphism is unclear. Here, we investigated the role of sex chromosomes in the development of SSc using the four-core genotype (FCG) mouse model that was created to identify effects of the sex chromosome complement without the confounding effects of differences in sex hormones. In this model, findings in the XX females (XXF) can be compared with XY females [XYF = XY−(Sry)], and in XX males [XXM = XX.SryTg] with XY males [XYM = XY−Sry.SryTg]. The FCG C57Bl/6 mice were administered with bleomycin to induce SSc and euthanized 2 weeks later. We found that regardless of the gender, mice with XY sex chromosome complement had more severe disease than mice with XX complement (p = 0.04, n= 37 and 48, respectively). When we compared the groups based on gender, XYF mice had higher fibrosis scores than XXF (p = 0.01). The differences between XY and XX were even more significant in males: XYM animals had much more severe fibrosis than XXM (p < 0.0001). Furthermore, the levels of soluble collagen in bronchoalveolar lavage fluid and of Col1a1 mRNA in lung tissue correlated with the extent of lung fibrosis. Animals with more fibrosis (i.e., XYM) had higher proportions and numbers of myeloid and plasmacytoid dendritic cells in the lungs. XYM mice also had more natural killer T cells, B cells, and T cells in the lungs than XXM mice at 2 weeks post-instillation. Thus, the presence of a Y chromosome, and not the male sex itself, predisposes to lung fibrosis; androgen may play an additive role in causing increased lung fibrosis, whereas the presence of the second X chromosome and/or estrogen might be protective.
Air Pollution Particulate Matter Exposure Modulates Human Peripheral Blood Mononuclear Cell Host Immunity to Mycobacterium tuberculosis (M.tb)
Authors: Sarkar Srijata , Rivas-Santiago Cesar , Munoz Felipe , Ohman-Strickland Pamela , Torres Martha , Schwander Stephan
Published: January 2023

Epidemiological studies indicate that indoor air pollution exposure increases susceptibility to M.tb infection and Tuberculosis (TB) development. Recently, we demonstrated that diesel exhaust particles and urban air pollution particulate matter (PM) impair human peripheral blood mononuclear cell (PBMC) and respiratory epithelial cell (A549) innate immune responses to M.tb via downregulation of TLR-dependent cytokines (IFN-γ, IL-1β IL-6, TNF-α), and antimicrobial peptides, respectively. Here we examined effects of real-world-derived air pollution PM from Iztapalapa, a municipality with high prevalence of air pollution and TB in Mexico City on innate and adaptive human immune responses to M.tb. Cytotoxic and inflammatory PBMC responses were examined upon exposure to PM2.5 (<2.5μm diameter) and PM10 (<10μm) from rainy, cold-dry and warm-dry weather seasons. PBMC from IGRA+ (immunodiagnostic evidence of M.tb exposure) and IGRA- donors were exposed for 20 hours to PM at 0.1, 1 and 10 mg/mL followed by additional 20-hour M.tb exposure. A PM dose-dependent inhibition of IFN-γ production was observed in M.tb-infected PBMC from IGRA+ but not IGRA- donors. Furthermore, the size range of PM, but not their seasonal source, affected the M.tb-induced IFN-γ production. No PM dose-dependent inhibition of IL-1β production was observed in M.tb-infected PBMC from either IGRA+ or IGRA− donors. PM10 from cold-dry and warm-dry seasons at high concentration caused cytotoxicity. This data suggests that urban ambient air pollution PM exposure (1) modulates M.tb-induced adaptive immune responses (suppression of IFN-γ but not IL-1β production in IGRA+ donors) and (2) potentially alters M.tb infection outcomes.

Funding NIEHS 5R01ES020382-05
Extensive characterization of islet-reactive CD8 T cells in type 1 diabetes by mass cytometry (CyTOF)
Authors: Wiedeman Alice E , James Eddie A , Greenbaum Carla , Long Sarah Alice
Published: January 2023

In type 1 diabetes (T1D), insulin-secreting beta cells of the pancreatic islets are targets of autoimmune destruction by T lymphocytes, resulting in insulin deficiency and dysregulation of glucose metabolism. CD8 T cells reactive to islet antigens have been implicated in this process, and phenotypic changes in CD8 T cells have been observed in the transition to clinical diabetes. While efforts have led to identification of peptide antigens targeted by autoreactive CD8 T cells, less has been done to characterize the phenotype and function of this population. We developed a 25+ marker mass cytometry (CyTOF) panel to identify and extensively characterize CD8 T cells reactive to pooled class I (HLA-A2) tetramer loaded with peptides of viral or islet antigens in the same sample. Results were confirmed with several smaller flow panels. In PBMCs of three healthy controls and four T1D patients, we found virus-specific CD8 T cells had a more memory- and exhausted-like phenotype compared to total CD8 T cells, including increased T effector memory (CD45RO+CCR7−), KLRG1, CD57, TIGIT, and PD1, and decreased naïve T cells (CD45RO−CCR7+). In contrast, islet-specific CD8 T cells (detectable in three T1D patients) did not vary significantly from total CD8 T cells. Given the differences in phenotype between viral and total CD8 T cells, we successfully used unsupervised clustering analysis software to reveal a distinct virus-specific CD8 T cell population. This approach of using pooled tetramer for detection of total viral and autoreactive CD8 T cells in the same sample, in combination with extensive phenotyping by mass cytometry will be useful in monitoring important phenotypic changes in this population during disease progression and in response to therapy.
AMPK-dependent and independent effects of AICAR and Compound C on T-cell responses
Authors: Rao Enyu , Zhang Yuwen , Li Qiang , Hao Jiaqing , Egilmez Nejat , Li Bing
Published: January 2023

As a master metabolic sensor, AMP-activated protein kinase (AMPK) is involved in different fundamental cellular processes. Regulation of AMPK activity either by agonists (e.g., AICAR) or by antagonists (e.g., Compound C) has been widely employed to study the physiological functions of AMPK. However, mounting evidence indicates AMPK-independent effects for these chemicals. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and Compound C is mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent roles of AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these “AMPK regulators”.
A checkpoint requiring cognate antigen recognition at the effector stage is necessary for protective memory CD4 T cell generation.
Authors: Bautista Bianca L , Devarajan Priyadharshini , McKinstry K. Kai , Strutt Tara M , Swain Susan L
Published: January 2023

Effective vaccination depends on the generation of memory T cells, B cells, and long-lived plasma cells. However, the mechanisms that govern memory CD4 T cell formation are not well understood. Antigen (Ag) recognition during priming drives effector generation, but it is unclear how Ag recognition at the effector stage impacts T cell fate. Using an influenza infection model, we find that effector CD4 T cells must re-encounter Ag at the effector phase to avoid default apoptosis and become memory. Cognate interaction at this defined “memory checkpoint” drives continued proliferation, enhances survival, upregulates memory markers CD25, CD127, CXCR3, and Bcl-6, and drives IL-2 production, resulting in the formation of long-lived memory cells with enhanced production of multiple cytokines and increased protective function. A cold-adapted influenza virus, like those used in live-attenuated influenza vaccines, fails to present Ag at this critical effector time point and fails to induce substantial CD4 memory. However, memory CD4 T cell formation was rescued by adding short-lived Ag-pulsed antigen presenting cells during the checkpoint. Importantly, the Ag alone without infection-induced inflammation, was sufficient to restore memory formation. Our findings imply that in order for vaccines to promote optimal memory CD4 T cell formation, they must provide Ag presentation long enough that effector CD4 T cells are effectively restimulated during the “memory checkpoint”.
Induction of B10 function on ligature-induced experimental periodontitis
Authors: Hu Yang , Yu Pei , Yu Xinbo , Hu Xingxue , Han Xiaozhe
Published: January 2023

Objective

IL-10 producing B cells (B10 cells) play an essential role in immune system balance by suppressing excessive inflammatory responses. This study is to investigate induction of B10 function in vitro and their effect on ligature-induced experimental periodontitis in vivo.

Methods

Spleen B cells were isolated from C57BL/6J mice and cultured for 48 hours under the following conditions: control, CD40L (1 μg/ml), IL21 (25, 50, 100, 1000ng/ml), anti-Tim1 (2.5, 5, 10, 20 μg/ml), CD40L + IL21, CD40L + anti-Tim1, CD40L + IL21 + anti-Tim1. The mRNA level and secreted protein level of IL-10 expression were detected by RT-qPCR and ELISA respectively. Silk ligatures were tied around both maxillary second molars of C57BL/6J mice for two weeks. Optimized combination of CD40L, IL21 and anti-Tim1 and vehicle were injected into contralateral side of palatal gingiva on days 3, 6 and 9. The palatal gingival tissues and maxillary bone were collected on day 14 to determine expressions of IL10 and RANKL and periodontal bone resorption respectively.

Results

IL-10 expressions of cultured spleen B cells were significantly increased in the presence of CD40L, IL21 and anti-Tim1 combination when compared with control groups. Gingival IL-10 expression were significantly increased after injection of CD40L, IL21 and anti-Tim1 combination, when compared to the control side (p<0.05). The gingival RANKL expression and periodontal bone loss were significantly decreased on the side with combined injection of CD40L, IL21 and anti-Tim1, as compared to the control side (p<0.05).

Conclusion

CD40L, IL21 and anti-Tim1 combination induced IL-10 production by B cells and inhibited periodontal bone loss in ligature-induced experimental periodontitis.
Mechanism of central and peripheral T cell tolerance to an antigen of the central nervous system
Authors: Wang Lei , Hinterberger Maria , Winnewisser Julia , Federle Christine , Klein Ludger
Published: January 2023

Autoreactive CD4+ T cells are censored by two mechanisms, the so-called clonal deletion and the deviation into regulatory T cells in the thymus both requiring the same stimulus, namely the interaction with self-peptide. In order to study the mechanisms of central and peripheral tolerance that operate to shape the CD4 T-cell repertoire, we have generated a TCR transgenic mouse specific for myelin proteolipid protein (PLP). We found that PLP-specific T cells undergo clonal deletion and Treg differentiation concomitantly upon encounter of the cognate self-antigen PLP. Medullary thymic epithelial cells (mTECs) express and present the endogenous antigen PLP and mediated tolerance in an autonomous manner, whereas thymic dendritic cells (DCs) were dispensable for central tolerance induction to PLP. Furthermore, the transcriptional regulator AIRE expressed by mature mTEC is essential for tolerance induction of PLP-specific CD4 T cells. Although central tolerance induction to PLP is very potent it is not complete as a proportion of autoreactive T cells also escape to the periphery. However despite the presence of potentially dangerous cells in the periphery, the mice do not develop autoimmunity. Thus, additional tolerogenic mechanisms promote tolerance in the periphery exist. We could further show that autoreactive PLP-specific T cells are deleted or become functionally inactivation (anergy) by recognition of self-peptide on DCs in the periphery. In a set of bone marrow transplantation experiments we found that PLP is expressed by radio resistant stromal cells and subsequently cross-presented by dendritic cells. Taken together our results indicate a complementing role of the thymus and the periphery for tolerance induction to PLP.
Etv5 promotes IL-10 production in Th2 cells
Authors: Koh Byunghee , Kaplan Mark H
Published: January 2023

The anti-inflammatory cytokine, IL-10, suppresses inflammatory responses at mucosal surfaces. Among T helper cell subsets, Th2 cells produce the highest concentrations. However, the transcription factors that regulate Il10 gene expression have not been completely defined. Etv5 is a member of the ETS transcription factor family that promotes production of IL-17 in Th17 cells and IL-9 in Th9 cells. To define the effects of Etv5 deficiency on cytokine expression in Th2 cells, T cells from CD4 specific Etv5 knockout (Etv5fl/flCD4-Cre+, termed Etv5 KO below) and littermate control (Etv5fl/fl CD4-Cre-, termed WT below) mice were differentiated under Th2 conditions. Etv5 was required for maximal IL-10 production and modestly repressed the Th2 cytokines IL-4, IL-5 and IL-13. In mice sensitized with Aspergilus fumigatus extract, IL-10 production in both BAL and lung was significantly reduced in Etv5 KO mice, consistent with in vitro data. Using ChIP and reporter assays, we showed that Etv5 directly binds to the Il10 locus, specifically at the CNS3 region. Etv5 promotes IL-10 production by recruiting IL-10 inducing transcription factors including Jun family members, IRF4 and E4BP4, and the binding of these factors to the Il10 locus, but not the expression of these factors, was decreased in the absence of Etv5. Ectopic GATA3 and E4BP4 expression recovers IL-10 production to wild type amounts, suggesting that Etv5 enhances the efficiency of function of these factors. Importantly, ectopic Etv5 expression in Etv5 KO Th2 cells restored binding of the factors to the Il10 locus. Thus, Etv5 promotes IL-10 production by enhancing the recruitment of IL-10 inducing transcription factors.
Regulation of β1 Integrin recycling, cell migration and focal adhesion turnover by CD13
Authors: Ghosh Mallika , Thangada Shobha , Devarakonda Charan , Shapiro Linda H
Published: January 2023

CD13 is a multifunctional cell surface peptidase that is expressed on a variety of cells where we have shown that it modulates receptor-mediated endocytosis and ligand internalization to control downstream signaling pathways. In fibroblasts and epithelial cells, CD13 is localized with β1-integrin at focal adhesions (FAs), the sites of communication between the extracellular matrix and the actin cytoskeleton, prompting our exploration of potential contributions of CD13 focal adhesion turnover, integrin endocytosis and trafficking. Phenotypically, FAs in CD13KO fibroblasts are elongated and irregular with displaced FA accessory proteins, markedly reduced actin stress fibers and fewer microtubule extensions, consistent with a link between CD13 and the control of FA dynamics. In wild type cells, CD13 and β1-integrin co-internalize, traffic to EEA1+ Rab5+ early endosomes and recycle to the plasma membrane via Rab11a+ recycling endosomes. Pulse-chase assays with wild type and CD13 phospho tyrosine mutant confirmed that tyrosine phosphorylated CD13 must associate with the ARF6 GTPase for β1-integrin recycling to the membrane to occur. Conversely, the absence of CD13 results in trafficking of internalized β1-integrin from early endosomes to Rab7+ late endosomes and lysosomes and a failure to return to the cell surface. Functionally, in migrating cells CD13 accumulates at the leading edge and co-localizes with IQGAP1 and focal adhesion protein, Paxillin to regulate cell migration and adhesion. In conclusion, CD13 is responsible for proper localization of focal adhesion proteins, coordination of integrin recycling and focal adhesion dynamics, thereby regulating integrin signal transduction and cell motility.
Insights into MHC-I peptide loading obtained from the structure of a TAPBPR/MHC-I complex
Authors: Natarajan Kannan , Jiang Jiansheng , Boyd Lisa F , Morozov Giora I , Mage Michael G , Margulies David H
Published: January 2023

The cell surface expression of MHC-I molecules displaying peptides derived from intracellular proteolytic processing is a critical requirement for T cell-mediated immunity. A complex series of steps, guided by the peptide loading machinery in the endoplasmic reticulum, ensures that only stably folded MHC-I molecules carrying peptides of proper length and high affinity are transported for display at the cell surface. TAP-binding protein, related (TAPBPR), has been recently shown to directly bind to certain MHC-I alleles and to facilitate peptide loading. To elucidate mechanistic details, we have examined the binding interaction between purified recombinant TAPBPR and MHC-I molecules loaded with truncated peptides that leave a portion of the peptide binding groove unoccupied. Such MHC-I molecules interact with TAPBPR with nanomolar affinities as measured by surface plasmon resonance and these TAPBPR/MHC-I complexes dissociate when offered a high affinity peptide. An extensive crystallization screen with purified TAPBPR/MHC-I complexes yielded crystals that diffracted to 3.5 Å. The structure of the TAPBPR/MHC-I complex, solved by molecular replacement, and refined at this resolution readily reveals the binding footprint and further structural details of the interaction. Data will be presented on the binding interaction and structural characterization of the TAPBPR/MHC-I complex that reveal insights into the mechanism of TAPBPR- and tapasin-mediated loading of peptides onto MHC-I molecules. (KN and JJ contributed equally to this work).
Cytokine-mediated germinal center reprogramming enhances vaccine efficacy
Authors: Pravetoni Marco , Laudenbach Megan , Baruffaldi Federico , Robinson Christine , Carter Philipp
Published: January 2023

Candidate vaccines against infectious agents and non-communicable diseases consist of purified proteins, proteins conjugated to synthetic peptides or haptens, and use adjuvants or novel delivery platforms. Despite pre-clinical proof of concept, vaccine clinical efficacy is often limited to a small subset of immunized subjects. Additionally, very few adjuvants meet regulatory approval. Thus, more effective immunization strategies are needed. This study tested the hypothesis that vaccine efficacy is enhanced by cytokine-based immunomodulators that guide B and T cells toward germinal center (GC) activation and generation of effective antibody (Ab) responses. To this end, a series of cytokines and monoclonal Ab against cytokines or cytokine receptors was co-administered with a candidate therapeutic vaccine for treatment of opioid abuse. Immunization with a drug-hapten conjugated to immunogenic carriers generates anti-drug Ab that block drug distribution to the brain, and reduce drug-induced behavioral effects. In this study, targeting IL-4 signaling enhanced vaccine efficacy in blocking opioid distribution to the brain in mice. Vaccine efficacy was associated with increased vaccine-specific GC B cell and GC-TFH cell differentiation, serum anti-opioid IgG Ab levels and IgG subclasses shift, and a balanced Th1/Th2 environment. No side effects were found. This strategy generalized to a model peptide-protein conjugate immunogen and an approved tetanus-diphtheria-pertussis vaccine. These data support targeting cytokine signaling to safely program vaccine-induced GC formation and achieve clinically effective Ab. This strategy provides a blueprint for rational development of more effective next-generation vaccines.
A novel vaccine vector: herpes simplex virus type-2 deleted in glycoprotein D (HSV-2 ΔgD) and expressing modified influenza A (IAV) hemagglutinin (HA) antigens
Authors: Dardick Joseph , Weiss Kayla , Lukose Regy , Weinrick Brian , Herold Betsy , Jacobs William R
Published: January 2023

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause significant global morbidity and mortality. In collaboration, the Jacobs and Herold laboratories generated an attenuated herpes simplex virus type 2 (HSV-2) strain deleted in glycoprotein D (HSV-2 ΔgD) and demonstrated that it triggers an FcγRIV activating response that elicits both antibody-dependent cell-mediated cytotoxicity and phagocytosis. The antibodies rapidly clear virus and prevent the establishment of latency in mice and guinea pigs following challenge with several clinical isolates of both HSV-1 and HSV-2. We therefore propose that HSV-2 ΔgD is an ideal vaccine vector to induce immunity against a wide array of pathogens.

The HSV genome is difficult to work with due to high GC content, repetitive regions, and tendency toward attenuation. Using Gibson cloning and homologous recombination we introduce exogenous genes such as red fluorescent protein (rfp) and influenza A (IAV) hemagglutinin (HA) into the HSV genome. IAV HA is a great model antigen to study the immunogenicity of proteins inserted into the HSV-2 ΔgD backbone. Moreover, cloning both full-length and stalk HAs into the HSV-2 ΔgD genome will test the influence of immunodominant antigens on the character of our antibody response.

Our expression vectors, verified by digestion and sequencing, encode a chimeric gD/HA protein that contains the signal sequence and cytosolic and transmembrane domains of HSV-2 gD. These modifications will promote trafficking of HA to the cell membrane and viral envelope. Our recombination system has produced an RFP+ HSV-2 ΔgD virus that we verified by fluorescence, digestion and sequencing. The kinetics of infection with the RFP+ virus match that its parental HSV-2 ΔgD strain.
Induction of Virus-specific Immune Tolerance in A Chronic Hepatitis B Virus Mouse Model
Authors: Lan Yu Hua , Lo Yu-Chieh , Wu Ping-Yi , Tao Mi-Hua
Published: January 2023

Hepatitis B virus (HBV) infection causes severe public health problem. There are estimated 250 million people being chronically infected by HBV and are at an increased risk of developing end-stage liver diseases, such as cirrhosis and hepatocellular carcinoma. It is well recognized that the chronicity of HBV infection is due to the impaired immune responses to HBV and thus not able to eliminate HBV from the infected hepatocytes. However, the immune mechanisms that lead to immune tolerance and HBV persistence have not been understood. A challenging question for HBV studies is lack of appropriate animal models due to the narrow host range of HBV. To address this challenge, we created a chronic HBV infection model in immunocompetent mice by transduction of adeno-associated virus serotype 8 (AAV8) vector which carries and these mice was transduced with carrying a replication-defective HBV genome with two point mutations in the polymerase(AAV/HBVp−). We showed that mice transduced with AAV/HBVp− produced HBV RNA and proteins, including serum HBV surface antigen and HBV e antigen as well as HBV core protein. The high levels of HBV RNA and proteins sustained for at least 1 year post AAV transduction. Viral transcripts and proteins were detected in the livers of the AAV-injected mice but not in other organs. DNA immunization of these AAV/HBVp− transduced mice failed to induce HBV-specific humoral and cellular immune responses, suggesting these mice developed immune tolerance toward HBV. Thus, the AAV/HBVp− transduced mouse model recapitulates many virological and immunological characteristics of chronic HBV in humans. This animal model could be useful for the development of new treatments and immune-based therapies for chronic HBV infections.
Zika virus infects human Sertoli cells and trespass the blood-testes barrier to migrate across the seminiferous epithelium
Authors: Siemann David N , Strange Daniel , Maharaj Payal , Verma Saguna
Published: January 2023

The 2015–2016 epidemic of Zika virus (ZIKV) has identified sexual transmission as a new route of disease spread. Presence of ZIKV in the semen when viremia is undetectable in humans suggests the ability of ZIKV to establish infection in the testes. Lack of specific therapeutic drugs to clear testicular infection warrants understanding of the associated mechanisms. Seminiferous tubules, the site of spermatogenesis, is an immune privileged site with an effective blood-testes barrier (BTB) constituted exclusively by specialized tight junctions proteins (TJP) between the adjacent Sertoli cells (SC) that protects germ cells from systemic immune attack. Increased inflammatory mediators such as TNF-α, IL-1β, matrix metalloproteinases and cell-adhesion molecules (CAMs) can disrupt BTB and facilitate testicular infections. We determined the infection kinetics of ZIKV in SC and its association with antiviral defense and BTB integrity. We demonstrate that SC were susceptible to ZIKV and induced cytokines (IFN-α, IFN-γ, TNF-α) and CAMs (VCAM), which correlated with peak virus titers. Interestingly, while ZIKV transmigrated efficiently across the in vitro BTB model, it did not affect barrier integrity, also supported by no difference observed in the levels of TJP such as ZO-1 and claudin between control and infected SC. However, the integrity of the BTB model decreased significantly following exposure to the supernatant from ZIKV-infected macrophages. Collectively, our data suggests infection of SC as one of the routes of virus transmigration into the seminiferous tubules and that inflammatory mediators from infected human macrophages may further compromise integrity of BTB thereby allowing persistent infection of ZIKV in the testes.
HVEM expression by intestinal epithelial cells modulates the microbiome and epithelial immunity
Authors: Seo Goo-Young , Shui Jr-Wen , Mikulski Zbigniew , Cheroutre Hilde , Kim Pyeung-Hyeun , Kronenberg Mitchell
Published: January 2023

The herpes virus entry mediator (HVEM), a member of the tumor necrosis factor receptor superfamily (TNFRSF), is highly expressed by intestinal epithelial cells (IEC). Previously, we reported that HVEM expression by epithelial cells was required for innate immune defense from acute infections in the lung and the intestine (Nature 488:222,2012). Here, we demonstrate a novel, constitutive role of HVEM expression by IEC in microbial homeostasis and epithelial immunity. Mice with an epithelial-specific deletion of the gene encoding HVEM (HvemΔIEC) had significantly increased segmented filamentous bacteria (SFB). This caused an increase in Th17 cells and IL-22+ILC3s in the intestine lamina propria. HvemΔIEC mice also exhibited increased goblet cell hypertrophy and hyperplasia, crypt hyperplasia, villous atrophy as they aged, and they showed a severe defect in fucosylation of cell surface proteins, even at younger ages. Treatment with the antibiotic vancomycin eliminated SFB and decreased Th17 cells and ILC3, but did not reverse the defect in fucosylation. Our findings suggest that epithelial cell expression of HVEM is important at steady state both for shaping the intestinal microbiota and for the homeostasis of IEC in the intestine.
Sequential activity of innate and adaptive lymphocytes supports non-inflammatory gut microbial commensalism
Authors: Mao Kairui , Baptista Antonio , Bouladoux Nicolas , Martins Andrew J , Tamoutounour Samira , Davis Jacquice , Huang Yuefeng , Gerner Michael Y. , Belkaid Yasmine , Germain Ronald N
Published: January 2023

The mammalian gut is colonized by trillions of microorganisms termed the “microbiota”, which have a mutually beneficial relationship with their host. In normal individuals, the gut microbiota matures after birth to a state of balanced commensalism that is marked by the absence of adverse inflammation. Both innate lymphoid cells (ILCs) and antigen-specific conventional T cells contribute to containment and clearance of microbial pathogens. But how these two major lymphoid cell populations help shape the mature commensal (non-pathogenic) microbiome and maintain tissue homeostasis has not been determined. Using advanced multiplex quantitative imaging methods, here we show that in the absence of adaptive lymphocytes, IL-23 induced by specific commensal bacteria such as Segmented Filamentous Bacteria (SFB) persistently activates RORgt+ group 3 innate lymphoid cells (ILC3s) in the ileum to produce IL-22, which induces STAT3 activation in virtually all epithelial cells, contributing to production of molecules such as anti-microbial peptides that protect the tissue from microbial damage. The distinct roles of ILCs in handling gut microbes play out in normal mice during development. The pSTAT3 signature is absent after birth, which is followed by microbial colonization and strong ILC3 activation and an extensive epithelial pSTAT3 signature upon weaning. This innate immune activity is subsequently extinguished as adaptive CD4+ T cell immunity develops in response to the expanding commensal burden. Our findings provide new insights into how innate and adaptive lymphocytes sequentially operate during normal development to establish steady state commensalism.
IL-17 promotes intestinal IgA response to intestinal infection but does not affect memory B cell development
Authors: Huang Xiangsheng , Sun Mingming , Chen Feidi , Xiao Yi , Chen Liang , Yao Suxia , Cong Yingzi
Published: January 2023

Secretory IgA is the principal immunoglobulin found in intestinal track and plays a crucial role in maintanence of intestinal homeostasis. We have previously demonstrated that Th17 cells, which produce IL-17, IL-21 and IL-22, promoted intestinal IgA responses to microbiota. However, the mechanisms involved are still largely unknown. In current study, we investigated the roles of Th17 cells in regulation of intestinal IgA response to enteric pathogen infection. We showed that adoptive transfer of CBir1-specific Th17 cells, which are specific for immunodominant gut bacterial antigen CBir1 flagellin, promoted intestinal IgA in T cell-deficient TCRβxδ−/− mice, which is inhibited by the blockade of IL-17 and IL-21. To determine whether IL-17 regulates memory IgA B cell responses to intestinal infection, we infect wild type (WT) and IL-17R−/− mice with Citrobacter Rodentium, and C. Rodentium in the intestines, as well as fecal C. Rodentium-specific IgA and lamina propria IgA+ memory B cells were assessed. IL-17R−/− mice shows less efficiency in C. Rodentium clearance, in that while there were almost no bacteria at week 2 after infection in WT mice, significant amounts of C. Rodentium were still present in IL-17R−/− mice. Consistently, in IL-17R−/− mice C. Rodentium-specific intestinal IgA production was impaired compare with WT mice, and the memory IgA development was diminished after re-infection. However, there was no difference in lamina propria IgA+ memory B cells between WT and IL-17R−/− mice. Collectively, our data demonstrated that the IL-17 promotes intestinal IgA responses to intestinal infection, however, it does not affect memory IgA B cell development.
HVEM expression by innate lymphoid cells protects against enteric bacterial infection
Authors: Seo Goo-Young , Shui Jr-Wen , Takahashi Daisuke , Song Christina , Mikulski Zbigniew , Kim Pyeung-Hyeun , Cheroutre Hilde , Colonna Marco , Kronenberg Mitchell
Published: January 2023

Yersinia enterocolitica causes food-borne disease and targets the small intestine, in which ILC3 are the predominant ILC subset. Here we show that ILC3 are required for protection of mice from oral Y. enterocolitica, as mice lacking ILC3 were more susceptible and transfer of ILC to recipients was protective. IFNγ is a cytokine required for host defense from this pathogen, and while ILC in the small intestine did not increase in number they greatly increased IFNγ production after infection. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily (TNFRSF), is expressed by all intestinal ILC subsets. Mice with a deficiency of HVEM expression in RORγt+ ILC had a reduced steady-state IL-22 production, and in vitro HVEM could signal to ILC3 to stimulate IL-22 secretion. Moreover, following oral infection with Y. enterocolitica, mice with HVEM deficiency mediated by Rorc-cre showed reduced survival and increased bacterial translocation early after infection. Cytokines are known to induce ILC activation, but our results demonstrate a novel role for the cell surface receptor HVEM in regulating ILC3 cytokine production, both at steady state, and as a critical survival factor during acute infection.
Cell-intrinsic TLR4-dependent bone marrow lympho-myeloid distortions in obesity
Authors: Borghesi Lisa A , Liu Ailing , Chen Minhui , Kumar Rashmi , Stefanovic-Racic Maja , O’Doherty Robert , Ding Ying , Jahnen-Dechent Willi
Published: January 2023

Obesity compromises bone marrow (BM) progenitor function and the mechanistic underpinnings are just beginning to be established. Here we show that following 16 weeks of high fat diet, mice exhibit poor emergency hematopoiesis in a toll-like receptor 4 (TLR4)-dependent manner, and that adoptive transfer of obese BM to lean recipients restores rebound capability. Genes associated with a myeloid signature (c-fms, pu.1, ikaros) are enhanced and lymphoid signature (flt3, meis1, e47) reduced. The skewed lympho-myeloid outgrowth of discrete BM subsets is apparent by 6 weeks of high fat diet, earlier than is commonly recognized. Using mixed BM chimeras we demonstrate that obesity-associated subversion of lineage-c-kit+Sca-1- (LKSneg) precursors to the myeloid lineage and common lymphoid progenitors (CLP) to the B lineage depends on BM cell subset-autonomous TLR4. The endogenous TLR4 ligands responsible for BM dysfunction in obesity are unclear. Here, we show that exposure of lean mice to lipopolysaccharide (LPS) at levels similar to that observed in obesity recapitulates key aspects of BM lympho-myeloid disruption. Together, our results establish a mechanistic role for BM cell-intrinsic TLR4 early in obesity-driven BM dysfunction, and demonstrate the biological importance of one obesity-associated TLR4 ligand.
MiRNAs are required for embryonic development of Langerhans Cells
Authors: Martin Carly Elizabeth , Yao Yi , Mi Qing-Sheng , Zhou Li
Published: January 2023

Langerhans cells (LCs) are epidermal-resident dendritic cells that play important roles in skin immunity and tolerance. Recent fate-mapping studies have demonstrated that adult LCs derive predominantly from fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. However, the role of epigenetic regulations in embryonic development of LCs remains unclear. MicroRNAs (miRNAs) are small, noncoding RNAs that negatively regulate gene expression of their mRNA targets. We and others recently reported that miRNAs are key players in regulating immune cell development and function, including LCs. To further study the role of miRNAs in LC ontogeny, we generated CSF1rCreDicerf/fKO mice in which miRNAs are deficient in myeloid lineage cells including LCs. As expected, the frequency of LCs was drastically reduced in Dicer KO adult mice. Interestingly, there was also a significant reduction (p=0.027) of epidermal LCs in Dicer KO embryos at E18.5, suggesting that miRNAs are crucial for embryonic development of LCs. Given the differential expression of miR-150 and miR-155 in embryonic LC development, we generated CSF1rCremiR-150f/fKI and CSF1rCremiR-155f/fKI mice in which expression of miR-150 and miR-155 is upregulated in LC-lineage, respectively. Surprisingly, neither strain caused defective LC development in fetal or adult epidermis. Overall, this is the first study to demonstrate that miRNAs are required for embryonic LC development. Furthermore, although overexpression of miR-150 or miR-155 is dispensable for LC ontogeny, the specific miRNAs responsible for proper LC development deserve to be further explored.