bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbiome-dependent modulation of mucosal immunity at the ocular surface.
Authors: Caspi Rachel R , Drummond Rebecca , Desai Jigarkumar , Silver Phyllis B , Lionakis Michail S , St. Leger Anthony J
Published: January 2023

Mucosal sites that interface with the environment and provide barrier function include the intestine, nasopharynx, lung, female reproductive tract and the ocular surface. Disruption of immune homeostasis at the ocular surface is associated with discomfort, inflammation and potential loss of vision. Immune cells are present within the mucosa of the ocular surface (conjunctiva) and are affected by environmental factors, potentially including microorganisms. However, proof that a resident ocular microbiome exists and influences local immunity has been elusive. We used a mouse model of ocular surface disease to address the question whether commensal microbes are present in ocular mucosa and modulate immunity. Our data indicate that IL-17 is constitutively produced within the conjunctiva-associated lymphoid tissue (CALT) and is necessary to recruit neutrophils to the ocular surface in the steady state and after a bacterial challenge. IL-17 sources in CALT include γδ T cells, αβ T cells and innate lymphoid cells (ILCs), in that order. Notably, conjunctival γδ T cells respond to a strain of Corynebacterium that colonizes the ocular surface by secreting IL-17, which modifies the inflammatory signature within the ocular mucosa. Furthermore, this interaction appears necessary to regulate local immunity at the ocular surface, since elimination of these bacteria by antibiotic treatment, or their introduction into non-colonized mice, correlated inversely with severity of an experimental Candida albicans infection. Our results indicate that a relationship exists between commensals and immune cells at the ocular surface, which is critical for maintenance of homeostasis and host defense within the ocular mucosa.
Group 3 innate lymphoid cells mediate intestinal selection of commensal bacteria-specific CD4+ T cells.
Authors: Sonnenberg Gregory F , Fung Thomas C , Goc Jeremy , Wang Xinxin , Hepworth Matthew R
Published: January 2023

Inflammatory CD4+ T cell responses to self or commensal bacteria underlie the pathogenesis of autoimmunity and inflammatory bowel disease (IBD), respectively. While selection of self-specific T cells in the thymus limits responses to mammalian tissue antigens, the mechanisms that control selection of commensal bacteria-specific T cells remain poorly understood. Here we demonstrate that group 3 innate lymphoid cell (ILC3)-intrinsic expression of major histocompatibility complex class II (MHCII) is regulated similarly to thymic epithelial cells, and that MHCII+ ILC3s directly induce cell death of activated commensal bacteria-specific T cells. Further, MHCII on colonic ILC3s was reduced in pediatric IBD patients. Collectively, these results define a selection pathway for commensal bacteria-specific CD4+ T cells in the intestine and suggest that this process is dysregulated in human IBD. On going studies are interrogating the role and regulation of MHCII+ ILC3s in the context of allergy, autoimmunity and tumorigenesis.
An enzyme from a human commensal bacterium triggers enhanced intestinal barrier function
Authors: Pedicord Virginia Anh , Rangan Kavita J. , Craig Jeffrey W. , Loschko Jakob , Rogoz Aneta , Hang Howard C. , Mucida Daniel
Published: January 2023

Intestinal infections affect billions of people worldwide. Commensal intestinal bacteria can prevent pathogenic infection, as demonstrated by drastically increased susceptibility to infection upon antibiotic use; however, the molecular mechanisms by which individual bacterial species contribute to pathogen resistance have yet to be fully elucidated. Using a human commensal Enterococcus faecium strain, we show that colonization of mice with E. faecium elicits improved intestinal epithelial barrier function and decreased antibiotic-induced susceptibility to Salmonella infection. This protection requires host antimicrobial peptide (AMP) expression and MyD88 and Nod2 signaling and corresponds to E. faecium secretion of a unique enzyme, secreted antigen A (SagA). Ectopic expression of SagA by non-protective bacterial species is sufficient to confer similar probiotic efficacy against Salmonella pathogenesis and induce AMP expression in the intestinal epithelium. These studies reveal a mechanism for E. faecium-mediated pathogen resistance and identify a specific factor produced by commensal bacteria that triggers a protective program in the mammalian intestine.
Periostin regulates fibrocyte function to promote myofibroblast differentiation and lung fibrosis
Authors: Ashley Shanna L , Wilke Carol A , Moore Bethany B
Published: January 2023

Fibrocytes are bone-marrow-derived circulating mesenchymal precursors (CD45+, col 1+) that are recruited to fibrotic areas. Fibrocytes secrete a number of profibrotic mediators including periostin; a matricellular protein that regulates cellular interactions with extracellular matrix (ECM) components. In bleomycin-induced fibrosis, loss of periostin by either structural or hematopoietic cells can limit the development of pulmonary fibrosis. To test the hypothesis that hematopoietic-derived fibrocytes might secrete soluble factors to signal structural myofibroblasts differentiation, wild-type (WT) fibroblasts were treated with conditioned medium from fibrocytes isolated from bleomycin-treated WT or periostin−/− mice. After 24 hours we saw marked decrease in α-smooth muscle actin protein expression in cells treated with the conditioned medium from periostin−/− fibrocytes. Adoptive transfer of WT fibrocytes augmented lung fibrosis to a greater extent than did transfer of fibrocytes from periostin−/− mice. We performed in vitro analyses of lung mesenchymal cells isolated from WT and periostin−/− mice by sorting fibroblasts and fibrocytes then treating with TGFβ1 or periostin. Additionally, connective tissue growth factor (CTGF), endothelin-1 (ET-1) and lysl oxidase (LOX) mRNA expression was downregulated in periostin−/− bleomycin-treated fibrocytes. Taken together these data suggest that fibrocytes may augment bleomycin-induced fibrosis via secretion of periostin and other soluble factors that promote myofibroblasts differentiation.
Innate Lymphoid Cell and Helper T Cell Subpopulations in Chronic Rhinosinusitis
Authors: Dietz Caroline Padro , Reed Robyn C , Luong Amber U.
Published: January 2023

Chronic rhinosinusitis (CRS) is an inflammatory disease of the upper airways subtyped by the absence (CRSsNP) or presence (CRSwNP) of nasal polyps. Allergic fungal rhinosinusitis (AFRS) is a clinical subtype of CRSwNP characterized by fungal growth within the sinuses. CRSwNP and AFRS are characterized as type 2 inflammatory diseases based on elevated IL-5 and IL-13 within sinus mucosa. In contrast, CRSsNP is classified as a type 1 disease with elevated type 1 cytokines such as IFN-γ. Although Th1 and Th2 cells have been traditionally described as type 1 and 2 cytokine producers, innate lymphoid cells (ILCs) have emerged as another significant source.

To determine the cell sources responsible for these cytokine elevations, the presence of type 1–3 ILCs (ILC1–3) and Th1 and Th2 cells in surgically-derived inflamed mucosa from CRSsNP (n=8), CRSwNP (n=14), and AFRS (n=8) patients were analyzed by flow cytometry. Overall, we found elevated total ILCs in CRSwNP (3.15 ± 0.66%) in comparison to CRSsNP (0.77 ± 0.22%, p=0.01). When further examined, all three types of ILCs were significantly elevated in CRSwNP (ILC1: 0.30 ± 0.09%, ILC2: 0.91 ± 0.31%, ILC3: 0.10 ± 0.02%) in comparison to CRSsNP (ILC1: 0.13 ± 0.05%, p=0.03, ILC2: 0.37 ± 0.18%, p=0.04, ILC3: 0.05 ± 0.01%, p=0.05), supporting a greater innate immune system focus in CRSwNP. In contrast, Th1 cells were significantly elevated in CRSsNP (72.23 ± 5.13%) in comparison to CRSwNP (52.72 ± 5.0%, p=0.009) and AFRS (45.19 ± 5.18%, p=0.003), indicating that the majority of IFNγ-secreting cells were of the adaptive immune system. The characterization of CRSwNP as an innate immune disease and CRSsNP as an adaptive immune system disease indicate potential therapeutic targets in the treatment of CRS.
Inducible bronchus-associated lymphoid tissue plays an important role in the induction of antigen-specific immune response by Ag85B-hPIV2-based anti-tuberculosis vaccine in mice
Authors: Nagatake Takahiro , Wada Yasuko , Matsumoto Naomi , Shimojou Michiko , Hirata Soichiro , Nasu Ayaka , Suzuki Hidehiko , Hosomi Koji , Ogami Kentaro , Tsujimura Yusuke , Kawano Mitsuo , Nosaka Tetsuya , Yasutomi Yasuhiro , Kunisawa Jun
Published: January 2023

Mycobacterium bovis bacilli Calmette-Guerin is the only licensed tuberculosis vaccine, which has been considered to be insufficient against adult pulmonary tuberculosis. As most of pathogens, including Mycobacterium tuberculosis, naturally enter the host through respiratory surface, nasal vaccine is an ideal method to prevent the diseases by inducing antigen-specific immune responses at respiratory sites as well as systemic compartments. Our group recently developed Ag85B-expressing human parainfluenza type 2 virus (Ag85B-hPIV2) as a nasal vaccine against tuberculosis. In this study, we aimed to elucidate the mechanisms underlying the induction of antigen-specific immune responses by Ag85B-hPIV2. We found that inducible bronchus-associated lymphoid tissue (iBALT) played an important role in the induction of antigen-specific immune responses. Intranasal administration of hPIV2 vector induced iBALT formation with induction of Th17 cells and CD11b+CD11c+ cells both of which are known to require for the formation and maintenance of iBALT structure, respectively. When iBALT structure was disrupted by depletion of CD11b+ cells in CD11b-diphtheria toxin receptor transgenic mice, Ag85B-specific immune response was diminished in the lung. These findings indicate that iBALT genesis is an essential process required in Ag85B-hPIV2 anti-tuberculosis mucosal vaccine.
Development and validation of a novel flow cytometric macrophage phenotyping method.
Authors: Welder Daniel A , Slief Sarah , Morton Rebecca , Baatar Dolgor
Published: January 2023

Macrophages differentiate into pro-inflammatory M1 or anti-inflammatory M2 phenotypes. M2 macrophages suppress anti-tumor responses, and phenotyping tumor-associated macrophages can be of prognostic value. Expression of mRNA of inducible nitric oxide synthase (iNOS) or arginase 1 is a well-accepted marker of murine M1 or M2 macrophages, respectively. However, macrophages need to be isolated and purified in order to evaluate the mRNA expression. There are no reliable and simple methods for detection of macrophage markers which would not require cell purification. Here we report the development and validation of a novel flow cytometric macrophage phenotyping method.

Murine macrophage RAW 264.7 cells were treated with endotoxin and IFN-γ to stimulate iNOS expression. Alternatively, RAW 264.7 cells were stimulated with 8-bromo-cAMP to induce arginase 1 expression. Our new technique allowed successful detection of iNOS expression in RAW 264.7 cells by flow cytometry, which correlated well with NO production measured by Griess assay. We also developed a method which allowed us to successfully detect the Arginase I expression by flow cytometry. Arginase I expression also correlated well with arginase activity as measured by an Abnova arginase assay kit.

Our results suggest that murine macrophage phenotype can be successfully determined by flowcytometric measurement of iNOS and arginase 1 expression.
Establishment of a humanized lupus model
Authors: Ren Jingjing , Liao Xiaofeng , Luo Xin M
Published: January 2023

Systemic lupus erythematosus (SLE), a systemic autoimmune disease, involves mild to severe manifestations in multiple organs. Currently, lupus-prone mouse models are used to understand the mechanisms of SLE development and discover new therapies. However, due to differences between rodents and humans, such as physiological development, disease progression, and response to therapies and drugs, many mechanisms and therapies discovered in mice may not be efficiently transferred from murine lupus models to SLE patients. Here we present a humanized mouse model of lupus as a new technology to bridge the gap between rodent models and patients. In our model, which involves vigorous selection of donors, we engraft peripheral blood mononuclear cells (PBMCs) from SLE patients into NOD/SCID/IL2Rgnull (NSG) mice to build a human-specific disease-based immune system in the mice. To avoid graft versus host disease, we stimulate PBMCs before transferring them into either female or male NSG mice. This method enhances the survival and maintenance of the engrafted cells. Human T cells and B cells that are critical to lupus development can be detected in the peripheral blood and spleen of humanized mice with no sex preference two weeks after engraftment. The model responds well to dexamethasone treatment with reduced proteinuria and decrease of spleen weight, indicating that our model could be used for preclinical studies for SLE. The therapeutic window can be as long as 3 weeks. This model allows for mechanistic studies and testing of potential therapies of SLE directly on human immune system in vivo without putting human life at risk.
Effect of methanolic extract of Annona muricta leaves on macrophage effector functions
Authors: Cetina-Dorantes Adan Josue , Arana-Argaez Victor , Torres-Romero Julio , Canul-Canche Jaqueline , Ramirez-Camacho Mario
Published: January 2023

Introduction

Annona muricata (guanabana), is a common plant in various tropical regions of the world. Annona muricata is widely used in traditional Mayan medicine for the treatment of gastrointestinal diseases, skin infections, inflammatory disorders, sedative, to reduce fever and as a treatment for cancer and diabetes. The reports of the traditional medicine about its properties and recent studies suggest that Annona muricata could enhance the effector mechanisms of the immune system.

Aim of the study

The aim of this study was to evaluate the immunomodulatory effect of methanolic extract of Annona muricata (AML-ME) leaves on macrophage function.

Materials and methods

The effect of Annona muricata on the immune system was tested in the monocyte THP-1 cell line. Prior to any test, the monocytes were differentiated to macrophages by the action of 100 ng/mL PMA for 24 hours The effect of AML-ME (75–800 μg/mL) on macrophage proliferation was measured using the MTT assay. The effect of AML-ME on production of nitric oxide, IL-6 and tumor necrosis factor alfa (TNF-α) production was assessed on macrophages treated with the AML-ME (75–800 μg/mL) for 24 hours and later activated with Escherichia coli lipopolysaccharide for another 24 hours.

Results

AML-ME showed to have no effect on THP-1 macrophages proliferation. However, it promotes, in a concentration-dependent manner, the production of nitric oxide, IL-6 and TNF-α.

Conclusion

The methanolic extract of Annona muricata leaves possesses immunomodulatory properties capable of modulate the effector functions of human macrophages. Further studies to elucidate the components of the extract as well as the mechanism of action this molecules are recommended
Stimulating antigen-specific regulatory T cells for an atherosclerosis vaccine
Authors: Benne Naomi , Jiskoot Wim , Kuiper Johan , Slutter Bram
Published: January 2023

Atherosclerosis is a chronic inflammatory disease of the large and medium sized arteries affecting millions of people world-wide. It is characterised by sub-endothelial retention of low density lipoprotein (LDL), which, over time, leads to the formation of an atherosclerotic plaque. Regulatory T cells (Treg) have been shown to reduce chronic inflammation in atherosclerosis and provide a protective effect. Whereas repeated immunisations with high doses of plaque-specific antigens can induce Treg and reduce the atherosclerotic plaques, particulate formulations may require fewer immunisations and less antigen by specific targeting of the immune system. Therefore, we optimised liposomal formulations to elicit potent Treg responses. Liposomes (average diameter 150–200 nm) consisting of the lipids DSPC, DSPG and cholesterol in different molar ratios were loaded with an ovalbumin-derived CD4+ T cell-specific peptide (OVA323). Murine BMDCs were pulsed with formulations and subsequently cultured with OT-II transgenic CD4+T cells. Compared to free OVA323 or OVA323 mixed with empty liposomes, encapsulation of OVA323 induced increased numbers of Treg as measured by FoxP3 expression. Interestingly, liposomes with the composition 4:1:2 DSPC:DSPG:cholesterol induced the highest Treg responses and also prevented Th1 differentiation as determined by IFN-γ production. Subsequent studies suggest that the 4:1:2 liposomes achieve the optimal balance of antigen loading, flexibility and surface charge to induce Treg. In conclusion, this indicates that liposome composition influences Treg differentiation and liposomes may be used to elicit antigen-specific Treg responses for atherosclerosis vaccination.
CD48 is upregulated on pathogenic CD4+ T cells and is a therapeutic target in experimental autoimmune encephalomyelitis
Authors: McArdel Shannon , Brown Daniel , Sharpe Arlene H
Published: January 2023

CD48 (SLAMF2) is an adhesion and costimulation molecule constitutively expressed on hematopoietic cells, and a member of the signaling lymphocyte activation molecule family of immune receptors. Polymorphisms in CD48 have been linked to susceptibility to multiple sclerosis (MS), and differential expression of its homolog CD58 (LFA-3), found exclusively in humans, is associated with disease remission in MS. We examined CD48 expression on lymphocytes during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, and found that a population of CD4+ T cells upregulated CD48 expression during disease. CD48++ CD4+ T cells were predominantly CD44+ and Ki67+, were most prominent in the CNS, and included IL-17+, GM-CSF+ and IFN-γ+ cells. Administration of an anti-CD48 mAb during EAE attenuated both clinical disease and accumulation of lymphocytes in the CNS. Treatment coincided with reduced expansion of effector CD4+ T cells and reduced numbers of pathogenic cytokine-producing CD4+ T cells in the spleen at early time points. These effects were partially dependent on FcγRs, as anti-CD48 could not limit disease in Fcɛr1γ−/− mice or in wild type mice receiving anti-CD16/CD32 mAb, and failed to reduce the number of cytokine-producing effector CD4+ T cells. Our data suggest that anti-CD48 mAb can both limit CD4+ T cell proliferation and preferentially deplete pathogenic CD4+ T cells during EAE, thereby preventing or attenuating disease. This highlights the potential of CD48 to serve as a marker of pathogenic effector T cells and a therapeutic target for autoimmunity.
A critical role of CD200R signaling in limiting the growth and metastasis of CD200 positive melanoma
Authors: Bai Xue-Feng , Liu Jin-Qing , Talebian Fatemeh , Wu Lisha , Markowitz Joseph , Wu Laichu , Carson William E , Basu Sujit
Published: January 2023

CD200 is a cell surface glycoprotein that functions through engaging CD200 receptor on cells of the myeloid lineage and inhibits their functions. Expression of CD200 has been implicated in a variety of human cancer cells including melanoma cells and has been thought to play a protumor role. However, our previous studies using B16 melanoma and J558 plasmacytoma models suggest that tumor expressed CD200 may inhibit tumor growth and metastasis via shaping tumor microenvironment. In this study, we have further evaluated the role of CD200R signaling in tumor growth and metastasis using CD200R-deficient mice. We found that CD200R-deficient mice exhibited accelerated growth of CD200-positive, but not CD200-negative B16 tumors. Strikingly, CD200R-deficient mice receiving CD200-positive B16 cells intravenously exhibited massive tumor growth in multiple organs including liver, lung, kidney and peritoneal cavity, while the growth of the same tumors in wild type mice is limited. CD200-positive tumors grown in CD200R-deficient mice contained higher numbers of CD11b+Ly6C+ myeloid cells, exhibited increased expression of VEGF and HIF-1a genes with increased angiogenesis and showed significantly reduced infiltration of CD4+ and CD8+ T cells. The Liver from CD200R-deficient mice under metastatic growth of CD200-positive tumors contained significantly increased numbers of CD11b+Gr1− myeloid cells, FoxP3+ regulatory T cells; liver T cells also had reduced capacity in the production of IFN-g or TNF-a. Taken together, we have revealed critical roles of CD200R signaling in limiting the growth and metastasis of CD200 positive tumors.
Use of an antigen specific T cell line to investigate unresponsiveness to PD-1 blockade.
Authors: Lodge P. Anne , Tjoa Benjamin A
Published: January 2023

Antibodies blocking the PD-1/PD-L1 interaction have shown great promise in cancer therapy. Inhibition of PD-1 signaling allows previously exhausted tumor specific T cells to resume proliferation and tumor elimination. Nivolumab is an antibody that binds to PD-1 and has been approved for treatment of melanoma, renal cell carcinoma and non-small cell lung cancer. In vitro studies show that nivolumab can enhance the secretion of IFNγ in a mixed lymphocyte reaction or recall antigen stimulation. To facilitate study of checkpoint inhibition, we attempted to use an antigen specific T cell line for an in vitro assay of PD-1 inhibition by nivolumab. We discovered that the myelin basic protein specific T cell line, BC3, is resistant to the effect of nivolumab in spite of expressing PD-1 and the presence of PD-L1 on the surface of the antigen presenting cells. Unresponsiveness to nivolumab is not donor specific but may reflect the nature of the response to specific antigens. In vitro responses to CMV and tetanus toxoid are enhanced by PD-1 blockade but reactivity to EBV nuclear antigen, as with the BC3 T cells, is not affected. Further study of these differences in sensitivity to nivolumab may allow better targeting of therapy to indications most likely to benefit.
hetIL-15 therapy results in tumor control by promoting tumor infiltration of CD8+ T cells with increased cytotoxic potential and decreased PD-1 expression.
Authors: Pavlakis George N , Ng Sin Man , Dimas Konstantinos , Valentin Antonio , Nagy Bethany , Alicea Candido , Felber Barbara K , Bergamaschi Cristina
Published: January 2023

Interleukin-15 (IL-15) is a common γ-chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha chain that are together termed heterodimeric IL-15 (hetIL-15). We investigated the anti-tumor efficacy of hetIL-15 in several mouse cancer models. Repeated injections of hetIL-15 were effective in delaying tumor growth in the MC38 colon carcinoma and the B16 melanoma models. The beneficial effects of hetIL-15 therapy included a significantly reduced onset of lung metastasis in 4T1 breast cancer-bearing mice. We study the mechanisms of antitumor effects of hetIL-15 in several tumor models. hetIL-15 administration promoted a dramatic increase of tumor infiltration and persistence of CD8+ T cells, including tumor specific T cells, and resulted in an increased CD8+/Treg ratio. Tumor-resident CD8+ T cells showed features of effectors cells and were characterized by increased proliferation (Ki67+) and high cytotoxic potential (Granzyme B+). In the absence of hetIL-15, the smaller population of tumor-infiltrating T cells exhibited high levels of the exhaustion marker PD-1, potentially limiting their anti-cancer effectiveness. Provision of hetIL-15 resulted in a significant decrease in lymphocyte expression of PD-1, alleviating one potential mechanism for the exhaustion phenotype. Preclinical cancer studies support the use of hetIL-15 in tumor immunotherapy approaches to promote the development of anti-tumor responses by favoring effector over regulatory cells.
Identification of B7-H4 as a functional surface marker for monocytic myeloid-derived suppressor cells
Authors: Li Limin , Diao Wenli , Liu Ying , Li Shan , Liu Yuan , Zen Ke
Published: January 2023

Myeloid-derived suppressor cells (MDSC), a heterogeneous population of immature myeloid cells, have a strong ability to suppress T cell proliferation and function. However, so far there is no universal surface marker of MDSC for mice and humans. Here we report that B7-H4 is expressed in monocytic myeloid-derived suppressor cells (M-MDSCs, CD11b+Ly-6G−Ly-6Chigh) but not granulocytic myeloid-derived suppressor cells (G-MDSCs, CD11b+Ly-6GhighLy-6Cmid). Both M-MDSCs and G-MDSCs are isolated from tumor-bearing mice. Cell sorting of B7-H4-positive and B7-H4-negative cells from the M-MDSC fraction isolated from tumor-bearing mice shows that B7-H4-positive M-MDSCs have significantly stronger T cell suppressive function than B7-H4-negative M-MDSCs independent of the expression level of iNOS and Arginase 1. Consistent with this, M-MDSCs from B7-H4-deficiency mice display a reduced immunosuppressive function compared to M-MDSCs from wild-type mice. Furthermore, by inducing B7-H4-positive M-MDSCs from mouse bone marrow-derived MDSCs or from human peripheral blood mononuclear cells, glucocorticoid treatment significantly suppresses T cell proliferation and function. In summary, our findings identify B7-H4 as a novel functional surface marker for both mouse and human M-MDSCs.
Reelin promotes the adhesion and drug resistance of multiple myeloma cells via integrin β1 signaling and STAT3 activation
Authors: Ge Qing , Lin Liang , Lu Jin
Published: January 2023

Reelin is an extracellular matrix (ECM) protein that is essential for neuron migration and positioning. The expression of reelin in multiple myeloma (MM) cells and its association with cell adhesion and survival were investigated. Overexpression, siRNA knockdown, and the addition of recombinant protein of reelin were used to examine the function of reelin in MM cells. Clinically, high expression of reelin was negatively associated with progression-free survival and overall survival. Functionally, reelin promoted the adhesion of MM cells to fibronectin via activation of α5β1 integrin. The resulting phosphorylation of Focal Adhesion Kinase (FAK) led to the activation of Src/Syk/STAT3 and Akt, crucial signaling molecules involved in enhancing cell adhesion and protecting cells from drug-induced cell apoptosis. Reelin-induced phosphorylation of STAT3 and Akt could be suppressed by inhibitors against Src and Syk. The myeloma cell survival promoted by reelin could be inhibited by STAT3-specific siRNAs. Together, these findings indicate reelin’s important role in the activation of integrin β1 and STAT3/Akt pathways in multiple myeloma and highlight the therapeutic potential of targeting reelin/integrin/FAK axis.
Cancer cell-derived transforming growth factor (TGF)-β and myeloid cell-derived interleukin (IL)-1β synergize to promote type 2 inflammation and breast cancer progression
Authors: Wu Te-Chia , Xu Kangling , Banchereau Jacques , Palucka Anna Karolina
Published: January 2023

Human breast cancer displays type 2 inflammation that promotes cancer progression and can be linked with interleukin (IL)-13 signature. The underlying molecular mechanisms include cancer-derived thymic stromal lymphopoietin (TSLP). Here, we found that IL-13 correlates with high levels of IL-1β in primary breast cancers. IL-1β levels correlate with disease stage indicating association with cancer invasiveness. IL-1β induces TSLP promoter activation, enhanced transcription and TSLP protein production from breast cancer cell lines. Administration of IL-1 receptor (IL-1R) antagonist Anakinra prevents breast cancer progression in a humanized mouse model of breast cancer. Conversely, breast cancer cells induce IL-1β transcription and secretion in dendritic cells (DCs) and monocytes. This is mediated by cancer cell-derived transforming growth factor (TGF)-β. TGF-β activated kinase 1 (TAK1) is involved in caspase-1 activation and subsequent IL-1β production. Administration of TGF-β neutralizing antibody prevents breast cancer progression in vivo and this is associated with a blockade of IL-13 and IL-4 production in the tumor. Thus, TGF-β - IL-1β cross-talk drives type 2 inflammation and contributes to breast cancer progression.
BET bromodomain inhibition synergizes with immune checkpoint blockade to facilitate anti-tumor response in a murine model of non-small cell lung cancer harboring activating KRAS mutation
Authors: Adeegbe Dennis O , Freeman Gordon J , Wong Kwok-Kin
Published: January 2023

KRAS mutations accounts for about 30% of human non-small cell lung cancer (NSCLC) and represent one of key oncogenic drivers of lung cancer. While recent advances in targeted therapy have shown promise, effective therapy for lung cancer still remains a challenge. Given the demonstrated efficacy of BET bromodomain inhibitors in treatment of hematologic malignancies and the emerging clinical benefit of immunotherapeutic agents such as anti-PD1 antibody, we sought to determine whether a combinatorial approach involving these agents could synergize to promote anti-tumor response in genetically engineered mouse model of NSCLC harboring activating kras mutation. Upon administration of the BET bromodomain inhibitor JQ1, and/or α-PD-1 antibody, we evaluated by FACS, the dynamics of tumor-associated immune cells. Notably, treatment with JQ1 alone or in combination with α-PD1 resulted in increased CD8:Treg ratio in the tumors, likely due to the reduced proportions of Treg cells. Interestingly, while JQ1 treatment alone led to remarkable down-regulation of PD-1, α-PD1 treatment resulted in augmented T cell activation evidenced by increased expression of CD69 on tumor-CD8+ T cells. When tested ex-vivo, T cells isolated from tumors of double agent therapy showed the most enhanced ability to secrete IFN-γ relative to either agent alone. Lastly, in survival studies, the combination of both drugs was superior in reducing tumor burden and prolonged the survival of tumor-bearing mice demonstrating a synergistic effect between the two agents. Collectively, our findings highlight the therapeutic potential of combining BET bromodomain inhibition with immune checkpoint blockade for treatment of solid malignancies such as lung adenocarcinomas.
Cancer Immunosurveillance by Tissue-resident Innate Lymphoid Cells and Innate-like T Cells
Authors: Dadi Saida , Chhangawala Sagar , Whitlock Benjamin M , Franklin Ruth A , Luo Chong , Oh Soyoung A , Toure Ahmed , Pritykin Yuri , Huse Morgan , Leslie Christina S , Li Ming O
Published: January 2023

Malignancy can be suppressed by the immune system in a process termed immunosurveillance. However, to what extent immunosurveillance occurs in spontaneous cancers and the composition of participating cell types remain obscure. Here we show that cell transformation triggers a tissue-resident lymphocyte response in oncogene-induced murine cancer models. Non-circulating cytotoxic lymphocytes, derived from innate, TCRαβ and TCRγδ lineages, expand in early tumors. Characterized by high expression of NK1.1, CD49a and CD103, these cells share a gene expression signature distinct from those of conventional NK cells, T cells and invariant NKT cells. Generation of these lymphocytes is dependent on the cytokine IL-15, but not the transcription factor Nfil3 that is required for the differentiation of tumor-infiltrating NK cells, and IL-15, but not Nfil3, deficiency results in accelerated tumor growth. These findings reveal a tumor-elicited immunosurveillance mechanism that engages unconventional type 1-like innate lymphoid cells and type 1 innate-like T cells.
Polyguanine-conjugated antigens target scavenger receptors in dendritic cells and induce antitumor immunity against melanoma
Authors: Ciccotelli Jo Erika Narciso , Erdos Geza , Falo Louis D
Published: January 2023

We have developed a vaccine design involving the conjugation of polyguanine (Poly(dG)) to protein or peptide antigens for the specific targeting of scavenger receptors in dendritic cells with intrinsic adjuvant capabilities. We have shown that Poly(dG) conjugation to ovalbumin generates stable particle conjugates (Poly(dG)-OVA) that induce greater T cell (helper, cytotoxic, and memory) and antibody responses. In this study, we show that Poly(dG)-OVA treatment of DCs in vitro induces greater antigen internalization and expression of activation markers CD86, MHC II, and CCR7. In vivo, intradermal injection of Poly(dG)-OVA resulted in the formation of an antigen depot in the skin lasting for several days and an increase in recruited immune infiltrates. Increased association of Poly(dG)-OVA is observed with MHC II- and CD11c-expressing cells in the skin compared to OVA. This is accompanied by the expression of pro-inflammatory CCL2 and IL-6 in the skin. Poly(dG)-OVA was also able to internalize more in skin migrating DCs and was transported more efficiently to draining lymph nodes. Poly(dG)-OVA immunization of B16-OVA-bearing mice contributed to delayed tumor progression and improved survival. We also developed Poly(dG)-Mgp10025–33 and Poly(dG)-TRP2180–188 to study melanoma-specific responses. Immunization with the these conjugates led to significant peptide-specific in vivo cytotoxic T cell responses compared to unconjugated peptides. Mice with B16 tumors immunized with combined Poly(dG)-Mgp100 Poly(dG)-TRP2 had delayed tumor progression and improved survival. Our results show that Poly(dG) conjugation of antigens is an effective strategy for the development of antitumor vaccines with improved immune responses.
Mechanisms of activity of the combination TLR4 agonist and emulsion adjuvant GLA-SE
Authors: Orr Mark T , Desbien Anthony L , Cauwelaert Natasha D , Reed Steven G
Published: January 2023

GLA-SE is a safe and potent clinical stage vaccine adjuvant consisting of the synthetic TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a squalene-in-water emulsion (SE). GLA-SE augments both TH1 and IgG2-skewed antibody responses to a variety of vaccine antigens. The adjuvanticity of GLA is critically dependent on its formulation in SE. Individually GLA promotes IgG2 responses with little detectable TH1 induction, whereas SE promotes IgG1 and TH2 responsiveness. The combination of GLA-SE mediates TH1 induction via MyD88 and TRIF signaling and activation of the inflammasome to produce IL-18. The TH1 induction activity also depends on an early burst of type I and II interferons, the latter of which is made by memory CD8 T cells, NK cells, and neutrophils.

In addition to TH1 responses GLA and SE synergize to promote the numbers of antigen-specific B cells and TFH cells quickly after immunization. This is reflected in a substantial increase in the IgG2-skewed antibody response. Promotion of B cell and TFH responses depends on IL-18 signaling and the presence of lymph node resident macrophages that take up the adjuvant. Disruption of either of these axes is sufficient to ablate the antigen-specific B cell and antibody responses. Surprisingly the generation of TFH cells did not depend on lymph node resident macrophages, whereas TH1 responses were dramatically reduced under these conditions. We identify GLA-SE as an adjuvant capable of inducing robust B cell and antibody response that operates through interaction with LN macrophages and dependent on IL-18 signaling but independent of the number of antigen-specific TFH cells.
Cutaneous vaccination with TLR2 agonists results in the suppression of Th1 responses
Authors: Johndrow Christopher T , Goldberg Michael F , Johnson Alison J , Porcelli Steven A
Published: January 2023

Novel effective vaccines are key to solving many global health problems. Past efforts to rationally design vaccines that elicit strong cell-mediated responses have largely failed. Vaccine adjuvants, particularly toll-like receptor (TLR) agonists, are a promising tool to shape immune responses by polarizing CD4 T cells to distinct fates. Use of these adjuvants to design novel vaccines requires the full characterization of their effects on CD4 T cell differentiation, particularly in regard to the route of vaccination. In a large scale in vitro screen of vaccine adjuvants, we demonstrated that Pam3CSK4, a synthetic mimic of bacterial lipoproteins with TLR1/2 agonist activity, strongly polarized CD4 T cells to the Th1 fate (IFN-γ+, T-bet+) supporting cell-mediated responses. We adapted a vaccination system to study CD4 T cell polarization in vivo using ovalbumin-specific transgenic CD4 T cells. This in vivo system showed a route-specific effect, with strong Th1 responses from intravenous immunization with ovalbumin plus Pam3CSK4, but a lack of Th1 responses with immunization through more clinically relevant cutaneous routes. Interestingly, Pam3CSK4 in combination with a known Th1 polarizing adjuvant, CpG ODN, showed little to no Th1 response suggesting a dominant suppressive mechanism. Furthermore, suppression of Th1 differentiation occurred in response to both TLR1/2 and TLR6/2 agonists. Using a variety of cell-ablative mouse models we were able to reverse suppression of Th1 responses and identify specific candidates of innate immune cells mediating suppression during vaccination with TLR2 agonists. Current work focuses on mechanisms of suppression by TLR2 agonists in the skin and relevance to improving vaccine design.
Performance of Keyhole Limpet Hemocyanin (KLH) as an Antigen Carrier for Protein Antigens Depends on KLH Property and Conjugation Route
Authors: Bi Shuguang , Bailey William , Brisson Catherine
Published: January 2023

KLH naturally exists either as high molecular weight (HMW) cylinders composed of 20 copies of subunits, or as separate subunits (Subunit). The molecular weight of KLH Subunit is about 400kDa. Owing mainly to its large size, and the abundant and complex carbohydrate structures on its peptide chains, KLH has been widely used for decades as an immune stimulant, antigen carrier, and vaccine component. But, how to characterize the properties of KLH as an antigen carrier through functional assays remains to be determined. At Stellar, we conjugated KLH of different formats and sources with ovalbumin (OVA) and fluorescence tag-conjugated OVA through different conjugation routes. Dendritic cells (DCs) derived from mouse bone marrow mononuclear cells and mouse DC line JAWSII were pulsed with these conjugates. By utilizing DC uptake assays and measuring the fluorescence intensity of DC lysate supernatant, the amount of KLH- OVA-fluorescence conjugates uptaken by DCs was quantitatively determined. With flow cytometry, both the DC maturation markers, and the amount of ovalbumin epitopes that are presented by MHC II molecules of DCs, were measured. To further compare the property of KLH-protein conjugates as potential vaccines, we vaccinated mice with OVA. OVA-specific memory T cells extracted from the vaccinated mice were stimulated with DCs that had been pulsed with KLH-OVA conjugates. T cell proliferation and cytokine release from these stimulated T cells were quantitatively measured and compared. To summarize, with the functional assays we designed, we found that the performance of KLH as a carrier for protein antigens depends on KLH property and conjugation route.
Development of a tri-agonist compound library used to determine optimal adjuvanticity of a Q fever vaccine
Authors: Tom Janine , Albin Tyler , Burkhardt Amanda M , Felgner Philip , Esser-Kahn Aaron
Published: January 2023

Vaccines are one of the most successful treatments for disease. However, the development of more effective vaccines is still necessary for diseases, such as malaria and HIV. Recently, researchers have demonstrated that administering a target antigen with multiple immune agonists, especially Toll-like receptor (TLR) agonists, can enhance specific immune responses. Thus, suggesting that vaccines will require synergistic activity of multiple agonists to effectively activate the immune system and eliminate disease. In addition, vaccine development would greatly benefit from understanding the effect distinct combinations of immune agonists have on immune activity. This information would allow us to more rationally design vaccines that elicit directed and prolonged immune responses. Previously, we probed immune system responses by conjugating three TLR agonists together. Testing our construct in vitro and in vivo, we were able to modulate innate immune and antibody responses, suggesting downstream changes in immune signaling and adaptive immune activation. Here, we synthesized a library of tri-agonist compounds and examined how different covalently linked TLR agonist combinations affected immune activation via NF-kB activity and cytokine production. We are currently testing our constructs on a Q fever vaccination model, where Q fever is a current bioterrorism threat, to examine changes in T and B cell populations, antibody responses, and efficacy of the tri-agonist adjuvants. From our studies, these tri-agonist scaffolds can inform us about immune system signaling and activation in order to study immune activation mechanisms. Therefore, allowing us to potentially design more effective vaccines.
Understanding the cellular impact of low dose rapamycin treatment.
Authors: Johnson Jenny Lynn , Thomas Paul Glyndwr , McGargill Maureen Ann
Published: January 2023

The need for improved influenza vaccines is highlighted every year through the infection of vaccinated patients and the emergence of new pandemic strains. Recent work from our lab showed that a low dose of rapamycin given during influenza vaccination significantly improved protection against subsequent infection with multiple strains. These data indicate that dampening aspects of the immune response paradoxically enhances immunity to multiple subtypes of influenza virus, which is the goal of a “universal” influenza vaccine. However, it is still unclear how the low dose of rapamycin enhances immunity to different influenza subtypes and what particular cellular population is important for this increased protection. In order to better understand how to generate the optimal immune response against multiple influenza subtypes, we are investigating how this low dose of rapamycin impacts B cell signaling. Interestingly, we found that a low dose of rapamycin blocks B cell class switching without having an impact on proliferation. Thus, we are investigating which components of the mTOR pathway are required for class switching, but not proliferation. Early work indicates that this dose of rapapmycin alters the speed and activation potential of several mTOR signaling components. Using this system, we hope to identify the signaling components and kinetics that generate the optimal immune response to multiple subtypes of influenza, which is important for generating a universal influenza vaccine.
Induction of Innate Immune Factor Expression in A549 Lung Alveolar Type II Cells by Cytokines Interferon-g and Tumor Necrosis Factor-α
Authors: Gutierrez Lucas , Yoon Alexander , Faull Kym Francis , Porter Edith
Published: January 2023

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a detrimental infectious disease that is estimated to infect one third of all people worldwide. The lack of TB control is in part due to the lack of protection of adults from the existing TB vaccine as well as an urgent need for novel vaccine design. Vaccines rely in part on interferon-γ (IFNγ), a cytokine secreted by T helper (TH) cells, which has been shown to be an important component of the adaptive host defense against Mtb. Tumor Necrosis Factor-α (TNFα) is also secreted by TH cells and has demonstrated protective effects on epithelial cells by increasing secretion of proinflammatory cytokines, chemokines such as IL-8, and antimicrobial effector molecules. We hypothesize that IFNγ and TNFα also affect expression of antimicrobial lipids including cholesteryl esters. A549 Alveolar Type II cells were stimulated with IFNγ and TNFα for 24 h. Gene expression of the IL-8, and sterol O-acyltransferase-1 (SOAT-1), a key enzyme for cholesteryl ester synthesis, was quantified by RT-PCR and SYBR green technology using ribosomal protein, large, P0 (RPLP0) as a reference gene. Cholesteryl esters in lipid extracts from culture supernatants were quantified by mass spectral analysis with multiple reaction monitoring. Our results showed a statistically significant upregulation of IL-8 and SOAT-1 gene expression in response to INFg (5 ng/mL) and for SOAT-1 to TNFa (10 ng/mL) (p < 0.05 in OneWay ANOVA), which was accompanied by increased cholesteryl ester content. Augmentation of epithelial antimicrobial lipid production by cytokines from resident TH cells may be incorporated in a novel vaccine design against Mtb.
Immunization with synthetic LbL microparticle vaccine administered using a microneedle patch elicits humoral and cellular immune responses and protects mice from challenge with respiratory syncytial virus
Authors: Powell Thomas Jeff , Mistillis Matt , Palath Naveen , Tang Jie , Jacobs Andrea , Cardenas Edwin , Boyd James G. , Prausnitz Mark
Published: January 2023

Synthetic vaccines made by layer-by-layer fabrication of microparticles (LbL-MP represent a novel method of delivering peptide antigens to elicit protective immune responses in the absence of complex adjuvant and without concomitant inflammation. We incorporated LbL-MP into a microneedle patch that delivers the vaccine directly to the immunologically rich environment of the skin in a minimally-invasive manner. BALB/c mice immunized with microneedle patches loaded with LbL-MP bearing two conserved epitopes of respiratory syncytial virus (RSV) generated humoral and cellular immune responses and were protected from virus challenge. The vaccine contained a conserved CX3C chemokine mimic epitope from the RSV attachment (G) protein that contributes to Th2-skewed inflammatory responses. Patch-immunized mice developed RSV-G-specific antibody titers comparable to the titers induced by direct injection of the vaccine into the footpad. The mice developed T-cell responses, including effector CTL, against a CD8 epitope of the RSV-M2 protein included in the vaccine. While footpad injection elicited both Th2 (IL-5) and Th1 (IFNγ) responses, microneedle patch immunization elicited a dominant Th1 response with very low Th2 response. A similar pattern of Th1>Th2 was seen in C57BL/6 mice immunized with a patch loaded with LbL-MP containing epitopes of the P. falciparum circumsporozoite protein, suggesting that the Th1 dominance was not antigen- or strain-specific, but rather an outcome of LbL-MP microneedle patch immunization. These studies demonstrate that LbL-MP retain their integrity and potency when delivered via microneedle patch, and favor Th1 immune responses that may be more effective against acute infection.