bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription factor Nrf2 (NF-E2 Related Factor 2) enhances myeloid-derived suppressor cell (MDSC) accumulation and tumor progression (46.13)
Authors: Beury Daniel , Nelson Cassandra , Ostrand-Rosenberg Suzanne
Published: January 2023

Previous studies have shown that MDSC block adaptive anti-tumor immunity by producing high levels of oxidizing agents such as reactive oxygen species (ROS), nitric oxide (NO), and peroxynitrite. Despite high levels of the toxic oxidizing agents, MDSC survive and suppress anti-tumor immunity. We hypothesize that MDSC survival is mediated by the antioxidant-regulating transcription factor Nrf2. To test this hypothesis, BALB/c wild type and Nrf2-/- mice were injected with 4T1 mammary carcinoma cells and assayed weekly for percentage of MDSC in the blood and for MDSC levels of ROS and glutathione. Mice were also followed for survival. Nrf2-/- MDSC had more ROS and less glutathione than wild type MDSC, indicating that Nrf2-/- MDSC were more oxidatively stressed. Increased oxidative stress is likely to lead to increased apoptosis of MDSC and reduced immune suppression. Tumor-bearing Nrf2-/- and wild type mice accumulated MDSC in the blood with increasing tumor-burden. However, tumor-bearing Nrf2-/- mice had less MDSC than wild type mice for a given sized primary tumor. Therefore, MDSC either accumulate more slowly or they apoptose more quickly in Nrf2-/- vs. wild type mice. Tumor-bearing Nrf2-/- mice lived longer than wild type mice demonstrating that Nrf2 contributes to tumor progression; consistent with our hypothesis that Nrf2 regulates MDSC survival, resulting in fewer MDSC in Nrf2-/- mice, thereby increasing anti-tumor immunity against metastatic disease.
Hypoxia can suppress T cell activation in a mechanism independent of A2A adenosine receptor (46.27)
Authors: Madasu Manasa Mahathi , Subramanian Meenakshi , Kini Radhika , Ohta Akiko , Sitkovsky Michail , Ohta Akio
Published: January 2023

Tissue hypoxia plays a key role in the establishment of immunosuppressive environment in vivo, most notably in tumors. Hypoxia promotes local increase of extracellular adenosine, and interaction of adenosine with A2A adenosine receptor (A2AR) strongly inhibits immune activation. The adenosine-A2AR pathway may represent a mechanism of the physiological immunoregulation initiated by hypoxia. However, in this study, we found that hypoxia can also exert its T cell inhibitory effects even in the absence of A2AR. When mouse T cells were activated by anti-CD3 mAb, hypoxia (1% oxygen) strongly impaired T cell activation. Hypoxia showed the same degree of inhibition against wild-type and A2AR-deficient T cells suggesting that hypoxia could inhibit T cells in an A2AR-independent mechanism. In comparison to A2AR stimulation, this A2AR-independent pathway strongly inhibited cell proliferation and cytotoxicity, while IFN-γ-producing activity was more susceptible to A2AR stimulation. In contrast to sustained functional impairment in T cells after A2AR stimulation, T cells activated under hypoxia immediately recovered from the immunosuppressed state and produced high levels of IFN-γ upon restimulation. Although previous in vivo studies have shown the A2AR-dependent immunosuppression by hypoxia, the current study suggests the involvement of multiple different mechanisms in T cell inhibition by hypoxia.
Chemotherapeutic drugs trigger rapid apoptotic induction and caspase-1/ inflammasome signaling in murine macrophages (46.33)
Authors: Antonopoulos Christina , Dubyak George
Published: January 2023

The success of certain cancer chemotherapy drugs relies critically on the host immune system. In some cancer models, induction of NLRP3/caspase-1/IL-1β signaling cascades in dendritic cells is required for effective anti-tumor immunity. Recent studies by Sauter et al. (Cancer Biol Ther 2010) reported (and we have confirmed) that the chemotherapeutic anthracycline, doxorubicin (DOX), induces NLRP3 inflammasome-dependent IL-1β secretion from murine macrophages. We have also tested several other chemotherapy drugs for their ability to induce IL-1β secretion and/or apoptotic induction in LPS-primed murine bone marrow-derived macrophages (BMDM). Staurosporine (ST), a pro-apoptotic kinase inhibitor, was identified as another rapid inducer of caspase-1 activation and IL-1β release; ST initially (over the first 3 h) activates a K+ efflux-independent casp-1 activation but secondarily engages a K+ efflux-dependent pathway at later (>3h) treatment times. Both DOX and ST also induce rapid (within 1-4 h) accumulation of casp-3/7 activity which coincides with, or precedes, the casp-1 activation and IL-1β secretion responses. Ongoing studies are testing whether: 1) casp-3/7-mediated cleavage of the pannexin-1 channel contributes to the K+ efflux required for NLRP3 inflammasome activation by DOX; and 2) NLRP3-independent inflammasomes mediate the initial K+ efflux-independent IL-1β release elicited by ST.
The expression of membrane-anchored GM-CSF reduces the tumorigenicity of murine breast cancer cells and correlates with a reduction in MDSCs in vivo (46.36)
Authors: Bozeman Erica , Shashidharamurthy Rangaiah , Selvaraj Periasamy
Published: January 2023

Currently under preclinical and clinical investigation are several cancer vaccines using whole tumor cells modified to secrete GM-CSF. However, a potential drawback of such vaccines is the risk of systemic toxicity. To address this concern, we generated a GPI-anchored form of mouse GM-CSF (GPI-mGM-CSF) to be stably expressed on the surface of tumor cells. To determine the immunogenicity of breast cancer cells expressing the GPI-mGM-CSF, we used the TUBO cell line, a highly tumorigenic, non-metastatic tumor cell line that constitutively expresses the HER-2/neu oncogene which is overexpressed in 30% of human breast cancer patients. BALB/c mice were challenged with 1e5 TUBO-WT or TUBO-mGM-CSF cells. At the conclusion of the experiment, mice were sacrificed, spleens were harvested and splenocytes were isolated for flow cytometry analysis following RBC lysis. Splenocytes were stained for the prevalence of immune suppressive cells including myeloid derived suppressor cells (MDSCs). We observed minimal, transient growth of tumors in mice challenged with TUBO-mGM-CSF compared to TUBO-WT. Additionally, we obsesrved a significant reduction in spleen weight and prevalence of CD11b+Gr-1+ MDSCs in these mice. These studies suggest that the addition of a membrane anchored form of GM-CSF to tumor cells can induce an antitumor immune response and inhibit the development of immune suppressive cells such as MDSCs.
Efficacy and mechanism-of-action of a novel superagonist IL-15:IL-15Rα/Fc fusion complex in murine multiple myeloma syngeneic mouse models (46.44)
Authors: Wong Hing , Xu Wenxin , Zhu Xiaoyun , Liu Bai , Edward Anna , Kong Lin , Jeng Emily , Hang Kaiping , Marcus Warren , Rhode Peter
Published: January 2023

ALT-803 is a fusion protein complex consisting of an interleukin-15 superagonist mutein (IL-15N72D) and a dimeric IL-15 receptor alpha (IL-15Rα)/Fc fusion protein. Previous studies have shown that IL-15:IL-15Rα complexes exhibit potent activity against murine melanoma in immunocompetent mice. In this study, we show that ALT-803 treatment significantly prolongs survival of C57BL/6 mice bearing syngeneic murine 5T33P myeloma. ALT-803-treated mice were also protected from paralysis and mortality due to subsequent rechallenge with 5T33P tumor cells. Consistent with these results, a single dose of ALT-803 was capable of significantly reducing the percentage of well-established 5T33P cells in the bone marrow of tumor-bearing mice shortly after ALT-803 infusion and promoted a substantial increase of CD8+ T cells and NK cells in peripheral blood and bone marrow. ALT-803 treatment also increased serum IFN-γ levels but did not affect the level of blood Tregs. Immune cell depletion studies suggest that activated effector and memory CD8+ T cells may play a vital role in the anti-tumor activity of ALT-803 against 5T33P myeloma cells. Similar potent anti-tumor effects of ALT-803 were also observed in BALB/c mice bearing MOPC-315 myeloma cells. The results of these studies support clinical development of ALT-803 as a novel immunotherapeutic agent against multiple myeloma.
Soluble CD80 restores T cell activation and overcomes tumor cell Programmed Death Ligand-1-mediated suppression (46.8)
Authors: Haile Samuel , Dalal Sonia , Clements Virginia , Tamada Koji , Ostrand-Rosenberg Suzanne
Published: January 2023

One of the mechanisms tumor cells employ to escape anti-tumor immunity is expression of Programmed Death Ligand 1 (PD-L1or B7-H1). PD-L1 protects tumor cells by binding to its receptor Programmed Death 1 (PD-1) on activated T cells, thereby causing T cell apoptosis. We previously reported that transfection of tumor cells to express the costimulatory molecule CD80 prevents PD-1 binding and restores T cell activation, consistent with the concept that CD80 binds to PD-L1 and inhibits PD-L1/PD-1 interaction. Since it is not therapeutically feasible to transfect tumor cells with CD80, we have tested a soluble form of CD80, consisting of the extracellular domains of the molecule, for its ability to overcome PD-L1-mediated suppression. Treatment of human lung adenocarcinoma, mammary carcinoma, and cutaneous melanoma cells with soluble CD80 prevents PD-L1-mediated suppression and restores T cell activation. Because on-going clinical trials have shown that mAbs to PD-1 facilitate tumor rejection, we have compared the efficacy of soluble CD80 vs. mAbs to PD-1 or PD-L1. Soluble CD80 treatment is more effective in restoring T cell activation than treatment with mAbs to either PD-1or PD-L1. Therefore, soluble CD80 may be a therapeutic agent for facilitating tumor-specific immunity by overcoming PD-L1-induced immune suppression, and is potentially more effective than mAbs to either PD-1 or PD-L1.
Functional abnormality of peripheral CD8 T cells in β5t-deficient mice (47.18)
Authors: Takada Kensuke , Takahama Yousuke
Published: January 2023

Proteasomes play an important role in the production of peptides presented by MHC class I. The thymoproteasome, which contains β5t catalytic subunit, is expressed specifically in cortical thymic epithelial cells. In β5t-deficient mice, the cellularity of CD8SP thymocytes and peripheral CD8 T cells is significantly decreased and their TCR repertoire is altered as characterized by defective responses to alloantigens and viral infections. In the present study, we functionally characterized CD8 T cells generated in β5t-deficient mice. We found that CD8 T cells in the secondary lymphoid organs of β5t-deficient mice were enriched with CD44hiCD122+ cells, which resemble memory T cells. This phenotypic alteration was not detectable in the thymus and was observed in the periphery of mice older than 1 week after birth, suggesting that CD8 T cells generated in the thymus of β5t-deficient mice are aberrantly maintained in the periphery. Enrichment of memory-like CD8 T cells was also seen in β5t-/-Rag-/-OT-I mice. In addition, CD44loCD122- cells obtained from β5t-/-Rag-/-OT-I mice showed enhanced proliferative responses to antigen stimulations. These results suggest that the thymoproteasome-dependent positive selection affects not only TCR repertoire but also functions of CD8 T cells, including the antigen reactivity and the homeostatic capability.
The use of IKK inhibitor as a lymphocyte depleting agent (47.4)
Authors: Lee Chih-Yuan , Lai Ting-Yu , Yeh Tzu-Wen , Chou Chih-Chung , Tsai Meng-Kun , Hsu Li-Chung
Published: January 2023

Purposes: Transcription factor NF-κB and its activating kinase IκB Kinase β (IKKβ) are critical for inflammation and innate immunity. In our previoius report, MLN120B, an IKKβ inhibitor, would cause neutrophilia in mice but their peripheral blood lymphocyte numbers were not affected. We tried other IKK inhibitors; however, we found that feeding with BMS-345541, a highly selective IKKβ inhibitor, would cause lymphopenia in mice. We explored the possibility of BMS-345541 as a therapeutic agent in depleting lymphocytes. Results: To characterize how BMS-345541 affects lymphocyte homeostatsis, C57BL/6 mice received vehicle, or BMS-345541 oral gavage once daily at different dose (10, 25, 50, and 100 mg/kg body weight in 3% Tween 80), and mice were sacrificed on day 3 or day 5. We collected the peripheral blood for blood cell counts and also collected spleen and lymphnodes for histology examination. The lymphocytes, but not neutrophils, were significantly decreased in mice received BMS-345541. Our preliminary results showed that the effect of BMS-345541 on lymphocytes were through apoptosis rather than trapping the lymphocytes in lymphoid organs. BMS-345541 affected both T cells and B cells. Conclusion: We conclude that oral administration of BMS-345541 would reduce lymphocyte counts and could be helpful in depleting lymphocytes in circulation, which would be useful as a therapeutic agent for lymphoproliferative disorder.
Integrin alpha-7+ pericytes are a novel, immunologically relevant lymph node stromal subset found in mice and humans (47.6)
Authors: Fletcher Anne , Malhotra Deepali , Lukacs-Kornek Veronika , Elpek Kutlu , Turley Shannon
Published: January 2023

Within lymph nodes, non-hematopoietic stromal cells attract, arrange, and promote the survival of naïve T cells, while deleting self-reactive T cells in a non-redundant fashion. Despite these important functions, subsets are poorly characterized. One major subset (gp38-CD31- double negative, termed DN) comprises 10-15% of stroma, with transcript for relevant peripheral tissue-restricted antigens (PTAs) and Aire, yet nothing was known of their lineage, localization, phenotype, or capacity to interact with leukocytes. We addressed this through in-depth transcriptomic, protein and functional analyses of DN stroma, in comparison to 5 other stromal subsets. DNs express CCL19, CCL21, CXCL13, CXCL9, CXCL10 and other chemokines, with high levels of Flt3L, BAFF, CSF-1, TGFb family members and lymphangiogenic growth factors. Phylogenetic analysis showed DNs were similar to but distinct from fibroblastic reticular cells, with a unique integrin profile, higher contractility in culture, and a lack of IL-7 expression. In situ, we found a unique surface protein, integrin α7, which identified DNs as lymph node pericytes in the cortex and medulla. Strikingly, DNs strongly alter PTA transcription following inflammation without increasing surface expression of MHC molecules. We subsequently identified DN cells in human lymph nodes. These data identify DNs as a unique, immunologically relevant subset of fibroblastic, contractile pericytes, which we term integrin α7+ pericytes (IAPs).
MicroRNAs and their role in the generation of myeloid derived suppressor cells (MDSC) by cannabidiol in vivo (48.16)
Authors: Hegde Venkatesh , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

The major non-psychotropic cannabinoid cannabidiol (CBD) induces G-CSF, CXCL1 and massive accumulation of functional CD11b+Gr-1+ MDSC in the peritonea of naïve mice. Role of miRNA in the regulation of MDSC function is unclear. Microarray analysis for 609 mouse miRNA using total RNA from purified cells revealed unique profile and 59 differentially expressed (>2 fold) miRNA in CBD-MDSC as compared to BM CD11b+Gr-1+ cells from control mice. MicroRNA signature of CBD-MDSC closely resembled with that of tumor-MDSC purified from EL4-bearing mice. CBD-MDSC showed significantly lower levels of miR-706 and miR-455 compared to BM controls as validated by qPCR. Target prediction using MicroCosm algorithm suggested that miR-706 and miR-455 bind to 3’ UTR of Stat1 and Stat6 mRNA respectively, which in turn have been shown to positively regulate arginase 1 (ARG1) crucial for MDSC immunosuppressive function. CBD-MDSC showed higher relative expression of Stat1 and Stat6 mRNA transcripts in qPCR as well as higher Stat1/ pStat1, Stat6 and ARG1 protein levels when compared to BM controls. Further, CBD-MDSC transfected with miR-706 mimic had decreased Stat1/ pStat1 and ARG1 protein levels whereas miR-455 mimic did not alter Stat6/ pStat6 or ARG1. In summary, induction of MDSC from BM precursors in response to CBD in vivo is associated with significant changes in global miRNA expression and miR-706 plays a role in the regulation of ARG1 and hence the functional MDSC phenotype by targeting Stat1.
ROLE OF ACCESSORY CELLS IN THE IVIG-MEDIATED INHIBITION OF T CELL ACTIVATION (48.5)
Authors: Padet Lauriane , Bazin Renée
Published: January 2023

Intravenous immunoglobulin (IVIg) is a therapeutic preparation of human IgG isolated from thousands of plasma and is currently used for the treatment of an increasing number of immune disorders. A decreased T cell activation was reported in some patients treated with IVIg. To understand the mechanisms responsible for this anti-inflammatory effect, several groups of investigators have performed in vitro assays in which PBMC were activated with immobilized anti-CD3. The main conclusion derived from these works is that IVIg inhibited the functions of activated T cell. However, direct effect of IVIg on activated T cells has been questioned recently. In the present study, we re-examined the mechanisms by which IVIg interferes with the functions of T cells activated with immobilized anti-CD3. First, the level of T cell activation after anti-CD3 stimulation of PBMC, purified T cells or PBMC depleted from monocytes was measured. Our results showed that in the absence of monocytes, T cells did not proliferate in response to anti-CD3 stimulation, suggesting that monocytes are essential to lead to full T cell activation. Second, we evaluated the effect of IVIg on T cell activation after anti-CD3 stimulation of PBMC. Our results showed that T cells were not activated in the presence of IVIg suggesting that not only T cells but also monocytes could be the target of IVIg. Work is underway to better define the role of monocytes in the effects of IVIg on T cell activation.
Mouse mesenchymal stem cell-derived exosome mediates a immunosuppressive function in Th17 cells (48.8)
Authors: Lee Sunho , Kim Sueon , Park Chung-Gyu
Published: January 2023

The immunoregulatory ability of MSCs is demonstrated in their ability to alleviate autoimmune diseases. In this study, by using purified exosomes from mesenchymal stem cell (MSC), we examined the immune-regulatory effect of exomes on activated T cells. Exosomes suppressed the proliferation of T cells by apoptosis and cell cycle arrest, which was confirmed by CFSE staining and FACS analysis. To confirm exosome’s immunoregulatory effect in vivo, EAE was induced in four groups of mice that were treated exosome and exosome-enriched, exosome depleted supernatant, and saline as a control from the point when the symptoms of EAE initiated. As expected, the symptoms were significantly alleviated in exosome and exosome-enriched treated groups. In addition, the level of IL-17 expression in exosome treated group evaluated by ELISPOT and ELISA was about 2 times lower than that in control group. Exosome significantly reduced the levels of stat3 phosphorlation which has been known to activate RORγt. In conclusion, these results indicate that exosomes alleviate EAE by suppressing IL-17 secretion and stat3 phosphorylation which has been known to play a crucial role in development of EAE.
A TNF family receptor regulates epithelial innate responses and host defense against pathogenic bacterial infections of the mucosa (49.1)
Authors: Kronenberg Mitchell , Shui Jr-Wen , Larange Alexandre , Kim Gisen , Vela Jose Luis , Cheroutre Hilde
Published: January 2023

Previously, in a mouse colitis model, we demonstrated an anti-inflammatory role for the herpes virus entry mediator (HVEM), a TNF superfamily receptor, when expressed by a radiation resistant cell population (J. Exp. Med. 205: 1463, 2008). However, the molecular mechanism for HVEM function remained to be determined. We therefore explored the role of HVEM in colonic epithelial cells, a radiation resistant cell type critical for preventing colitis and enhancing host defense. During Citrobacter rodentium infection, Hvem-/- mice had impaired colonic epithelial responses, resulting in higher bacterial burdens, inflammation and increased mortality. HVEM stimulation induced epithelial responses by NIK-dependent Stat3 activation, resulting in the expression of genes important for mucosal immunity. HVEM signaling also induced IL-22R1 expression, which boosted IL-22 signaling, leading to enhanced Stat3 activation in the epithelium. While HVEM or IL-22R signaling alone induced Stat3 activation in epithelial cells in vitro, during infection in vivo, both Stat3-activating pathways were required. Likewise, in Streptococcus pneumoniae infection, HVEM was required not only for survival and bacterial clearance, but also for Stat3 activation and induction of innate responses in the lung. Collectively, our findings establish HVEM as a crucial regulator of innate defense at mucosal surfaces via directly enhancing epithelial Stat3 activation and by inducing epithelial IL-22R1 expression.
Intestinal epithelial cells-derived exosomes provide a bridge between the gut and the liver that prevents liver inflammation through prostaglandin E2 induction of NK T cell anergy (49.11)
Authors: Deng Zhongbin , Ju Songwen , Zhuang Xiaoying , Xiang Xiaoyu , Miller Donald , Zhang Huang-ge
Published: January 2023

The in vivo regulation/induction of NTK anergy has been associated with inhibition of autoimmune and anti-tumor response, but the underlying mechanisms are poorly understood. In this study, we demonstrated that exosomes released from intestinal epithelial cells migrated into liver and induced liver NKT anergy. Unexpectedly, these effects were mediated by an exosomal PGE2 , which were demonstrated in a NKT-mediated liver injury model induced by ConA. Consistent with these findings, blocking PGE2 synthesis attenuated intestinal epithelial cells exosomes mediated inhibition of induction of IFN-γ and IL-4 of α-GalCer stimulated liver NKT cells as a PGE2 receptors mediated and PKA dependent manner. Pro-inflammation condition increased permeability of intestine and thus enhanced accumulation of intestinal epithelial cells exosomes migrated into liver where PGE2 induced NKT anergy in response to subsequently α-GalCer stimulation. Thus, these findings demonstrate a previously unappreciated role for intestinal epithelial cell exosomes served as immune modulator between liver and intestine and maintaining liver NKT homeostasis. Our findings have also important implications for the development of NKT cell-based vaccines and immunotherapies.
Campylobacter jejuni induces mixed Type 1 and 17 responses in a C57BL/6 IL-10−/− mouse model of colitis. (49.12)
Authors: Malik Ankit , Mansfield Linda
Published: January 2023

Campylobacter jejuni is the leading cause of food-borne enteritis and has also been linked with peripheral neuropathy and arthritis. C57BL/6 IL-10+/+ and congenic IL-10−/− mice serve as C. jejuni colonization and colitis models respectively. We hypothesized that in the absence of regulatory cytokine IL-10, uncontrolled induction of proinflammatory cytokines and infiltration of inflammatory cells is responsible for tissue obliteration in colitis. Colon cytokine analysis revealed significant time-dependent up regulation of IFN-γ, IL-17A, IL-6, TNF-α and IL-1β. A similar panel of cytokines were upregulated in the plasma and draining lymph node cultures of infected mice. Colon flow cytometric and immunohistochemical analysis revealed time-dependent increases in numbers of infiltrating macrophages, neutrophils, T cell subsets, NK cells and Thy1+ CD3- innate lymphocytes. Intra-cellular cytokine staining of colon and lymph node lymphocytes further linked IFN-γ, IL-17 and IL-22 upregulation to specific T cell subsets and innate lymphocytes. Time - dependent up regulation in anti-C. jejuni reactive IgG2b, IgG2c and IgG3, but not IgG1 or IgM were also observed in plasma of infected mice. These data demonstrate a mixed Type 1 and Type 17 induced colitis in C. jejuni 11168 challenged C57BL/6 IL-10-/- mice. Future work is directed towards understanding the relative importance of Type 1 and 17 pathways, and innate and adaptive lymphocytes in C. jejuni induced colitis.
Immune ignorance of a commensal bacterial antigen by endogenous antigen-specific CD4+ T cells in the gut. (49.14)
Authors: Legoux Francois , Moon James
Published: January 2023

The immune system must discriminate between beneficial and harmful microbes in the gut, and failure to maintain a proper balance of immune responsiveness and immune tolerance to gut microbes can lead to intestinal pathology. In particular, inflammatory bowel diseases such as Crohn's Disease and ulcerative colitis are thought to be the result of aberrant immune responses targeting commensal bacteria normally present in the gut lumen. The early events that break steady-state tolerance and trigger an immune response directed to commensal bacterial antigens remain poorly understood. Using a peptide:MHC-II tetramer-based enrichment approach, we characterized endogenous CD4+ T cells specific for Cbir1, a flagellin antigen from commensal bacteria present in the gut of normal mice. Our data show that endogenous Cbir1-specific T cells develop in the thymus of normal mice at the double positive stage. These cells survive negative selection and populate the peripheral lymphoid organs without becoming activated. Following immunization with their cognate antigen, they undergo massive proliferation and upregulate activation markers. Despite the presence of Cbir1 in the gut lumen, Cbir1-specific T cells in the lamina propria do not differentiate into regulatory T cells, nor do they induce colitis in normal mice. These data suggest that immune ignorance, rather than regulatory T cell induction, is a major mechanism of immune tolerance to commensal bacteria flagellin in the gut.
FcRn-mediated transport of IgG can either enhance or neutralize chlamydial infection (49.17)
Authors: Beagley Ken , Armitage Charles , Kuo Timothy , Blumberg Richard , Wijburg Odilia , Timms Peter
Published: January 2023

IgG is the dominant antibody in the female and male reproductive tracts (RT). Furthermore, the neonatal Fc receptor (FcRn) is expressed by epithelial cells in both these tissues. The acidic environment (pH 4.5-6.5) in the RT is optimal for FcRn uptake of IgG by epithelial cells, which suggests that IgG could either enhance or protect against infection by pathogens such as Chlamydia, depending on antibody specificity. We show that IgG is indeed internalized by reproductive tract epithelial cells in an FcRn and pH-dependent manner. Furthermore, IgG specific for the chlamydial major outer membrane protein (MOMP), expressed predominantly on the extracellular elementary body (EB) actually enhanced infection, whilst IgG specific for an antigen expressed during intracellular chlamydial replication (IncA) partially neutralized infection. Both enhancement (IgG-MOMP) and protection (IgG-IncA) were abrogated by knockdown of FcRn expression. Chlamydiae inhibit lysosomal activity to promote infection and the presence of intracellular IncA-IgG enhanced lysosomal activity in infected cells. IgA is also found in RT secretions as is the IgA transport molecule the polyimmunoglobulin receptor (PIgR). Unlike IgG, IgA-MOMP and IgA-IncA both neutralized chlamydial infection in a PIgR-dependent manner. Thus for antibody-mediated protection against Chlamydia, IgG specific for intracellular chlamydial antigens, together with IgA specific for both stages of the replicative cycle may be optimal.
Effect of human corneal resident cells on the immune modulation of CD4 T cells (49.19)
Authors: Ryu Jin Suk , Jeong Hyun Jeong , Lee Young Eun , Kim Mee Kum , Wee Won Ryang
Published: January 2023

Fibroblasts which are terminally differentiated stromal cells have been reported to share immunosuppressive effect on the lymphocyte activation with mesenchymal stem cells. However, the immunomodulatory role of the corneal residential stromal cells(keratocytes) have not been known. We investigated the immunomodulatory effect of human keratocytes on the CD4+ T cells as a part of ocular immune privilege. The study was performed in accordance with the guidelines of the Declaration of Helsinki. Human peripheral blood mononuclear cells were obtained from the healthy voluntary donors with informed consents. CD4+ T cells were purified through MACS separator with the human CD4+ T Cell Isolation Kit II (Miltenyi Biotec). The human keratocytes were collected from the remaining donor graft. CFSE stained CD4+ T cells were stimulated with anti-CD3/CD28, and were then co-cultured with keratocytes with or without 1-methyltryptophan. The proliferation of T cells and the secretion of IFN-γ were evaluated. Changes of cytokine expression were also evaluated using ELISA after co-culture. We found that keratocytes suppressed the proliferation and the secretion of IFN-γ of CD4 T cells, and enhanced IL-10 and TGF-β, which were partially reversed with 1-methyltryptophan treatment. It implied that human keratocytes might have an immune modulatory effect on the CD4 T cells through indoleamine 2,3 dioxygenase-dependent in part, which seemed likely to contribute to ocular immune privilege.
Influenza infection generates memory CD4 T cells that occupy a specific lung resident niche and mediate early in situ responses (49.21)
Authors: Turner Damian , Bickham Kara , Thome Joseph , Yudanin Naomi , Farber Donna
Published: January 2023

Lung-resident T cells are uniquely poised to mediate early in situ responses to respiratory pathogens. We have previously identified a subset of lung-resident CD4 T cells that mediate optimal protective immunity to respiratory infection. Here, we have examined the mechanisms for the establishment of lung-resident memory CD4 T cells and their role in anti-viral responses. Using intravenous (iv) infusion of antibody to differentiate between cells in the lung interstitium and those accessible to the circulation, we discovered that a minor subset of lung αβ CD4 T and most γδ T cells are protected from iv antibody staining (protected subset) while the remaining αβ CD4 T cells are readily stained (accessible subset). The protected αβ CD4 T cell subset and γδ T cells similarly expressed increased levels of CD11a, CD69, CD103 and CCR6 as compared to the accessible subset. The protected population of αβ CD4 T cells is established through respiratory infection as it comprises less than 10% of αβ CD4 T cells in naïve animals but represents approximately 30% of lung cells in influenza immune mice during the memory stage. Once established, the protected memory αβ CD4 T cells along with γδ T cells produces IFN-γ and IL-17 in situ early after influenza rechallenge. Our results show that the lung CD4 T cell population is comprised of a circulating subset and a tissue-resident subset with the tissue resident αβ CD4 T and γδ T cells acting as early responders to respiratory infections.
Hallmarks of adaptive immunity in γδ T cells following oral Listeria infection (49.7)
Authors: Sheridan Brian , Pham Quynh-Mai , Fu Han-Hsuan , Schubert Wolf-Dieter , Freitag Nancy , Lefrancois Leo
Published: January 2023

Traditionally, γδ T cells have been thought of as an arm of the innate immune system. Our findings suggest that γδ T cells also have an important role in adaptive immunity and anamnestic responses. The ability of γδ T cells to generate bona-fide memory populations has not been well established. Surprisingly, oral Listeria monocytogenes (L.m.) infection induced a unique population of γδ T cells in the mesenteric lymph nodes and lamina propria that was maintained into immunologic memory. They were mobilized into the blood and upregulated the gut homing receptor α4β7. This adaptive γδ T cell response differs from innate γδ T cells in their function, proliferation, and Vγ usage. The responding mucosal γδ T cell subset was comprised of both IL-17A and IFNγ producers. Notably, all cytokine production was limited to γδ T cells which did not express CD27 suggesting distinct regulation of cytokine production by adaptive γδ T cells. This population expanded more rapidly and more robustly to a secondary oral L.m. infection but not to a secondary intravenous L.m. infection or a secondary oral Salmonella infection suggesting contextual specificity to the priming pathogen. Further, this population is capable of providing protection in collaboration with αβ T cells following secondary oral challenge demonstrating their physiologic significance in vivo. Thus, γδ T cells are capable of generating a bona-fide immunologic memory population following oral L.m. infection.
Effective prevention and treatment of CD8 T cell-mediated graft-versus-host-like disease (GvHD) using a JAK inhibitor. (51.3)
Authors: Okiyama Naoko , Furumoto Yasuko , Villarroel Vadim , Gutermuth Jan , Ghoreschi Kamran , Gadina Massimo , Katz Stephen
Published: January 2023

A Janus kinase (JAK) inhibitor, tofacitinib (tofa), inhibits JAK 1, 2 and 3 of some cytokine receptor pathways, affects CD4+ T cell respnses, and is being tested in more than 20 clinical trials in autoimmune diseases.To determine if tofa affects CD8+ T cells, we analyzed its effect on a CD8+ T-cell-dependent murine model of GvHD (OT-I T cell transfer into Tg mice expressing ovalbumin (OVA) in their skin and mucosae). Tofa at 50 mg/kg/day (50) p.o. completely prevented GvHD development in our model vs. vehicle-treated mice with final weights: -0.6%±5.6 and -15.4%±4.1 (p<0.01; 2-way ANOVA) and clinical scores: 0 and 7.3±0.52 (p<0.01; U test) 13 days post OT-I cell transfer. Mice treated with tofa 50 starting at day 5 were completely rescued, with no clinical signs of GVHD. FACS analysis demonstrated reduced expression of OVA-specific TCR and reduced activation of OT-I cells in skin-draining lymph nodes from mice transferred with GFP/OT-I cells and treated with tofa 50 vs. vehicle alone. Tofa inhibited proliferation of OT-I cells stimulated by SIINFEKL (OVA peptide) in vitro. qRT-PCR showed increased chemokine mRNA (CXCL9; 39-fold and CXCL10; 22-fold) in the epidermis of vehicle-treated mice vs. tofa 50-treated mice 4 days post OT-I cell transfer. The effect of tofa could be explained by its inhibition of JAK signaling in IL-2 and IL-15 receptor-pathways of OT-I cells and in IFN-γ receptor-pathways of keratinocytes and tofa may thereby be effective in autoimmune diseases.
Dietary fish oil results in a microenvironment with high anti-inflammatory potential, but does not inhibit mice from mounting an effective immune response to Borrelia burgdorferi. (51.9)
Authors: Cunningham Anna , Dumlao Darren , Wax Laura , Norris Paul , Hughes-Hanks Jennifer , Halpin Rachel , Lett Kawasi , Blaho Victoria , Mitchell William , Fritsche Kevin , Dennis Edward , Brown Charles
Published: January 2023

Dietary fish oil is suggested to alleviate a variety of inflammatory disorders by altering eicosanoid production. However, the extent of changes resulting from a diet high in omega-3 fatty acids is unknown, and any benefits resulting from these changes are unsubstantiated. Several studies conclude that the lack of phenotypic effects of fish oil result from an inability to consume sufficient quantities for any benefits to be evident. We fed mice either a control diet containing 18% by weight soybean oil or fish oil and infected the mice with Borrelia burgdorferi in order to quantify changes in temporal eicosanoid production throughout the course of disease. Using a lipidomics approach, we detected dramatic temporal and compositional changes in the majority of the 103 eicosanoids monitored. LOX metabolites produced in fish oil-fed mice were primarily generated non-enzymatically, and arachidonic acid metabolism was shunted from PGE2 synthesis to PGD2 synthesis resulting in the production and accumulation of 15d-PGJ2. Despite the fact that the fish oil diet led to a highly anti-inflammatory microenvironment, mice were able to mount an immune response and clear infection as well as mice fed a control diet. This study confirms that a diet high in fish oil results in a microenvironment with high anti-inflammatory potential but does not necessarily affect the ability of the organism to mount an effective immune response to infection with Borrelia burgdorferi.
Vaccine Dose Alters Immunodominant Epitopes and Cytokine Profile in Response to the Acinetobacter baumannii rOmpA Vaccine (52.12)
Authors: Lin Lin , Baquir Beverlie , Tan Brandon , Ho Tiffany , Pantapalangkoor Paul , Hujer Kristine , Bonomo Robert A. , Spellberg Brad
Published: January 2023

Background: The rOmpA vaccine protects mice from lethal infection caused by extreme-drug-resistant (XDR) Acinetobacter baumannii. The role of dose and epitope in immunology of the rOmpA vaccine was explored. Methods: The rOmpA vaccine was epitope-mapped using overlapping peptides to stimulate splenocytes from mice administered varying vaccine doses. B cell epitopes were mapped by peptide immunoblotting. Results: Vaccination with rOmpA plus Al(OH)3 adjuvant induced IFN-γ, IL-4, and IL-17+ splenocytes. Epitope mapping revealed distinct T cell epitopes that induced IFNγ+, IL-4+, and IL-17A+ splenocytes. B cell and T cell epitopes had only partial overlap, and the overlapping epitopes induced more potent IL-4 responses from splenocytes than IFN-γ responses. More than twice as many peptides induced IFN-γ, IL-4, and IL-17 responses when used to active splenocytes from high dose (100 µg) vaccinated mice versus low dose (3 µg) vaccinated mice. The IFN-γ:IL-4 ratios induced by each peptide varied depending on the dose of whole protein used to vaccinate the mice. Conclusions: rOmpA dose alters the epitope profile of responding lymphocytes, with higher vaccine doses resulting in epitope spreading. Furthermore, each individual epitope may stimulate a different cytokine profile from responding lymphocytes depending on the dose of vaccine used to immunize the mice.
Pattern recognition receptor SRA/CD204 restricts T cell activation and antitumor immunity (52.13)
Authors: Wang Xiang-Yang , Yi Huanfa , Yu Xiaofei , Guo Chunqing , Qian Jie , Zuo Daming , Subjeck John
Published: January 2023

Scavenger receptor A (SRA), also termed CD204, is an innate immune receptor primarily expressed in dendritic cells (DCs) and macrophages, and displays a broad ligand-binding specificity. SRA/CD204 has been implicated in multiple physiological and pathological processes, including atherosclerosis, Alzheimer's disease, and host defense against pathogen infection. Although initial studies identified SRA/CD204 as a binding receptor for large heat shock proteins (HSPs), our subsequent studies revealed an unexpected feature of SRA/CD204 in attenuating antitumor immune response generated by vaccines formulated with adjuvants of mammalian and foreign origins. SRA/CD204 is capable of dampening both Th1 and CTL responses by limiting the intrinsic immunogenicity of DCs during interactions with T cells. SRA/CD204 may also serve as a molecule bridging antigen-presenting cells (APCs) and activated T cells for immune regulation. Dual effects-mediated by SRA/CD204 indicate pleiotropic functions of this molecule in regulating immune homeostasis and host defense. Furthermore, genetic manipulation of SRA/CD204 expression markedly enhances the immunostimulatory activities of DCs in driving antigen-specific T cell immunity, leading to profoundly improved control of tumor and metastases in preclinical models. Therefore, our studies also provide a rationale for improving vaccine potency and cancer immunotherapy through SRA/CD204-targeting approaches.
Glycosylation Status and Presentation Format Determines Immune Responses Induced by Keyhole Limpet Hemocyanin (KLH) (52.16)
Authors: Bi Shuguang , Baum Mary , Morse Daniel
Published: January 2023

KLH is a heavily glycosylated protein. It can exist as 8MDa cylinders composed of 20 copies (didecamer) of a 400KDa subunit, or as separate subunits. KLH is a well-established immune stimulant, hapten carrier and generalized vaccine component. It is also used as a therapeutic vaccine against bladder cancer. Besides its application in schistosomiasis diagnosis, chronic stress such as post-traumatic stress disorder (PTSD) recently has been found to suppress KLH-induced adaptive immune responses, suggesting the potential for KLH in PTSD diagnosis. However, literature reports cite dramatically different immune responses to different preparations of the molecule, thus limiting the predictive outcome of using KLH as antigen carrier, vaccine adjuvant, vaccine or diagnostic agent. Mechanisms responsible for this variation remain largely unknown. Using lectin array and dendritic cell (DC) maturation assay, we found that KLH didecamers and subunits from different sources exhibit different carbohydrate profiles, and that the potency of KLH-induction of DC maturation varied with glycosylation status. We also found that conjugating KLH to poly (ethylene glycol)-based dendrimers significantly boosted DC maturation. Using endocytosis inhibitors, glycosylation status and presentation format was found to determine KLH uptake pathways in DCs. We thus propose that KLH can be tailored by manipulating glycosylation status and presentation format for optimal activity to meet different needs.
Ii- tumor cells present novel MHC II peptides that are tumor specific and immunogenic (52.18)
Authors: Chornoguz Olesya , Gapeev Alexei , O'Neill Michael , Ostrand-Rosenberg Suzanne
Published: January 2023

We have generated tumor cell-based vaccines to activate patients’ CD4+ T-cells to facilitate tumor immunity. The vaccines are based on the hypothesis that invariant chain negative (Ii-), MHC II+ , CD80+ human breast cancer cells (MCF10) present novel MHC II peptides and circumvent patients’ tolerance to their cancer. We sequenced MHC II peptides from Ii- (MCF10/DR7/CD80 and MCF10/CIITA/CD80/Ii siRNA) and Ii+ (MCF10/DR7/CD80/Ii and MCF10/CIITA/CD80) cells using LC-MS/MS. 64/116 peptides identified were unique to MCF10/DR7/CD80 and 176/228 peptides were unique to MCF10/DR7/CD80/Ii cells. 102/108 and 22/28 peptides were uniquely identified for MCF10/CIITA/CD80 and MCF10/CIITA/CD80/Ii siRNA cells, respectively. Therefore, the absence of Ii enables the presentation of unique peptides that are not presented by Ii+ or professional antigen presenting cells. Five peptides unique to Ii- and 2 peptides shared by Ii- and Ii+ cells and with the highest MHC II binding affinity, as predicted by artificial neural net analysis, were tested for their ability to activate T-cells from the blood of healthy donors or breast cancer patients. All peptides expanded tumor-reactive T-cells and induced IFNγ production. This is the first study to compare the human MHC II peptide repertoire in the absence or presence of Ii and to identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.
Acute intrahepatic CD8 T cell activation by plasmid DNA induces defective CD8 T cell immune response. (52.3)
Authors: Obeng-Adjei Nyamekye , Choo Daniel , Weiner David
Published: January 2023

The fate of CD8 T cells primed in the liver has been a subject of controversy in the attempt to explain the failure of immunity in chronic liver infections such as HBV, HCV and malaria. While most models show activation of CD8 T cells when liver resident cells present antigen, there are differences in opinion as to what becomes of these T cells after their initial priming. In this study, we used hydrodynamic injection that mimics acute HBV/HCV infection to express antigen in the liver, while taking advantage of the well-established LCMV P14 system, to induce acute intrahepatic T cells activation. We observed comparable CD8 T cells proliferation, up-regulation of CD44 and CD25, down-regulation of L-selectin, to the control (activation in lymphoid tissues). However, CD8 T cells primed in the liver expressed higher levels of pro-apoptotic markers such as Bim and active caspase 3, resulting in the loss of activated cells in all tissues. These liver activated CD8 T cells were incapable of protecting mice from lethal intracranial LCMV challenge. Our results confirm the up-regulation of ‘death markers’ during CD8 T cell priming in the liver, which leads to deletion of activated CD8 T cells needed to control viral infections in the liver. Thus, the failure of T cells to control chronic HBV/HCV is more likely due to defective liver priming of antigen-specific T cells.
Targeting Her-2/neu breast cancers with a plant derived natural compound, Withaferin A (53.15)
Authors: Smith Annalise , Marin Susana , Andreansky Samita
Published: January 2023

Over expression of oncogenic Her-2/neu protein in breast cancer cells correlates with poor prognosis and patients initially responsive to Her-2/neu targeted treatments often experience recurrence within a year of treatment. Thus, new strategies to combat these cancers should be explored. Our research focuses on the development of Withaferin A (WA), a steroidal lactone derived from the Indian ayurvedic medicinal plant Withania somnifera as an immunotherapeutic agent. The benefit of using natural plant derived products is their low toxicity, multimodal action and potential ability to enhance the immune system. Our goal is to establish WA as a chemoimmunotherapeutic agent in the treatment of Her-2/neu breast cancers. We provide evidence that WA is effective in vitro against both human and murine Her-2/neu breast cancer cells. WA induces cell death by apoptosis and increases expression of immunogenic stress proteins. Furthermore, WA up-regulated costimualtory molecules, cytokine production and antigen presentation in cultured bone marrow derived murine dendritic cells (BMDC). Additionally, in vivo administration of WA in a therapeutic setting significantly delayed tumor growth with a 10% survival benefit. Our long-term goal is to elucidate the anti-tumor immunomodulatory mechanisms of WA. Such agents that can deliver multimodal action towards cancer at both a chemotherapeutic and immunotherapeutic level might prove to be the best alternative to current treatment strategies.
APC Migration and antigen uptake is increased by the TLR2 ligand Neisserial Porin PorB (53.4)
Authors: Platt Andrew , Liu Xiuping , Berg Gretchen , Wetzler Lee
Published: January 2023

TLR ligands play an essential role in identifying threats to the innate immune system, which can then activate the adaptive arm. Our lab has worked with the N. meningitidis outer membrane protein Porin B (PorB), a TLR2 ligand, and shown that it increases the adaptive immune response as measured by antibody production and T cell activity. In examining the adjuvant activity of PorB we now investigate the ability of PorB to increase APC antigen uptake and migration to effector draining lymph nodes. Using an in vitro model with a fluor-linked antigen we show that PorB increases the quantity of the model antigen ovalbumin taken up by the APC in a rate dependent manner. We are in the process of characterizing the intracellular compartmentalization of the processed antigen by fluorescent microscopy of BMDM. In vivo experiments were conducted using a hock injection model and tracking the movement of the antigen through its fluorescent tag. Draining lymph nodes were examined through flow cytometry and immunofluorescence of frozen node sections. Using the latter technique, we are studying changes to lymph node morphology with the inclusion of the TLR ligand. Confirming the in vitro results, the addition of PorB to a vaccine injection increases the degree of APC migration to draining lymph nodes and the carriage of the antigen by these cells. With these results we have identified characteristics of the TLR2 ligand PorB that may account for some of its efficacy as a vaccine adjuvant.