bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-22 inhibits CCL20 expression induced by Helicobactor pylori infection (44.28)
Authors: Chen Jia-Perng , Liao Fang
Published: January 2023

Helicobactor pylori is a Gram-negative bacterium that infects the human gastric mucosa and causes various gastric diseases including gastritis, gastric ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. Given that Helicobactor pylori infection induces CCL20 production in gastric mucosa and leads to gastric inflammation, and that IL-22/IL-22R axis has been shown to play a critical role in regulating homeostasis and inflammation of epithelial cells at barrier surfaces, we investigate the effect of IL-22 on CCL20 expression induced by Helicobactor pylori. We demonstrated that Helicobactor pylori infection in AGS, a gastric epithelial cell line, significantly induces CCL20 expression. Functional analysis of the CCL20 promoter revealed that activation of NF-κB is required for CCL20 expression induced by Helicobactor pylori. Of interest, we found that IL-22 is able to reduce Helicobactor pylori-induced CCL20 expression by attenuating NF-κB activation. We further demonstrated that the suppressive effect of IL-22 on CCL20 is mediated by STAT3 since IL-22 fails to inhibit CCL20 production in AGS cells with STAT3 knockdown. Our findings suggest that STAT3 suppressing NF-κB activation is an anti-inflammatory mechanism for IL-22 to reduce inflammatory chemokine gene transcription during Helicobactor pylori infection.
Inhibitory Effect of Sargassum muticum (Yendo) Fensholt on Adipogenesis in 3T3-L1 Cells (44.29)
Authors: Ahn Eun-Kyung , Lee Jung A , Baik Seung Ho , Hong Seong Su , Oh Joa Sub
Published: January 2023

In this study, we investigated the ethanol extract of Sargassum muticum (Yendo) Fensholt (ESMF) inhibits adipocyte differentiation and expression of adipogenic marker genes, such as PPARγ (peroxisome proliferator activated receptor γ), C/EBPα (CCAAT-enhancer-binding protein α), GLUT4 (glucose transporter 4), adiponectin, aP2 (adipocyte fatty acid-binding protein 2) and ADD1/SREBP1c (adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c) in 3T3-L1 cells. We demonstrated that ESMF induced a significant decrease in lipid accumulation and expression of adipogenic marker genes in a dose-dependent manner. In addition, ESMF reduced the transcripitional activity of PPARγ induced by troglitazone, a potent diabetes agent; it also suppressed expression of PPARγ and C/EBPα protein levels. Our findings provide that ESMF inhibits adipocyte differentiation through downregulation of various adipocytokines by blocking PPARγ and C/EBPα expression.
Generation and characterization of a CXCR3-EGFP bicistronic reporter (CIBER) mouse reveals phenotypically and functionally distinct populations of innate CD8+ T cells (44.9)
Authors: Oghumu Steve , Dong Ran , Shawler Todd , Kampfrath Thomas , Rajagopalan Sanjay , Sharpe Arlene , Satoskar Abhay
Published: January 2023

The chemokine receptor CXCR3 is expressed mainly on activated T cells, NK cells and NKT cells. CXCR3 is implicated in a host of immunological conditions and it contributes either to disease resolution or pathology. Here, we report the generation and characterization of a novel CXCR3 IRES bicistronic EGFP reporter (CIBER) mouse in which EGFP expression correlates with surface levels of CXCR3. Using CIBER mice, we have identified two distinct populations of innate CD8+ T cells, one which constitutively express CXCR3 and the other which does not. We demonstrate that CXCR3+ innate CD8+ T cells preferentially express higher levels of Ly6C and CD122 but lower levels of CCR9 compared to CXCR3- innate CD8+ T cells. Furthermore, we show that CXCR3+ innate CD8+ T cells express high transcript levels of anti-apoptotic and low levels of pro-apoptotic co-stimulatory molecules, respond more robustly to IL-2 and IL-15, and produce significantly more IFN-γ and granzyme B as compared to CXCR3- innate CD8+ T cells. Interestingly, CXCR3+ innate CD8+ T cells do not respond to IL-12 or IL-18 alone, but produce significant amounts of IFN-γ upon stimulation with a combination of these cytokines. Taken together, these findings demonstrate that CXCR3+ and CXCR3- innate CD8+ T cells are phenotypically and functionally distinct. CIBER mice could be a novel tool for studying the role of CXCR3 and CXCR3-expressing cells in vivo during infectious, autoimmune and neoplastic diseases.
Balance of Positive and Negative Roles of Alpha-beta and Gamma-delta CD1d-reactive T Cell Populations in Th1-type Anti-Viral and Anti-Tumor Responses (45.15)
Authors: Exley Mark , Simon Yue , Varghese Bindu , Lynch Lydia , Nowak Michael
Published: January 2023

iNKT can enhance anti-pathogen and anti-tumor immunity via NK and DC, leading to lymphocyte activation. Th1-biased NKT can contribute to protective responses against cancer and viruses. iNKT respond like CD1d+ DC to immunotherapy, but reciprocally to NK and Treg. Patient iNKT defects are reversible in vitro, so we are investigating correcting such defects in models and clinic. NKT are required for optimal resistance to certain viruses. CD1d KO mice are impaired in lymphocyte activation and in picornavirus clearance. Conversely to acute infection, chronic viral stimulation of NKT can lead to immunopathology. CD1d-specific gamma-delta T cells respond to CD1d induced by infection, causing viral myocarditis and potentially contributing to hepatitis C, since human dominant liver gamma-delta T cells Vd3+ T cells were largely CD1d-reactive. We also found CD1d is up-regulated and high levels of hepatic CD1d-reactive NKT in chronic hepatitis C (CHC) produce pro-inflammatory Th1 cytokines and their Type 2 responses (which can suppress anti-tumor responses and promote fibrosis) increase in cirrhosis. NKT in CHC, instead of being protective against virus and hepatocellular carcinoma, may promote inflammation and fibrosis while suppressing anti-tumor responses. Cancer model iNKT have functional defects similar to patients, apparently due to iNKT:CD1d+ tumor interactions, but reversed by alpha-Galcer+IL-12. Models are being utilized to optimize immuno-monitoring of an iNKT clinical trial.
Activation of murine γδ T-cells by mitochondrial damage-associated molecular patterns (DAMPS) (45.4)
Authors: Schwacha Martin , Rani Meenakshi , Zhang Qiong
Published: January 2023

Gamma delta T-cells have been shown to be important in the early immunoinflammatory response to injury, which can be independent of infection. This sterile inflammatory response is in part associated with DAMPs. Mitochondrial DAMPs (MTDs) have been shown to be important in trauma-induced neutrophil activation, but it is unknown whether MTDs activate other innate immune cells, such as γδ T-cells. To study this, splenic CD3+ γδ T-cells were isolated from αβ T-cell deficient C57BL/6 mice and mitochondria isolated from wildtype mouse livers. MTDs were isolated from mitochondria by sonication and centrifugation. Gamma delta T-cells were incubated with various concentrations of MTDs (0-1000 µg/ml) for 24 hr. T-cells were phenotyped for Toll-like receptor (TLR) expression by flow cytometry and the supernatants assayed for cytokine and growth factor content. MTDs caused a dose-dependent increase in TLR-2 and TLR-4 expression by γδ T-cells. Both the percentage of cells positive for TLRs and the degree of expression increased. MTDs also induced the dose-dependent production of the chemokine RANTES and the growth factors FGF and VEGF. These findings support the concept that the MTDs released after tissue/cellular injury are capable of activating γδ T-cells, thus initiating sterile inflammation as well as subsequent healing processes.
Essential roles of heat shock protein gp96 (Hsp90b1, grp94) in melanoma growth and melanosome development (46.1)
Authors: Zhang YONGLIANG , Liu Bei , Sun Shaoli , Li Zihai
Published: January 2023

Heat shock protein gp96(HSP90b1,grp94) is an endoplasmic reticulum chaperone of the HSP90 family. It is a master molecular chaperone for Toll-like receptors and integrins. Accumulating clinical and pathological evidences indicate that enhanced gp96 expression in cancer tissue is a poor prognostic factor. However, the underlying mechanism is unclear. To address this question, we knocked down gp96 from melanoma cell line B16F1 (B16-KD) using lentivirus-mediated shRNA strategy. We found that knockdown of gp96 did not interfere with the in vitro growth of B16F1, but robustly suppressed the growth of orthotopically inoculated tumor in mice. Mechanistic study demonstrates that gp96 deletion compromises VEGF-A expression and inhibited tumor-associated angiogenesis. These results suggest that altered tumor microenvironment upon gp96 knockdown was responsible for the delayed tumor growth in vivo. Unexpectedly, we also observed that melanin production was significantly inhibited in B16-KD, which correlated with the reduced tyrosinase activity but not its expression in B16-KD cells. Thus, our study has uncovered two novel roles of gp96 in regulating tumor angiogenesis and in facilitating melanin synthesis. The molecular mechanisms of these findings are under active investigation.
Is lymphatic pumping safe for breast cancer patients?: The role of lymphatic flow in tumor formation and metastasis (46.19)
Authors: Odeh Adam , McKee Courtney , McCauley Lyndsey , Jones Harlan , Hodge Lisa
Published: January 2023

Lymphedema, a common side effect of breast cancer, can only be treated effectively with techniques such as pneumatic compression, exercise, massage, and osteopathic manipulation. However, the effect of such therapies on tumors and metastases is unknown. We have developed a murine model to test the effect of abdominal lymphatic pump treatment (LPT) on cancer. F344 rats were injected intravenously or subcutaneously with MADB106 mammary carcinoma cells. Rats were then given 4 minutes of LPT under anesthesia, 4 minutes of light touch under anesthesia (sham), or no treatment (control) for 7 days. At day 8, lung tissues were analyzed for metastases, and for leukocyte number/activity. LPT on intravenously-inoculated rats reduced tumor metastases in the lungs by more than 40% compared to sham and control. This corresponded with an increase in IFN-γ, and in CCR9+CD4+, CCR9+CD8+, and CCR9+ NK cells in the lungs, suggesting that these cells were actually gut-derived. Interestingly, in the case of subcutaneous solid tumors, LPT may be detrimental, as tumor size and number of metastases were more than doubled. These data suggest that increasing lymphatic flow may mobilize gut-derived leukocytes, which can traffic to the lungs and confer protection against tumor metastases, possibly through IFN-γ. To our knowledge, this is the first time gut-derived cells have been shown to be protective in the lungs. However, these therapies may cause harm when performed in proximity to solid tumors.
Multiple mechanisms for ATP release from apoptotic tumor cells (46.21)
Authors: Boyd Tressler Andrea , Dubyak George
Published: January 2023

Extracellular ATP released from dying tumor cells treated with pro-apoptotic chemotherapeutic drugs plays a key role in activating anti-tumor immune responses. However, the mechanisms by which ATP is released from such tumor cells remain poorly defined. Physiological apoptosis induced by Fas signaling can elicit ATP release via pannexin 1 (panx1) channels gated by caspase 3/7 (casp3/7)-mediated proteolytic cleavage of the panx1 C-terminus. We used human Jurkat leukemia and murine EG7 lymphoma cells to test whether the pro-apoptotic drugs doxorubicin (Dx) and staurosporine (STS) elicit the same casp3/7> panx1> ATP release pathway. Cells were treated with Dx or STS for 1-18 h in the presence or absence of the pan-caspase inhibitor zVAD and then assayed for accumulation of extracellular adenine nucleotides (ANex) and intracellular active casp3/7. Accumulation of ANex in response to Dx required prolonged (> 8h) treatment in both cell models and was insensitive to zVAD inhibition of casp3/7. Similar results were observed in EG7 cells treated with STS. In contrast, STS-treatment of Jurkat cells induced rapid ANex accumulation within 2-4 h that was markedly suppressed by zVAD. Thus, while ATP release is a common response of tumor cells to chemotherapeutic and other pro-apoptotic stimuli, its mediation via proteolytic gating of panx1 channels requires cell-specific and drug-specific factors that are differentially expressed in tumor cell models.
Progression of acute lymphoblastic leukemia (ALL) results in compartment-restricted T cell expression of PD-1 and T cell dysfunction (46.22)
Authors: Su Paul , Burk Chad , Fix William , Qin Hai-Ying , Fry Terry
Published: January 2023

ALL is the most common cause of cancer related mortality in children. Although immune-based therapies for B cell malignancies have recently demonstrated promising results, the limited efficacy of donor lymphocyte infusions after allogeneic transplantation suggest that ALL may compromise immune responses. Using a murine model of pre-B-cell ALL, we have shown that leukemia progression is associated with PD-1 and TIM3 expression on CD4+ and CD8+ T cells evident at early infiltration of ALL with eventual expression on 80% of T cells with advanced disease. This was apparent in all leukemia-containing compartments including bone marrow, spleen and lymph nodes. PD-1 expressing CD8+ cells in leukemia compartments are predominantly CD44+CD62L- consistent with effector memory phenotype and a subset also express KLRG1 and LAG3. Finally, we assessed functionality of T cells from ALL mice including expression of the degranulation marker CD107a as well as production of TNFα, IFNγ, and IL-2 cytokines. Preliminary data suggests global impairments of functionality in the presence of leukemia. Currently, we are investigating the antigen-specificity of PD-1 acquisition, the translatability of our findings to clinical samples, and the effects of anti-PD-1 blockade on tumor progression. Given the pervasiveness of PD-1 and evidence for T cell dysfunction with ALL progression, elucidating the mechanism by which this process occurs could provide therapeutic targets for advancing ALL immunotherapy.
IL-33 promotes type 1 cell-mediated immunity and has a potent antitumor function (46.25)
Authors: Li Gang , Yang Qianting , Zhu Yibei , Gao Xin , Zhang Xueguang , Finn Olivera J. , Lu Binfeng
Published: January 2023

Interleukin-33 is a member of the IL-1 family of cytokines and is released by stressed cells, necrotic cells or activated innate immune cells such as macrophages during trauma or infection. Thus, IL-33 is thought to serve as an endogenous “danger signal” to trigger inflammation and promote cell-mediated immune responses. Prior studies have supported the role of IL-33 in promoting type 2 immune responses but little was known for its role in type 1 immune responses such as the anti-tumor immune response. We have found that that ST2, a receptor for IL-33, is highly expressed in CD8 T cells cultured in Th1 conditions. We have further revealed that, T-bet, a critical transcriptional factor for Th1 differentiation and effector functions, is required for ST2 expression by CD8 T cells, suggesting that ST2 is an integral part of the Th1 gene network in CD8 T cells. Furthermore, we have demonstrated that IL-33 synergized with TCR or IL-12 in increasing both human and mouse CD8 T cell functions such as IFN-γ production. Thus IL-33 can greatly enhance type 1 cell-mediated immune responses in both human and mice. The in vivo relevance of this pathway is examined in a preclinical model of tumor immunity. Our results support a potent function of IL-33 in inhibiting tumor growth and provide a novel therapeutic approach for tumor immunity.
Dendritic cells transduced with T-bet and IL-12 effectively restrict immunosuppressive activity of myeloid-derived suppressor cells and regulatory T cells in a murine tumor model (46.26)
Authors: Chen Lu , Qu Yanyan , Taylor Jennifer , Storkus Walter
Published: January 2023

Despite recent attempts to take advantage of dendritic cell (DC)-based vaccines for cancer immunotherapy, clinical results for such approaches have been disappointing, partly due to tumor-associated immune suppression in the tumor-bearing host. Successful DC-based cancer vaccines must overcome the immunosuppressive functions of myeloid-derived suppressor cells (MDSC), regulatory T cells (Treg) or inhibitory soluble factors and promote the survival of protective central memory T cells. We have recently shown that DC adenovirally-engineered to express Tbet and IL-12p70 (i.e. DC.Tbet/IL12) mediate superior anti-tumor effects in mice. The dominant mechanism of action associated with the therapeutic efficacy of DC.Tbet/IL12 remains unknown, although preliminary gene array analyses suggest these antigen-presenting cells may be coordinately enriched in NOTCH- and depleted of IL-10/TGF-β-signaling potential. In the current study, an analysis of TIL suggests that DC.Tbet/IL-12-based therapy downregulates MDSC and Treg numbers in tumor microenvironment (TME), and promotes higher levels of central memory T cells (Tcm) that are capable of mediating durable anti-tumor effects. This potential may be all related to a selective silencing of p38 MAPK signaling, and a coordinate reinforcement of ERK-signaling in DC.Tbet/IL12 cells, which has been suggested to dramatically enhance the immunogenicity of DC based vaccines in supporting Type-1 immune responses.
Enhanced levels of endogenous common γ chain receptor cytokines influence CD8 T cell subpopulation numbers and phenotype in cancer patients receiving CD4 T cell immunotherapy: Observations In Four Cases Of Patients With Ovarian Cancer (46.31)
Authors: Dobrzanski Mark , Rewers-Felkins Kathleen , Samad Khaliquzzaman , Quinlin Imelda , Phillips Catherine , Robinson William , Dobrzanski David , Wright Stephen
Published: January 2023

The common γ chain receptor family of cytokines have been associated with T cell homeostasis, development and survival. In this study, we assessed the therapeutic effects of adoptively transferred CD4 T cells and their affects on endogenous expression of such cytokines in patients with ovarian cancer. Using MUC1 peptide and IL-2 for ex vivo CD4 effector cell generation, we show that 3 monthly treatment cycles of autologous T cell restimulation and local intraperitoneal reinfusion resulted in diminished serum CA125 tumor marker levels and enhanced patient survival. One patient remains disease free, another patient experienced prolonged survival for nearly 16 months with recurrent disease and two patients expired within 3-5 months following treatment. Prolonged survivors showed lower levels of systemic memory (CD8+CD45RO+)/naïve (CD8+CD45RA+) CD8 T cell ratios when compared to that of short-term survivors. Such subpopulations among prolonged survivors were predominantly FoxP3-NEG and at relatively greater levels when compared to that of corresponding pre-treatment conditions. Lastly, peptide-restimulated PBMCs from these patients showed variable gene expression levels of pre- and post-treatment IL-15, IL-21 and IL-9 that were further associated with prolonged and/or disease-free survival. This suggests that CD4 cell transfer may induce levels of endogenous cytokines associated with CD8 survival and/or homeostasis that may contribute to enhanced CD4 therapeutic potentials
TLR7 and -9 activation of human plasmacytoid dendritic cells enhance endocytosis and processing of membrane material from a Her2/neu positive breast cancer cells (46.32)
Authors: Pierog Piotr , Singh Sukhwinder , Fitzgerald-Bocarsly Patricia
Published: January 2023

pDC detect viral nucleic acids and mount an immune response by secretion of IFN-α as well as TNF-α and chemokines which results in recruitment and activation of the adaptive immune system. Over-expression of Her2/neu in breast cancer patients results in poor prognosis; however, CTL specific to Her2/neu have been identified in patients indicating an active immune response towards this antigen. Unfortunately, Her2/neu is a self-antigen and therefore has a low immunogenicity. Therefore, we hypothesize that by making tumor cells more immunogenic for human pDC through introduction of HSV-infection, we will be able to efficiently activate Her2/neu specific cytotoxic T lymphocytes (CTL) through the process of antigen cross-presentation. Here we report that pDC take up membrane material from cancerous MCF-7 cells and that viral infection of MCF-7 cells significantly enhances this process. Interestingly, pDC activated with other TLR7 or TLR9 agonists had higher nibbling and endocytic capacity. The membrane nibbled from HSV-infected MCF-7 cells was endocytosed and processed through CD71 and CD107 positive compartments, as assessed by AMNIS Imaging Flow Cytometry. As expected, the nibbling of membrane from HSV-infected MCF-7 cells induced production of IFN-α by pDC as measured by FACS and ELISA. HSV-infected but not uninfected MCF-7 cells induced maturation of pDC as determined by loss of CD62-L and upregulation of CD40 and CD80 co-stimulatory molecules.
Intravenous administration of a novel IL-2 fusion protein, ALT-801, inhibits bladder cancer in mouse models (46.35)
Authors: Wong Hing , Wen Jinghai , You Lijing , Xu Wenxin , Edwards Ana , Rhode Peter
Published: January 2023

ALT-801 is a fusion protein between interleukin-2 and a T cell receptor domain capable of recognizing tumors presenting human p53 peptide (aa264-272)/HLA-A*0201 complexes. Previously, we have reported that ALT-801 exhibits significantly better antitumor activity than IL-2 in various mouse tumor models. Substantial efficacy in human malignancies has been observed. In this study, we found that intravenous administration of ALT-801 significantly prolonged survival of C57BL/6 mice bearing MB49luc orthotopic muscle invasive and superficial bladder cancer when compared with PBS treatment. The ALT-801-treated mice also survived rechallenge with MB49luc tumor cells, indicating long-lasting immune response and long-term memory. Additionally, ALT-801 exhibited potent antitumor activity against human bladder cancer HLA-A*0201+/p53+ UMUC-14 and HLA-A*0201-negative/p53+ KU7 xenografts in nude mice, suggesting its targeting activity is not required for efficacy. ALT-801 combined with gemcitabine showed better antitumor effects and less toxicity than gemcitabine + cisplatin (GC) chemotherapy in the UMUC-14 and KU7 xenograft models, despite the different sensitivity of these tumor cells to GC. We’ll discuss the mechanisms of action of ALT-801 in this report. Based on these preclinical findings, we have already initiated a clinical study of ALT-801 on human metastatic bladder cancer.
A Polymorphism in CD1e is associated with enhanced immune responses in African American Prostate Cancer Patients (46.40)
Authors: Webb Tonya , Dorsey Tiffany , Goloubeva Olga , Klyushnenkova Elena , Alexander Richard , Ambs Stefan , Mann Dean , Strome Scott , Moudgil Kamal , Sun Wenji
Published: January 2023

Prostate cancer is the second leading cause of cancer mortality in men older than 40 years in the United States. African American (AA) men and men with a family history of prostate cancer are at a significantly increased risk for developing prostate cancer. A recent study found highly significant differences in the expression of immune regulatory genes and cytokines between tumors from AA and European-American (EA) patients. Therefore, we tested the hypothesis that genetic variations in immune regulatory genes including the CD1 locus may confer an increased disease risk and a poor outcome prostate cancer phenotype in African-American men. Natural Killer T (NKT) cells produce large amounts of cytokines following activation vis-à-vis recognition of lipid antigen presented in the context of CD1d molecules. Prostate tumors commonly express CD1 family members, thus polymorphisms in CD1 genes may be responsible for differences in tumor biology between AA and EA patients. We report that a polymorphism in CD1e occurs commonly in AA but not EA men. When the distribution of this polymorphism was examined in a large cohort (n=562), we found that it is ethnicity specific and occurs primarily in men of African ancestry (P<0.0001). We also observed that this polymorphism strongly influences IFN-γ production following stimulation. Thus, our data indicate that an ethnicity-related polymorphism affects a key function of NKT cells which in turn may modulate prostate cancer biology.
Mouse tumor vasculature expresses NKG2D ligands and can be targeted by chimeric NKG2D-modified T cells (46.45)
Authors: Zhang Tong , Sentman Charles
Published: January 2023

Tumor angiogenesis plays an important role in the development and progression of solid tumors. Targeting the tumor vasculature has emerged as a strategy to prevent growth and progression of solid tumors. In this study, we showed that murine tumor vasculature express Rae1, a ligand for a stimulatory natural killer receptor NKG2D. By genetic modification of T cells with a chimeric NKG2D receptor (chNKG2D), in which the NKG2D receptor is fused to the signaling domain of CD3ζ chain, we generated T cells that are capable of targeting tumor vasculature, leading to reduced tumor angiogenesis as well as tumor growth in tumors where the tumor cells do not express NKG2D ligands themselves. H5V, an endothelial cell line, expresses Rae1 and can be lysed by chNKG2D-bearing T cells in a perforin-dependent manner. In vitro capillary tube formation by H5V was inhibited by both soluble factors (IFN-γ) and cell-cell contact by chNKG2D T cells. The in vivo anti-tumor angiogenesis efficacy mediated by chNKG2D T cells was also dependent on IFN-γ and perforin. In addition, chNKG2D T cells could inhibit VEGF expression by myeloid derived suppressor cells (MDSCs). In summary, these results provide a novel mechanism for NKG2D-targeting of solid tumors.
ROS-p38 MAPK-Fas/Fas Ligand-Linked Cascade Regulate Bax Translocation and Calcium Redistribution in Salubrinal-Induced Apoptosis of EBV-Transformed B Cells (46.49)
Authors: Park Ga-bin , Kim Yeong Seok , Lee Hyun-Kyung , Song Hyunkeun , Kim Seonghan , Cho Dae-Ho , Lee Wang-Jae , Hur Dae Young
Published: January 2023

The small molecule salubrinal has antiviral activity against herpes simplex virus-1 (HSV-1) and inhibits dephosphorylation of eIF2 alpha. Salubrinal prevents vulnerable cells from going down the cell-death pathway and is deeply associated with ER stress. The Epstein-Barr Virus (EBV), also called human herpesvirus 4 (HHV-4), is a virus of the herpes family (which includes herpes simplex virus) and is one of the most common viruses in humans. However, maintaining hyper-phosphorylated eIF2α state with high doses of salubrinal treatment promotes apoptosis in some cancer cells. In this report, salubrinal inhibited proliferation and induced apoptosis of EBV-transformed B cells in both dose and time dependent manner. Notably, salubrinal induced ROS generation and p38 MAPK activation, which then induced expression of FasL. Moreover, salubrinal subsequently led to activation of caspases, calcium redistribution, Bax translocation, cytochrome c release, and apoptosis. Caspase inhibitors (z-VAD, z-IETD, and z-DEVD), p38 MAPK inhibitor (SB203580) and ROS inhibitor (NAC) recovered salubrinal-induced apoptosis. ZB4, an antagonistic anti-fas Ab, and SB203580 effectively blocked apoptosis without exerting any influence on ROS generation. These findings suggest that salubrinal may be a novel therapeutic approach for EBV-associated malignant diseases.
FoxP3+Tim-3+ Tregs are site-specific suppressors supporting tumor growth by influencing the functional phenotype of tumor-infiltrating lymphocytes (46.5)
Authors: Sakuishi Kaori , Sullivan Jenna , Kuchroo Vijay , Anderson Ana
Published: January 2023

In prior studies, we and others have found Tim-3 to be expressed on exhausted CD8+ tumor infiltrating lymphocytes (TILs). It was shown that anti-Tim-3 antibody treatment together with anti-PD-1/PD-L1 antibody could abrogate tumor growth and effectively restore T cell effector function to exhausted CD8+ T cells (Sakuishi et al 2010, Fourcade et al 2010, Zhou et al 2011, Ngiow et al 2011). Tim-3 is also expressed on CD4+ TILs but its role in tumor immunity has not been addressed. We have found that the majority of FoxP3+ TILs express Tim-3 in multiple tumor models. Kinetic studies in B16F10 melanoma show that the accumulation of FoxP3+Tim3+ cells parallels tumor growth. Interestingly, this accumulation of FoxP3+Tim-3+ cells is not observed in either the tumor draining lymph node or spleen of tumor-bearing mice, indicating site-specific expansion. FoxP3+Tim-3+ cells are higher producers of IL-10 and are more efficient in suppressing FoxP3- effector T cells in vitro when compared to FoxP3+Tim-3- cells. Depleting FoxP3+ regulatory T cells (Tregs) from established B16 melanoma results in tumor regression and modulation of Tim-3 expression on both FoxP3- CD4 and CD8 TILs. Furthermore, Treg depletion influences the development of exhausted phenotype in CD8+ TILs. This is the first report revealing that the Tim-3+ subset of Tregs predominates within tumor and plays an important role in tumor progression in situ by regulating the functional phenotype of FoxP3- CD4+ and CD8+ TILs.
Long-term memory CD4+ T cells - a survival network (47.12)
Authors: Khoruzhenko Stanislav , Jie Chunfa , Sadegh-Nasseri Scheherazade
Published: January 2023

Memory CD4+ T cells specific for a particular antigen can survive for a lifetime of an individual through undefined mechanisms. Previously we have demonstrated that memory CD4+ T cells undergo a state of dormancy during the resolution of infection, and dormant memory T cells survive for the life of mice. To gain an understanding of the genetic programs involved, we examined gene expression profile of memory DO11.10 CD4+ T cells in mice infected with Vaccinia-OVA virus 10.5 months post infection and compared it with gene expression in age-matched old mice. Extensive literature search allowed reverse engineering of genetic network of differentially expressed genes in long-term memory T cells. Pattern of gene expression pointed to the induction of anti-apoptotic and genomic stability pathways. We found that in long term memory CD4+ T cells a shift from glucose to lipid metabolism occurs, along with upregulation of ceramide synthesis and utilization that may favor memory T cell survival. Insulin resistance appears to play a major role in downregulation of glucose metabolism. In conclusion, our data show that in long-term memory CD4+ T cells several major regulatory genes that regulate anti-apoptotic, genomic stability, glucose and lipid metabolism pathways are upregulated and possibly coordinate an overall pro-survival effect.
The tyrosine phosphatase CD45 regulates lymphopenia-induced T cell proliferation (47.15)
Authors: Saunders Amy , Johnson Pauline
Published: January 2023

Lymphopenia-induced T cell proliferation (LIP) occurs naturally in neonates and in patients with depleted T cell numbers due to infection or immunosuppression. LIP occurs in response to an interaction between TCR with self-MHC as well as to cytokines such as IL-7 and IL-15. However, the precise factors that induce and limit LIP are not well understood. CD45 is a leukocyte specific tyrosine phosphatase that reduces the TCR signaling threshold in T cells, modulates TLR signaling in dendritic cells and can negatively regulate cytokine signaling in leukocytes. Given that CD45 deficient mice have severely reduced numbers of T cells in the periphery, we sought to determine whether CD45 might regulate LIP. CD45 deficient mice were crossed with the RAG1 deficient mice to generate CD45RAG deficient mice and LIP was examined in a CD45 sufficient and CD45 deficient environment. LIP was significantly reduced in the spleen and lymph nodes of lymphopenic hosts lacking CD45. This was attributed to a reduced signal to induce T cell proliferation rather than increased cell death or delayed kinetics. Reduced levels of IL-7 but not IL-15 were evident in the spleens of CD45RAG1 deficient mice and CD45 deficient splenic dendritic cells were less efficient at stimulating antigen-induced T cell proliferation. Together this suggests that dendritic cell CD45 is required for optimal LIP and experiments are ongoing to evaluate this possibility.
Regulation of A1 and Mcl-1 from costimulatory signals affects T cell survival (47.2)
Authors: Song Jianxun , Lei Fengyang , Haque Rizwanuel
Published: January 2023

Costimulation is critical for T cell activation. In recent years, advances have been made in studies of costimulation as potential therapies in the treatment of diseases. However, the intracellular signals by which costimulation affects the function of T cells are not fully understood. A1 and Mcl-1, two bcl-2 family proteins are up regulated in effector T cells by ligation with an agonist antibody to the costimulatory molecule OX40 (CD134), which is essential for long-term T cell survival. To determine whether A1 and Mcl-1 are intracellular molecules of OX40, we examined A1 and Mcl-1 expression in primary T cells from wild-type and OX40-deficient mice, and found A1 and Mcl-1 were up-regulated after early T cell activation, but not be maintained in T cells from OX40-deficient mice. In addition, we used retrovirus-mediated transduction to introduce A1 or Mcl-1 into antigen (Ag)-responding T cells from OX40-deficent mice. We found that exogenous expression of A1 or Mcl-1 reversed the survival defect of OX40-deficient T cells both in vitro and in vivo. Moreover, sustained expression of A1 or Mcl-1 in CD8+ T cells from OX40-deficient mice reversed their defect in preventing tumor growth in a murine model. These results indicate that A1 and Mcl-1 are intracellular molecules of OX40, which regulate T cell survival. The data provide new insights into the mechanisms by which costimualtion regulates T cell survival.
Phenotypic and functional immune attributes predicting survival in very elderly people (47.7)
Authors: Pawelec Graham , Maier Andrea , Hähnel Karin , Zelba Henning , de Craen Anton , Roelofs Helene , Slagboom Eline , Westendorp Rudi , Derhovanessian Evelyna
Published: January 2023

The low level of naïve T-cells commonly observed in the elderly is thought to be causally associated with mortality. However this has not been tested in longitudinal studies. Here, we present correlations between peripheral T-cell subsets and 8-year survival in individuals from the population-based prospective Leiden 85-plus Study. Counter-intuitively, a lower frequency of naïve (CD45RA+CCR7+CD27+CD28+) CD8+ T-cells at baseline (>88 years) correlated with significantly better survival, while there was a tendency for the reciprocal accumulation of late-differentiated effector memory cells (CD45RA-CCR7-CD27-CD28-) also to associate with better survival, suggesting that better retention of memory cells specific for previously encountered antigens provided a survival advantage in this particular population. Given the prevalence of Cytomegalovirus (CMV) and its reported association with immunosenescence, we tested whether memory for this potential pathogen was relevant to survival. We found that individuals mounting an exclusively pro-inflammatory ex-vivo response (TNF, IFN-γ, IL-17) to the major CMV target molecules pp65 and IE1 had a significant survival advantage over those also having anti-inflammatory responses (IL-10). These findings suggest that higher levels of naïve T cells are not necessarily associated with a survival advantage and imply that the nature of immunosurveillance against CMV may be crucial for remaining longevity, at least in the very elderly.
Defective CD4 T cell cognate helper functions in response to influenza immunization and infection in aged mice (47.8)
Authors: Lefebvre Julie
Published: January 2023

A poor immune response is a hallmark of aging. Older individuals are more susceptible to infections and respond poorly to new immunization. This is caused, at least in part, by a defective antibody response. We have shown that age-associated defects impair CD4 T cell functions. Since an optimal antibody response requires good CD4 cognate help through T follicular helper (Tfh) cells, we aimed to determine whether the impaired cognate help resulted from defects in the generation and/or functions of Tfh cells in aged mice. The work presented herein shows that the generation of Tfh cells in response to influenza immunization is delayed, reduced and shortened in aged mice compared to young mice. Also fewer Tfh cells are generated in aged mice than in young mice following influenza infection. The Tfh cells generated in aged mice, however, express similar levels of IL-21, BCL-6, CXCR5 and IL-4 mRNA than Tfh cells from young mice. These data suggest that the generation rather than the functions of Tfh cells is impaired in aged mice. The addition of poly(I:C) as an adjuvant to the influenza vaccine increases Tfh generation and antibody production in young and aged mice. We are investigating the mechanisms by which TLR ligands improve the Tfh response.
Human CD1c+ DCs drive the differentiation of CD103+ intraepithelial CD8+ T cells via TGF beta (48.12)
Authors: Yu Chun , Becker Christian , Marches Florentina , Garcia-Sastre Adolfo , Banchereau Jacques , Merad Miriam , Palucka A. Karolina
Published: January 2023

Mucosa is a major site of invasion and replication of pathogens, including influenza virus. However, our understanding of cellular immune responses and their regulation at mucosal sites in the human is limited due to inherent difficulties in assessing cellular interactions using ex vivo systems. Here we analyzed the response of human myeloid dendritic cells (DCs) to live-attenuated influenza virus (LAIV) and the type of CD8+ T cell responses that were generated using human lung tissue samples and humanized mice. Upon LAIV inoculation, the number of CD1c+ (BDCA-1) DCs in the lungs had increased by 16 hours; whereas the number of CD141+ (BDCA-3) remained stable up to three days post inoculation. Both DC subsets acquire flu antigens in vivo and expand specific memory CD8+ T cells ex vivo. However, at an early time point CD141+ DCs are more efficient in expanding CD8+ effector T cells. CD1c+ DCs are able to expand CD8+ T cells at a later time point, and they uniquely expand CD8+ T cells expressing CD103, a receptor for the epithelial cell-specific E-cadherin. CD1c+ DCs induce CD103+CD8+ T cells with the characteristics of cytotoxic intraepithelial T cells owing to the expression of membrane TGF-β. Our results suggest that lung-resident CD141+ DCs launch the effector response early after viral exposure while the role of migratory CD1c+ DCs is to retain the T cells in the epithelium. Thus, myeloid DCs have a major role in the regulation and expansion of memory T cells in mucosa.
p38α signaling programs dendritic cells to drive TH17 cell differentiation and function (48.14)
Authors: Huang Gonghua , Wang Yanyan , Chi Hongbo
Published: January 2023

Dendritic cells (DCs) bridge innate and adaptive immunity, but how DC-derived signals regulate T cell lineage choices and function remains unclear. Our previous study showed that DCs employed the intracellular phosphatase MKP-1 pathway to instruct multiple T cell lineage choices and anti-microbial and inflammatory responses (Huang et al. Immunity 2011). Since MKP-1 is a potent inhibitor of p38α MAP kinase, we investigated the function of p38α in DCs using the CD11c-Cre deletion system. DCs deficient in p38α were unable to drive the differentiation of responding T cells into the TH17 lineage. Mechanistically, p38α controlled IL-6 and IL-27 and further shaped CD86 expression in DCs, thereby establishing a gene-expression program in DCs to direct TH17 development. We further determined the roles of DC-derived p38α in autoimmune, inflammatory and infectious diseases. We found that mice lacking p38α in DCs showed diminished TH17-dependent inflammation in three disease models: experimental autoimmune encephalomyelitis (EAE), colitis and fungal infection. Moreover, inhibition of the p38 activity either by deleting p38α expression or by using the chemical inhibitor markedly ameliorated the disease severity in mice with ongoing EAE, suggesting a key therapeutic effect of p38α in TH17-dependent diseases. In summary, our results identify p38α MAPK signaling as a central pathway in DCs to program TH17 differentiation and inflammation.
CXCR2 and CXCL5 Regulate the IL-17/G-CSF axis and Neutrophil Homeostasis (48.15)
Authors: Mei Junjie , Liu Yuhong , Dai Ning , Hoffmann Christian , Hudock Kristin , Zhang Peggy , Guttentag Susan , Kolls Jay , Oliver Paula , Bushman Frederic , Worthen G. Scott
Published: January 2023

Neutrophil homeostasis is critical for maintaining innate immune surveillance under normal conditions. CXCR2 plays a critical role in neutrophil homeostasis, as Cxcr2-/- mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. We found that neutrophil CXCR2 inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis, while enterocyte-derived CXCL5 in the gut, regulates the IL-17/G-CSF axis and contributes to CXCR2-regulated neutrophil homeostasis. CXCL5-regulated neutrophil homeostasis was completely dependent on G-CSF, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5-/- and Cxcr2-/- mice. Cxcr2-/- mice express IL-17A, and show increased IL-17A-producing cells in the lung, terminal ileum and spleen, which include conventional Th17 cells, γδ T cells, and innate immune cells. Most IL-17 producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment markedly decreased IL-17A and G-CSF expression, neutrophilia and marrow myeloid hyperplasia in Cxcr2-/- mice. These data provide insights regarding the critical role of CXCR2, CXCL5 and commensal bacteria in regulating the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites, which keeps the set point of innate immunity in tight control.
Inhibition of Indoleamine 2,3-dioxygenase (IDO) enhances the memory CD8 T cell response and modulates the immunodominance hierarchy following influenza infection (48.17)
Authors: Klonowski Kim , Sage Leo , Rose David , Fox Julie , Huang Lei , Barber Jaime , Tompkins S. Mark , Mellor Andrew , Tripp Ralph
Published: January 2023

The generation of heterosubtypic CD8 T cell responses is important for cross-protective immunity against unrelated strains of influenza. The need to maintain tolerance in the lung, however, limits the overall breadth and duration of CD8 T cell responses, warranting a better understanding of mechanisms of immunoregulation at this site. Influenza infection induces pulmonary expression of the tryptophan catabolizing enzyme IDO. Depletion of tryptophan results in suppression of T cell expansion or effector function, T cell anergy, or deletion. We hypothesized that ablation of IDO activity during CD8 T cell priming would enhance the subsequent CD8 T cell memory pool. To test this in vivo, we conditionally ablated IDO activity during the priming phase using the inhibitor 1-methyl tryptophan (1MT). 1MT-treated animals challenged with a lethal dose of a heterosubtypic influenza virus generated a more robust Th1 response, with enhanced migration to the lung airways. Interestingly, the immunodominance hierarchy was also shifted to favor subdominant CD8 T cell epitopes in both 1MT-treated and IDO-deficient animals, likely a result of observed decreased regulatory T cell function. While 1MT treatment did not affect cytotoxicity or viral clearance, pulmonary pathology was reduced in the absence of IDO. Together these data provide evidence that modulation of IDO activity in the context of influenza infection could be exploited in vaccine development to enhance memory CD8 T cell responses.
Ability of LPS to regulate expression of tolerance-related molecules on dendritic cells is blocked by IL-10 deficiency (48.18)
Authors: Zhang Guag-Xian , Zhou Fang , Ciric Bogoljub , Li Hongmei , Yan Yaping , Li Ke , Cullimore Melissa , Lauretti Elisabetta , Dimeco Antonio , Gonnella Patricia , Rostami Abdolmoham
Published: January 2023

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays an important role in regulating the local inflammatory immune response, but regulatory mechanisms of this cytokine have not been fully elucidated. Here we demonstrate that IL-10 deficiency renders LPS treatment ineffective in regulating expression of CD40, CD80, CD86, B7-H2 and B7-DC on dendritic cells (DCs) and blocks up-regulation of IL-27. This inability to respond to LPS was found in both IL-10-/- bone marrow-derived and splenic DCs. Compared to wild type DCs, IL-10-/- DCs expressed similar levels of TLR4 and CD14, but produced less LPS binding protein (LBP). The deficiency in LBP production may explain the failure of IL-10-/- DCs to respond normally to LPS. Moreover, lack of IL-10 modulated the proportions of CD11c+CD8+ and CD11c+B220+ DCs, which play an important role in local inflammatory responses and tolerance. IL-10 deficiency also blocked expression of galectin-1, CD205 and CD103, which are necessary for central and peripheral tolerance. While they did not respond to LPS, IL-10-/- DCs produced increased levels of IL-6 and CCL4 after TNF-α treatment. Together, our results demonstrate that IL-10 deficiency affects the immune functions of DCs, which may contribute to the increased severity of autoimmune diseases seen in IL-10-/- mice.
Distinct LLO-specific CD4 T cells infiltrate the intestinal mucosa following oral Listeria monocytogenes infection. (49.16)
Authors: Fu Han-Hsuan , Sheridan Brian , Pham Quynh-Mai , Schubert Wolf-Dieter , Lefrançois Leo
Published: January 2023

We analyzed the mucosal CD4 T cell response to oral Listeria monocytogenes (LM) infection. Early after infection, a subset of blood-borne LLO-specific CD4 T cells expressed α4β7. Subsequently, antigen-specific CD4 T cells accumulated in the lamina propria and intraepithelial lymphocyte compartment where they were retained long-term. These data suggested that priming in the mucosal lymphoid tissues generated mucosa-seeking CD4 T cells with high memory potential. While naïve CD4 T cells contained both Ly6Chigh/CD27high and Ly6Clow/CD27high subsets, most primary and memory mucosal LLO-specific CD4 T cells were Ly6Clow/CD27low. In contrast, LLO-specific CD4 T cells in the spleen and MLN were comprised of four subsets based on CD27 and Ly6C expression. Upon challenge, mucosal CD4 T cells phenotypically resembled those in the primary infection, except for the appearance of a PD-1+ subset. Mucosal CD27low Ly6Clow and splenic CD27lowLy6Clow or high CD4 T cells either produced both IFN-γ and IL-2 or IFNγ alone and little IL-17 was detected. These results indicated that antigen-specific intestinal effector and memory CD4 T cells were phenotypically and functionally distinct from their lymphoid counterparts. Furthermore, the distinct phenotype of mucosal memory CD4 T cells implied that these cells may not recirculate. The generation of mucosal memory CD4 T cells in response to oral infection suggested a potential protective role for these cells in maintaining barrier infection.