bluuebunny/crossref_metadata_2025 huggingface dataset:
Host-derived IL-6 utilizes TGF-β of tumor origin to modulate the differentiation of Treg cells and Th17 against cancer cells (101.26)
Authors: Chiang Hsin-Chien , Chu Rea-Min
Published: January 2023

TGF-β is a key inducer of Treg differentiation, and the presence of both TGF-β and IL-6 is vital to Th17 generation, which plays an important role in autoimmunity. Canine transmissible venereal tumor (CTVT) produces TGF-β during the progression (P) and regression (R) phases; however, during the R phase, tumor-infiltrating lymphocytes (TIL) secrete IL-6 to antagonize TGF-β. We have demonstrated that the level of Treg in peripheral blood mononuclear cells (PBMCs) dramatically increased in the P phase and decreased in R phase, and that exogenous IL-6 reduced Treg generation in TGF-β-containing medium but increased Th17-related mRNA expression. Further, upon addition of supernatant from a P-phase culture, the percentage of Treg cells significantly increased in PBMCs; however, the level decreased when R-phase supernatant was added, and the Th17-associated mRNA level increased. Moreover, using Affymetrix microarray analysis, we identified an autoantigen, YKL40, that was highly-expressed in the R phase. Antibodies against YKL40 were found to decrease the number of IFN-γ-producing cells in PBMCs by ELISPOT. These results suggested TIL-derived IL-6 utilizes TGF-β originating from the tumor, resulting in an increase in Th17, which may enhance the host autoimmunity and consequently break the tumor tolerance against YKL40. These complex interactions may explain how the tumor regresses in canine hosts. Further, YKL40 has the potential for use in the development of a treatment for cancer.
Characterization of a tumor-associated antigen on 9L gliosarcoma (101.31)
Authors: Gaffrey Matthew , Redd Joan
Published: January 2023

Cell surface antigens play a distinct role in the process of cell transformation and proliferation. The malignant, transformed counterparts of glial cells are referred to as glioma cells. Tumors or cancers resulting from the proliferation of these glioma cells constitute some of the most lethal types of cancer. We have characterized a tumor-associated antigen (TAA) expressed on the cell membrane of the 9L rat glisoarcoma (9L-Ag). Previous work has identified this antigen as a glycoprotein. In this study, we further characterize expression of 9L-Ag by normal brain cells, 9L cells, and a non-glial tumor cell line (P3X3Ag8.653). Characterization of 9L-Ag may provide a better understanding of the transformation processes from a normal to a malignant cell, and the role that 9L-Ag and similar TAAs may play in these processes.
Influence of intermolecular disulfide bonds on assembly of mouse MHC class I molecules (130.25)
Authors: Simone Laura , Wang Xiaojian , Tuli Amit , Solheim Joyce
Published: January 2023

Within the major histocompatibility (MHC) class I peptide-loading complex, tapasin and ERp57 are joined by a stable disulfide bond which is necessary for the efficient assembly of MHC class I molecules. In our investigation of murine tapasin, we identified a large covalently bound complex involving wild type mouse tapasin and ERp57. This complex, which migrates at ~150 kDa, is noticeably larger than the 110 kDa tapasin-ERp57 complex previously identified in human cells. Additional data suggest that the mouse MHC class I molecule Kd may be a part of this ~150 kDa complex. In order to investigate the importance of tapasin-ERp57 disulfide bonding for the assembly of murine MHC class I molecules, we introduced a tapasin C95S mutant into tapasin knockout mouse fibroblasts. Although this tapasin mutant is unable to disulfide bond with ERp57, mouse tapasin C95S was found to maintain a non-covalent association with ERp57. Mutation of tapasin C95 impaired the maturation and stability of MHC class I Kd molecules to a greater extent than was previously observed for the human MHC class I molecule, B*4402. Furthermore, the maturation and stability of mouse MHC class I Kb molecules was also impaired in cells expressing mouse tapasin C95S. In total, these results highlight inter-species differences in the MHC class I assembly pathway and emphasize the importance of studying diverse MHC class I allotypes.
Adjuvanted Flu Vaccination Using a Novel High-Efficiency Hollow Microprotrusion Skin Patch (130.37)
Authors: Chang Stella , Kaspar Roger , Fairman Jeff , Speaker Tycho
Published: January 2023

Influenza spreads around the world in seasonal epidemics, resulting in the deaths of hundreds of thousands worldwide annually, and millions in pandemic years. Hypodermic needle-based administration of current vaccines to large underserved populations is hampered by refrigeration requirements, painful injections, and infectious sharps waste disposal issues. We have developed a prototype micro-protrusion array device (PAD), comprising water-soluble polymer microneedles loaded with a dry-stable adjuvanted flu antigen vaccine for topical application as a patch. Three groups of mice were tested to evaluate the efficacy of PAD vaccination: Non-treatment, Fluzone (Flz) and Flz adjuvanted with JVRS-100. PADs were applied to anesthetized mice with a primary dose, followed by a second boost dose applied two weeks later. Four weeks after the primary dose, the animals were challenged with 1x LD50 of HKx31 (H3N2) Flu virus. Serum antibody levels were assessed before the challenge. Total IgG of Anti-Flz was 6 fold higher in the Flz/JVRS-100 than Flz. Control and Flz mice had 60% mortality while the Flz/JVRS100 group had a 100% survival rate. We conclude that the dry PAD adjuvanted vaccine format may provide a powerful new approach to transdermal vaccine delivery. Immunization potency is significantly increased through adjuvanted antigen which generating a robust immune response that was protective against lethal influenza virus challenge.
Enhanced IL-2 signaling renders IL-12 primed CD8+ T-cells superior in adoptive immunotherapy. (131.20)
Authors: Lisiero Dominique , Soto Horacio , Liau Linda , Prins Robert
Published: January 2023

Sustained persistence and function of adoptively transferred T-cells are critical parameters for effective anti-tumor activity. To address this, our group has explored the mechanisms by which TLR agonist-induced cytokines, such as IL-12, are capable of efficiently priming CD8+ T-cells. IL-12 primed CD8+ T-cells upregulate the expression of the high affinity IL-2 receptor (CD25) and STAT5. Enhancement of these signaling components increases IL-2 mediated signaling events at low concentrations of IL-2. Blockade with anti-CD25 antibody effectively terminates this enhanced IL-2 signaling in IL-12 primed CD8+ T-cells. More importantly, IL-12 priming of CD8+ T-cells significantly increases in vivo proliferation as measured by CFSE dilution, and ex vivo recovery from lymphoid organs. Utilization of in vivo bioluminescent imaging of firefly luciferase transduced IL-12 primed T-cells demonstrated their ability to traffic into the tumor area, through lymph nodes and bone marrow. Finally, adoptive transfer of IL-12 primed CD8+ T-cells decreases tumor burden in a subcutaneous melanoma model and increases survival in an intracranial tumor model. This anti-tumor activity can be abrogated by addition of an anti-CD25 blocking antibody. These results demonstrate the importance of pro-inflammatory signals in the tumor setting, and their ability to render CD8+ T-cells functional and responsive to proliferative signals.
Vaccination with tumor cell lysate plus CpG oligodeoxynucleotides elicits tumor-specific T cells with potent antitumor efficacy in adoptive immunotherapy (131.31)
Authors: Shindo Maki , Chen Wei
Published: January 2023

This study investigated whether tumor-specific T cells can be elicited by vaccination with tumor cell lysate plus CpG oligodeoxynucleotides (CpG/lysate) for adoptive immunotherapy. Vaccination of C57BL/6 mice with syngeneic EL-4 tumor cell lysate mixed with a CpG-B ODN (CpG 685) significantly increased the number of T cells and activated DCs in the draining lymph nodes (DLN). Mice vaccinated with CpG/lysate developed protective immunity against EL-4 tumor challenge with a significant longer median survival time (P<0.01). The CpG/lysate-primed DLN T cells were expanded in a 5-day culture with anti-CD3 mAb and IL-2, and adoptively transferred into EL-4 tumor-bearing mice following cyclophosphamide (CY) treatment. Therapy with CY plus CpG/lysate-primed T cells induced an 80% tumor-free survival rate whereas EL-4 mice in the control groups all died within 30 days. When the mice cured by CpG/lysate-primed T cells were re-challenged with EL-4 tumor 2 months later, all the mice (100%) survived without tumor growth, suggesting the persistence of T cell-mediated antitumor immunity. The efficacy of CpG/lysate-primed T cells was comparable to DC/lysate vaccine-primed T cells in adoptive immunotherapy. Cell-depletion studies showed that CD4 T cells play a pivotal role in CpG/lysate vaccine-induced antitumor effects. The results suggest that CpG/lysate vaccine provides an effective approach to induce host antitumor immunity and tumor-reactive T cells for adaptive immunotherapy.
Investigating the involvement of Foxo1 in T cell ageing (132.1)
Authors: Gubbels Bupp Melanie
Published: January 2023

Advanced age is associated with increased susceptibility to and mortality from infectious diseases, especially influenza and pneumonia; and vaccination responses in the elderly are diminished. Declining resistance to infection in aging individuals is correlated with a progressive decline in the size and functionality of the naïve T cell pool. A better understanding of the mechanisms underlying age-related deterioration of T cell immunity could inform therapies that enhance general immune function and vaccination effectiveness in this vulnerable population. The forkhead transcription factor, Foxo1, has recently been identified as a major regulator of naïve T cell homeostasis. Moreover, a Foxo1 homolog, DAF-16, promotes longevity in C. elegans. Therefore, these studies documented the decline of the naïve T cell pool in mice selectively deficient for Foxo1 in the T cell compartment (Foxo1 cKO mice) to determine the involvement of Foxo1 in ageing T cells. Our studies indicate that naïve CD8+ CD122- T cells decline at a more rapid rate in the blood of Foxo1 cKO mice as compared to WT controls. Together, these observations suggest the intriguing possibility that in mice, Foxo1 may be involved in retarding age-related deterioration of T cell immunity.
Upregulation of B220 expression correlates with sensitivity to arsenic trioxide (As2O3)-induced cell death in different leukemic T-cell lines (132.11)
Authors: Bobé Pierre , Benbijja Mohcine
Published: January 2023

As2O3 kills a variety of leukemia cells, but with different efficacy. Although cellular redox status is considered essential for As2O3 sensitivity, we show here that murine and human leukemic T-cell lines display dramatic differences in As2O3 sensitivity independently of their GSH content and O2− production. Unexpectedly, As2O3 differently upregulated expression of phosphatase B220 on these T cells in dose-dependent manner. Since we previously reported that As2O3 selectively eliminates pathogenic B220+ T cells in autoimmune MRL/lpr mice (Bobé et al, BLOOD, 2006, 108:30967), we have studied the possible correlation between B220 expression and As2O3-induced T-cell death in leukemic T-cell lines. In the presence of As2O3, we found low (HPB-ALL), intermediate (Jurkat) and high (EL-4, BW5147) levels of both B220 expression and cell death. Ca2+ ionophore A23187 also upregulated B220 expression and cell death, but with opposite efficiencies in the T-cell lines compared to As2O3. A23187 only upregulated the T-cell activation marker CD69 before B220 expression and cell death, suggesting that it kills by an activation-induced cell death mechanism. In L1210 cells, we found constitutive B220 expression. L1210 cells were the most resistant to cell death among As2O3-treated T-cell lines whereas they were the most sensitive to A23187. As2O3 and A23187 probably trigger different signaling pathways through B220 leading to T-cell death, suggesting a checkpoint role for B220 in death pathways.
An evaluation of techniques to efficiently induce in vitro T-cell expansion. (132.29)
Authors: Langsdorf Chris , Bantly Andrew , Bradford Jolene , Buller Gayle , Liu Jixiang
Published: January 2023

The current study evaluated three in vitro methods of inducing proliferation in human T lymphocytes. The first method involved treatment with mouse anti-human CD3 antibody and interleukin-2. The second approach used the lectin Concanavalin A, a mitogen which preferentially activates T cells. The third technique made use of mouse anti-human CD3 and CD28 antibodies immobilized on the surface of superparamagnetic polystyrene microspheres. Human lymphocytes were isolated from fresh whole blood and stained with CellTrace™ Violet, a novel cell proliferation tracing dye with a mode of action similar to CFSE. Samples of stained cells were stimulated and cultured for 7 days. T cell subsets induced to proliferate with each treatment were characterized in multicolor flow cytometry. Cells treated with anti-CD3/IL-2 or Concanavalin A showed a moderate, heterogeneous level of stimulation. Microsphere-bound antibodies provided a much stronger, more uniform stimulus. This strong, homogeneous stimulus is useful when massive numerical expansion of T cells is desired for subsequent research or adoptive transfer in vivo, but does not appear to be as useful when a population containing several discrete generations of cells is desired. The more moderate stimuli resulting from treatment with anti-CD3/IL-2 or Concanavalin A result in a less efficient numerical expansion, but provide an excellent tool to evaluate the phenotype and pedigree of cells as they proliferate through several generations.
Influence of partial agonist expression on CD8 T cell responses for non-self antigens (132.35)
Authors: Ryu Su Jeong , Sproule Tom , Christianson Gregory , Roopenian Derry , Choi Eun Young
Published: January 2023

Differences in affinity of TCR for peptide/MHC complex induced by single amino acid substitutions in the peptide dramatically alter recognition by T cell in the periphery. With H60 as an antigenic model, we investigated the influence of transgenic expression of natural H60 antigen (H60N Tg) or of H60 antigen with single amino acid substitution at position 4 to histidine (H60H Tg) on H60H- or H60N-specific CD8 T cell responses, respectively. Immunization of B6 female mice with splenocytes from male H60NTg or H60H Tg mice induced strong CD8 T cell responses against each epitope, H60N or H60H, in CD4 T cell help dependent ways. While the immunization with splenocytes from male H60H Tg induced proliferation of H60-specific CD8 T cell responses in the female H60N Tg mice, CD8 T cell response for H60H induced in female H60N Tg showed significant reduction in the magnitude of H60H-tetramer-binding CD8 T cells at the peak time. And differences in TCR V beta usages of H60H-reactive CD8 T cells were also found between in the female B6 female and female H60N Tg hosts. Our data suggest that expression of proteins with amino acid changes influences the antigen-specific CD8 T cell responses and repertoire of TCRS involved in the responses.
Regulation of Surface Free Thiol Levels During CD8+ T Cell Activation, Proliferation and Differentiation (132.7)
Authors: Pellom Samuel , Michalek Ryan , Grayson Jason
Published: January 2023

Upon both B and T cell activation the level of reactive oxygen intermediates (ROI) increases. Prior studies in the literature demonstrated that activated lymphocytes have increased total levels of surface free thiols, potentially as a compensatory mechanism to prevent surface oxidation. In this study, we determined how surface free thiol levels were regulated during the activation, proliferation, and differentiation of naïve CD8+ T cells both in vitro and in vivo. In vitro, we found that the levels of surface free thiols increased within 24 hours regardless of the activation stimulus and were maintained for at least three divisions. To understand how levels were modulated in vivo, we examined antigen-specific CD8+ T cells using lymphocytic choriomeningitis virus (LCMV) infection of mice and MHC Class I tetramers. On day 5 postinfection, the levels of surface free thiols were 8-fold higher on effector cells compared to those found on naïve CD8+ T cells. By day 8 postinfection this had decreased to 4-fold. After the infection was cleared, memory CD8+ T cells had 1.5-fold higher levels of surface free thiols than naïve cells. This contrasted with the results during chronic LCMV-Clone 13 infection. On day 8 postinfection surface free thiol levels were 11-fold higher and remained elevated until day 35 postinfection. These results suggest that surface free thiol levels are elevated most highly in rapidly proliferating cells and do not correlate with lymphocyte function.
STAT3 controls the neutrophil migratory response to CXCR2 ligands by direct activation of G-CSF-induced CXCR2 expression and via modulation of CXCR2 signal transduction (133.2)
Authors: Nguyen-Jackson Hoainam , Panopoulos Athanasia , Zhang Huiyuan , Li Haiyan , Watowich Stephanie
Published: January 2023

Neutrophil mobilization, the release of neutrophils from the bone marrow reserve into circulating blood, is important to increase peripheral neutrophil amounts during bacterial infections. Granulocyte colony-stimulating factor (G-CSF) and chemokines, such as MIP-2 (CXCL2), can induce neutrophil mobilization, but the mechanism(s) they employ remain unclear. The transcription factor STAT3 is the principal intracellular signaling molecule activated upon G-CSF-ligation of its receptor. Using a murine model with conditional STAT3 deletion in bone marrow, we demonstrated previously that acute G-CSF-responsive neutrophil mobilization and MIP-2-dependent neutrophil chemotaxis is mediated by STAT3. In this study, we show that STAT3 is also necessary for MIP-2-elicited neutrophil mobilization. STAT3 appears to function by regulating the amplitude of Raf/MEK/Erk activation, a signaling pathway that is important for MIP-2-mediated chemotaxis. In addition, we demonstrate that G-CSF stimulates the expression of the MIP-2 receptor via STAT3-dependent transcriptional activation of Il8rb. G-CSF treatment also induces changes in bone marrow chemokine expression levels in a STAT3-dependent manner, which may further affect neutrophil retention and release. Taken together, our study demonstrates that STAT3 regulates multiple aspects of chemokine and chemokine receptor expression and function within the bone marrow, indicating a central role in the neutrophil mobilization response.
Atypical protein kinase C (aPKC) isoforms ζ and λ/ι synergically control Th2 development and function (134.13)
Authors: Yang Jun-Qi , Diaz-Meco Maria , Moscat Jorge
Published: January 2023

The protein kinase C (PKC) family consists of three subfamilies: classical, novel, and atypical PKCs. The atypical PKC (aPKC) consists of two isoforms, PKCζ and PKCλ/ι. We have recently presented in vitro, ex vivo, and in vivo genetic evidence that PKCζ is necessary for optimal activation of the IL-4 signaling cascade upstream of Jak1. Since whole-body genetic deletion of PKCλ/ι is lethal, we have used a conditional gene knockout system by loxP-Cre recombination to generated conditional PKCλ/ι KO mice to selectively delete PKCλ/ι in activated T cells. With this system, we found that PKCλ/ι was necessary for Th2 cytokine production. Upon anti-CD3/CD28 stimulation, IL-4 and IL-13 production from CD4+ T cells was significantly reduced in conditional KO mice as compared to their WT littermates. The activation of the transcription factors NFAT and NF-κB was impaired in PKCλ/ι-deficient activated T cells. Furthermore, deficiency of PKCλ/ι inhibited OVA-induced allergic airway inflammation in vivo. Recently, we have introduced the activated T cell-specific conditional PKCλ/ι deficiency into a PKCζ-/- background to generate mice with PKCζ and PKCλ/ι double knockout (DKO) in activated T cells. From these T cells, the secretion of Th2 cytokines, IL-4, IL-5 and IL-13, was more dramatically reduced and the proliferation was more significantly impaired compared to T cells with single PKCζ or PKCλ/ι deficiency. PKCλ/ι, like PKCζ, emerges as a new critical regulator of Th2 cell activation.
Dendritic cells in the skin of autoimmune mice display an inability to migrate: a novel mechanism and role in skin inflammation (135.12)
Authors: Eriksson Anna , Kim Peter , King Jennifer , Okereke Chetachi , Singh Ram
Published: January 2023

Tissue-resident DC carry antigens to tissue-draining lymph nodes (LN) to induce immunity. To investigate the role of this DC function in the pathogenesis of autoimmune disease, we analyzed the migration of skin-resident DC, namely Langerhans cells (LC) and dermal DC, in lupus dermatitis. LC and dermal DC, but not blood-derived DC, from lupus-prone MRL-lpr and MRL+/+ mice exhibited defects in their ability to emigrate from the epidermis, to migrate through dermal lymphatics, and to immigrate into LN (JI 2008). Since conventional DC migration assays require DC activation, we used Lang-EGFP knock-in (KI) mice to visualize LC at steady-state. EGFP+ cells (LC) were lower in LN of KI MRL mice than of KI B6 mice, thus confirming what we found using inbred mice. Importantly, skin DC migration defect precedes disease and correlates with the severity of dermatitis. Conversely, treatment with αGalCer restores skin DC migration and ameliorates dermatitis. Surprisingly, αGalCer-mediated increase in skin DC migration does not require NKT cells, but is associated with epidermal γδ T cell (eγδT) expansion in a CD1d-dependent manner. Finally, eγδT are reduced in MRL mice, and deficiency of CD1d or eγδT, but not of NKT, reduces skin DC migration and worsens dermatitis. Thus, we elucidate a novel mechanism, whereby CD1d-dependent eγδT normally facilitate skin DC migration. This regulatory mechanism is impaired in lupus dermatitis, providing evidence for a novel mechanism of autoimmune disease.
Dominant epitope p21-40 of snRNP D protein recognized by both autoreactive T and B cells in lupus-prone MRL lpr/lpr mice (135.22)
Authors: Ding Chuanlin , Cai Yihua , Yan Jun
Published: January 2023

Aberrant T and B cell responses to multiple nuclear and cytoplasmic Ags such as small nuclear ribonucleoparticles (snRNPs) are a hallmark of systemic lupus erythematosus. Although Th1 response is considered to be predominant in lupus pathogenesis, the identification of Th17 cells may challenge Th1 paradigm. To investigate the respective roles of Th1, Th17, and Treg cells in lupus development, we analyzed different T cell subsets in spleen and kidney of lupus-prone MRL lpr/lpr mice. With increasing age and disease severity, the IFN-gamma-producing T cells were significantly increased. No significant change was observed in the IL-17-secreting CD4 T cells. Furthermore, the ration of Th1/Treg was significantly increased when mice aged while Th17/Treg ratio remained unchanged. To determine the antigenic epitopes recognized by autoreactive T and B cells, synthesized peptides of autoAg snRNP D protein were used. Two epitopes were recognized by autoreactive T cells while three epitopes were recognized by autoantibodies in MRL lpr/lpr mice. Interestingly, peptide 21-40 of snRNP D protein was recognized by both autoantibodies and T cells. Furthermore, one of T cell hybridomas generated from snRNP D-immunized B10.A Tg mice was also responsive to peptide 21-40. Taken together, our studies demonstrate that Th1 response remains dominant in lupus-prone MRL lpr/lpr mice and p21-40 of snRNP D protein appears to be a pathogenic epitope that can be recognized by both autoreactive B and T cells.
Chronic Stimulation of the DNA Repair Enzyme DNA-PKcs Impairs T-Cell Survival in Rheumatoid Arthritis (RA) (135.7)
Authors: Shao Lan , Goronzy Jörg , Weyand Cornelia
Published: January 2023

Patients in RA have premature aging of the immune system, with accumulation of pro-inflammatory and tissue-destructive T cells. Accelerated immune senescence has been attributed to chronic T cell loss. Naïve T cells in RA have a defect in repairing damaged DNA. We have examined whether chronic stimulation of DNA repair mechanisms affects T cell survival in RA. Naïve CD4+CD45RA+ T cells from RA patients had a higher load of double-stranded DNA breaks (p=0.0005) and spontaneous apoptosis rates (p=0.03) than in healthy controls. High DNA damage failed to upregulate the ATM-p53 axis. Instead, the transcript and protein levels of the DNA repair enzyme DNA-PKcs were elevated. Induction of DNA-PKcs was disease-specific and not found in patients with SLE. Knocking down DNA-PKcs transcription or inhibiting enzyme activity with a kinase inhibitor normalized T cell apoptosis rates in patients. Activation of DNA-PKcs was correlated with stimulation of SAPK/JNK pathway. Inhibition of either DNA-PKcs or JNK suppressed the pro-apoptotic protein Bim and protected T cells from apoptotic death. T cells in RA patients accumulate damaged DNA, causing chronic induction of repair activity. DNA double-strand breaks are recognized by a ATM-independent pathway involving DNA-PKcs. Due to the activation of the JNK-Bim death signaling cascade, RA T cells are apoptosis sensitive. Therapeutic blockade of DNA-PK-dependent cell death machinery could help rejuvenate the immune system of RA patients.
Nod1 deficiency inhibits autophagy against Salmonella typhimurium in macrophages (136.21)
Authors: Anand Paras , Kanneganti Thirumala-Devi
Published: January 2023

Autophagy is a conserved bulk-degradation process involving double-membraned structures called autophagosomes that degrade damaged organelles. Recently, it has been reported that autophagy is important in immunity to microbial pathogens whereby intracellular bacteria are targeted for clearance by autophagy. However, the exact mechanism of how these microbes are identified by autophagy within host cells is still not clear. Innate immune cells rely on pathogen recognition receptors such as nucleotide-binding oligomerization-domain (NOD) like receptor (NLR) family to mount an appropriate immune response against pathogens. The NLR proteins Nod1 and Nod2 were shown to be important for bacterial clearance in dendritic cells. In this context, we found that Nod1 regulates autophagy in macrophages infected with Salmonella typhimurium . Infection of wild-type bone marrow-derived macrophages with Salmonella increased LC3-I lipidation to LC3-II as observed by Western blot. However, Nod1 deficient macrophages showed reduced LC3-II expression and thus autophagy. There were fewer cells with LC3-GFP autophagomes in macrophages deficient for Nod1 as compared to infected wild-type macrophages. Activation of Nod1 and Nod2 initiates a pro-inflammatory response by NF-kB pathway through the recruitment of adaptor protein RICK. We found a similar result in RICK deficient macrophages infected with Salmonella . Our results show a potential role for Nod1 in antibacterial autophagy activation.
Reprogramming of TLR4 signaling in endotoxin tolerance: altered IRAK4 and TAK1 activation, K63-linked polyubiquitination and signalosome assembly of IRAK1, TRAF6, IKKγ and increased A20 expression (136.23)
Authors: Xiong Yanbao , Qiu Fu , Piao Wenji , Song Chang , Wahl Larry M , Medvedev Andrei E
Published: January 2023

Prior exposure to LPS induces endotoxin tolerance that impairs TLR4-elicited pro-inflammatory cytokines without inhibiting anti-inflammatory or anti-microbial mediators, and is important for pathogenesis of the anti-inflammatory response syndrome and secondary infections in septic patients. The impact of endotoxin tolerance on the earliest TLR4 signaling events is largely unknown. We report that endotoxin tolerization impairs LPS-mediated activation of IRAK4 and TAK1 in human monocytes and THP1 cells. Overexpression of wild-type and mutant ubiquitin variants and the use of antibodies discriminating K48- vs. K63-linked polyubiquitin chains revealed that endotoxin tolerization compromises LPS-induced K63-linked, but not K48-linked polyubiquitination of IRAK1 and TRAF6. Diminished K63-linked polyubiquitination of IRAK1 and TRAF6 correlated with compromised LPS-inducible IRAK1-TRAF6 and IRAK1-IKKγ complex assemblies observed in endotoxin-tolerant cells. Alterations in these proximal signaling events manifested by LPS-tolerant cells correlated with increased expression of A20, an essential de-ubiquitination enzyme that targets TRAF6 to remove K63-linked polyubiquitin chains. Thus, deficiencies in LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6 and IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased expression of A20 represent new critical molecular determinants of endotoxin tolerance.
The role of TLR3 in protection of diabetes by PolyI:C in NOD mice (136.31)
Authors: Xiang Yufei , Wen Li , Zhou Zhiguang , Wong F. Susan
Published: January 2023

We aim at dissecting the timing and the role of TLR3 in the PolyI:C protective effect in NOD mice using adoptive transfer models. Irradiated young WT or TLR3-/- NOD mice were used as recipients. We treated the recipients with PolyI:C before or after adoptive transfer. Splenocytes from diabetic wild type or TLR3-/- NOD mice were transferred into non-diabetic, young WT or TLR3-/- NOD recipients in a criss-cross approach. Our results showed that treatment with PolyI:C either before or after adoptive transfer in the NODàNOD system significantly protected from diabetes development (p=0.022).This is particularly evident in treatment after adoptive transfer. Interestingly, in the NOD to TLR3-/- system, treatment with Poly I:C after adoptive transfer accelerated diabetes development. In the TLR3-/- to NOD groups PolyI:C administration again showed a protective effect and it appears that treatment with Poly I:C before adoptive transfer had a better protective effect (p=0.047).Finally, to further differentiate the role of TLR3 in hematopoietic from non-hematopoietic cells, we performed bone marrow chimera experiments without any exogenous treatment. Interestingly, the onset of diabetes was faster and the overall disease development was also higher in NOD to TLR3-/- chimeras (p=0.04). In summary, our results suggest that the Poly I: C protective effect is likely to be time dependent and expression of TLR3 in non-hematopoietic cells may be important in diabetes development.
Papillomavirus virus-like particles trigger IL-1beta release through the canonical inflammasome activation signaling pathway (136.33)
Authors: Zhong Zhenyu , Zhai Yougang , Qiao Liang
Published: January 2023

Papillomavirus virus-like particles (VLPs), formed by self-assembled caspid L1 protein, are extremely immunogenic. They can be used both as a mucosal delivery vector and an adjuvant for treating human diseases. However, the mechanism(s) by which papillomavirus VLPs function as an adjuvant is still unclear. We hypothesized that papillomavirus VLPs activate inflammasome in antigen-presenting cells to function as an adjuvant. In order to test our hypothesis, we cocultured HPV16, BPV-1 VLPs or a BPV-1 VLP presenting HIV-1 gp41 neutralizing epitope (chimeric HIV-BPV VLPs) respectively with PMA-primed human THP-1 cells and determined IL-1beta release in the presence or absence of inhibitors of the Nlrp3-inflammasome pathway. We found that HPV-16, BPV-1 VLPs and chimeric HIV-BPV VLPs are capable of activating inflammasome in PMA-primed THP-1 cells, which eventually results in the release of IL-1beta. The release of IL-1beta by the VLPs requires the uptake of VLPs by macrophages as inhibition of clathrin-mediated endocytosis blocked IL-1beta release. Furthermore, the inhibition of cathepsin B or caspase-1 abolished IL-1beta release in the presence of the VLPs. Finally, we also found that generation of reactive oxygen species (ROS) is necessary for activating inflammsome since inhibition of ROS production reduced the level of released IL-1beta. Thus, our results demonstrated that papillomavirus VLPs can activate inflammasome through the canonical inflammasome activation pathway.
BK channel controls the TLR-induced pro-inflammatory activation of macrophages via IκBα/NFκB and p38 MAPK signaling. (136.35)
Authors: Dolganiuc Angela , Nelson Oranmiyan
Published: January 2023

Background: Macrophages (Mf) are key in innate immunity and inflammation. Toll-like receptors (TLR) sense bacterial-derived products and trigger the inflammation. Big-conductance (BK) channels contribute to cell repolarization and participate in signal transduction. The role of BKs in innate immunity is largely unknown. Methods: We stimulated RAW246.7 Mf with ligands of pro-inflammatory TLRs (TLR2-PGN, TLR4-LPS or TLR7/8-R848)± BK blocker iberiotoxin or opener NS1619 and analyzed RNA by PCR, protein by ELISA and western blot, DNA-binding by EMSA. Results: TLRs augmented BK RNA and protein levels. Chemical BK block inhibited Mf activation via TLR2, TLR4 and TLR7/8, indicated by reduced production of TNFα, IL-1, IL-6, IL-8 at the levels of both RNA and secreted protein. The inhibitory effect of BK block was time- and dose-dependent and was specific to stimulation with TLR ligands (not observed upon Mf stimulation with TNFα). Upstream in signaling, BK block prevented TLR-induced activation of the transcription factor NFκB in an IκBα-dependent manner. Further, BK block prevented MAPK p38 phosphorylation but spared activation of ERK. In contrast BK opener augmented LPS-induced NFkB activation and TNFα production with minimal effect of MAPK; its effect on TLR2- and TLR7/8-induced Mf activation was minimal. In conclusion, we identified that BK channels are key for TLR-induced pro-inflammatory activation of macrophages and could be useful for the control of TLR/Mf function.
Monophosphate Lipid A's TRIF-biased TLR4 signaling results in weak NLRP3 inflammasome priming and reduced IL1beta production, leading to decreased inflammation in vivo compared to Lipid A. (136.38)
Authors: Eaves Chelsea , Franchi Luigi , Roy Eron , Nunez Gabriel , Mitchell Thomas
Published: January 2023

Monophosphate Lipid A (MLA), a PAMP variant of the Lipid A moiety from LPS, exhibits 100-fold reduced toxicity than its parent molecule. We previously characterized MLA as TRIF-biased agonists of TLR4, meaning that while inductions of TRIF-dependent transcripts are comparable to Lipid A, MyD88-dependent transcripts are greatly reduced. Previous studies showed that MLA fails to induce IL1beta production from monocytic cell culture, and it was suggested that the lack of production of this pro-inflammatory cytokine could be responsible for MLA’s reduced toxicity. Our initial goal was to reconcile TRIF-biased TLR4 signaling and IL1beta loss with MLA’s reduced induction of inflammatory mediators. We find that MLA’s TRIF-biased TLR4 signaling leads to weak NLRP3 inflammasome priming and reduced IL1beta production from murine bone marrow derived dendritic cells. Comparison of inflammatory transcripts from spleen and liver of MLA and Lipid A treated WT or IL1RImutant mice suggests that IL1beta plays a critical role in the toxicity of Lipid A in vivo. We propose that TRIF-biased TLR4 signaling by MLA results in weak inflammasome priming, reduced IL1beta production and decreased induction of inflammatory mediators in vivo.
The Heat Shock Protein gp96 play a multifaceted role in regulating Toll-like receptor 9 (136.40)
Authors: Leifer Cynthia , Brooks James , Cameron Jody , Chiosis Gabriela
Published: January 2023

Inappropriate activation and failure to inactivate Toll-like receptors (TLRs) contribute to immune mediated pathology. Nucleic acid sensing TLRs, including TLR9, are tightly regulated to prevent self DNA and RNA recognition, which can contribute to autoimmune disease. The overall goal of our research is to determine the molecular mechanisms governing nucleic acid sensing TLRs using TLR9 as a model. Regulation of TLR9 includes control of intracellular trafficking by association with proteins such as the endoplasmic reticulum chaperone gp96. Obligate proteolytic cleavage of TLR9 to an 80 kDa form, p80, precedes TLR9 recognition of CpG DNA. Here we show that in the absence of gp96, there is a failure to generate p80. Furthermore, gp96 traffics with TLR9 to the endosomal compartment. Dissociation of gp96 from TLR9 using specific inhibitors leads to rapid degradation of the receptor and inhibition of signaling. CpG DNA pull down experiments demonstrate that gp96 is associated with the ligand-bound form of the receptor suggesting it contributes to CpG DNA recognition. We provide a model where gp96 plays a multifaceted role in TLR9 biology. These studies have major implications for pharmacologically manipulating the levels of active and inhibitory forms of TLR9 and other nucleic acid sensing TLRs, which will likely uncover novel therapeutic interventions for autoimmune diseases. AI076588 and AI076588-S1 (CAL).
Identification of dTOR as a signaling component in the Drosophila immune response (136.41)
Authors: Duan Yukan , Powell Jonathan
Published: January 2023

We have previously demonstrated a critical role for the mammalian Target Of Rapamycin (mTOR) in regulating T effector and regulatory cell differentiation. In light of the fact that TOR is highly evolutionarily conserved, we hypothesized that this signaling pathway would play a role in Drosophila immune responses. In Drosophila the Toll pathway, which is characterized by the induction of drosomycin, is responsible for fighting fungal and gram positive infections while the immune deficiency (IMD) pathway, which is characterized by attacin induction, responds to gram negative infection. Drosophila S2 cells were transfected with either drosomycin or attacin reporter constructs and appropriately stimulated in the presence of rapamycin. For both signaling pathways, rapamycin inhibited lucierase induction in a dose dependent fashion. Alternatively, S2 cell phagocytosis of either E. coli or S. aureus which is not dependent on these pathways was not inhibited by rapamcyin. Furthermore, western blot analysis of LPS stimulated S2 cells demonstrated increased Drosophila TOR (dTOR) activity as measured by phoshorylation of S6K-1. Overall our data identifies dTOR as playing a heretofore unappreciated role in the Toll and IMD pathways in Drosophila. Dissecting the precise role of dTOR in Drosophila immunity should provide important insight into the downstream mechanisms by which mTOR directs adaptive immune responses.
IL-10- and PD-1-dependent regulation of CD8+ T cell effector function in an adenoviral murine model for liver tropic viral infection (137.12)
Authors: Dolina Joseph , Hahn Young
Published: January 2023

Immune responses to pathogens in the liver are uniquely characterized by a tolerogenic feature as a result of constant exposure to bacterial constituents and food-derived antigens. Little is known about the molecular basis of the immune regulation to persistent viral infection in the liver. In these studies, we employed a model comparing subcutaneous (SC) and intravenous (IV) adenovirus infection, which result in the trafficking of CD8+ T cells to the liver with effective or defective antiviral function, respectively. During IV infection, intrahepatic CD8+ T cells display an inability to produce proinflammatory cytokines and lyse targets. IV adenovirus infection distinctively results in local intrahepatic antigen presentation responsible for limiting the CD8+ T cell immune response. Importantly, CD8+ T cells mounted by the IV infection demonstrate drastic up-regulation of PD-1 on their surface and produce IL-10 at the peak of the CD8+ T cell response. In addition, IL-10 producers elevate PD-1 expression, and PD-L1 blockade attenuates CD8+ T cell IL-10 production but increases IFN-γ production. These results suggest that the PD-1 and IL-10 signaling pathways appear to be dependent on each other. Interestingly, IL-10-producing CD8+ T cells have also been found in the livers of patients harboring hepatitis C virus, implicating a common regulatory role for these cells in liver tropic viral infections.
Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites (137.16)
Authors: Cockburn Ian , Chen Yun-Chi , Overstreet Michael , Lees Jason , Farber Donna , Zavala Fidel
Published: January 2023

Immunization with irradiated sporozoites is the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization - a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate into liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite antigen.
Galectin-1 reduces the severity of herpes simplex virus induced ocular immunopathological lesions (137.20)
Authors: Rajasagi Naveen , Reddy Pradeep , Suryawanshi Amol , Rouse Barry
Published: January 2023

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye caused by herpes simplex virus-1 (HSV-1) infection and one of the most common causes of blindness in humans. The inflammatory lesions are primarily perpetuated by IFN-γ producing CD4+ T cells. Therefore, targeting activated CD4+ T cell subsets represents a potentially valuable form of therapy to reduce the severity of disease. Galectin-1, an endogenous lectin, was recently shown to suppress certain autoimmune disorders through the selective inhibition of T helper type1 (TH1) and interleukin-17 producing T helper cells (TH-17). In the present report, we determined if galectin-1 administration begun at different times after ocular infection with HSV could influence the severity of HSK lesions. Treatment with recombinant galectin-1 (rGal-1) significantly reduced the extent of angiogenesis and SK lesions that occurred. Treated mice had reduced numbers of inflammatory cells that included TH1 and TH-17 cells as well as neutrophils in the cornea. The mechanisms by which galectin-1 acts will be described. The many effects include apoptosis of effector CD4+ T cells, induction of regulatory T cells, increased production of anti-inflammatory cytokines and inhibitory effects on molecules involved in neovascularization and SK pathogenesis. Our findings are the first to show that rGal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.
IL-2 therapy synergizes with PD-L1 blockade to rescue exhausted CD8+ T cells during chronic viral infection (137.24)
Authors: West Erin , Ha Sang-Jun , Tan Wendy , Jin Hyun-Tak , Freeman Gordon , Smith Kendall , Ahmed Rafi
Published: January 2023

Blocking PD-1 signaling, by administering anti-PD-L1 or anti-PD-1 antibodies, has proven to be effective in partially restoring CD8+ T cell function during multiple chronic infection models. However since PD-L1 blockade does not completely rescue CD8+ T cell function, we have focused on finding treatments that synergize with PD-1 blockade. Our lab has previously shown that IL-2 administration can increase the frequency of LCMV-specific CD8+ T cells and decrease viral load. Using a chronic model of lymphocytic choriomeningitis virus (LCMV) we demonstrate that combined IL-2 administration and PD-L1 blockade induces massive increases in LCMV-specific CD8+ T cells and an increased ability to produce inflammatory cytokines to multiple epitopes, including subdominant epitopes, over PD-L1 blockade alone. Furthermore, combined treatment results in greater reduction of viral loads. Importantly, IL-7Rα (CD127), a marker of functional memory cells that is not seen on exhausted cells, even after anti-PD-L1 treatment, was up-regulated on a population of LCMV-specific CD8+ T cells after IL-2 or, even more drastically, after combined treatment. These results indicate that combined IL-2 and PD-1 blockade may prove a potential therapy for increasing CD8+ T cell responses and viral control in human chronic infections.
Regulatory T cell expression of herpes virus entry mediator (HVEM) following HSV-1 infection and its functional significance. (137.25)
Authors: Sharma Shalini , Sundararajan Aarthi , Mulik Sachin , Rouse Barry
Published: January 2023

In many infections especially those that are chronic, the outcome may be influenced by the activity of one or more types of regulatory T cells (Tregs), particularly those that express FOXP3. In many instances, for example the response that follows HSV infection, the majority of expanded Tregs are not antigen specific. In this report, we demonstrate one possible mechanism by which HSV-1 infection could act to signal and expand the Treg population. We show that HVEM, a tumor necrosis factor receptor superfamily member which interacts with the gD protein of HSV-1 to facilitate cell entrance, is expressed by FOXP3+ T cells and that engagement of HVEM by its ligand expands the Treg population. To demonstrate this, FOXP3-GFP knock in mice were infected with HSV kos and change in cell numbers of Tregs and their expression levels of HVEM were recorded in draining popliteal lymph node (DLN). Tregs expansion occured following HSV-1 infection, peaking at around day 8 compared to the peak numbers of CD8+T cells (day 5 p.i). Both FOXP3+ and FOXP3- CD4 T cells expressed HVEM in the resting state but Tregs upregulated HVEM reaching highest MFI at day 7 p.i. In-vitro stimulation of DLN cells with UV inactivated HSV kos, further up regulated HVEM on Tregs (possibly through gD) and down regulated on FOXP3_ cells suggesting a possibility that engaging this receptor with gD expressed by infected cells results in Treg activation and expansion which in turn modulates CD8+ effector responses.
Expression of CD35 on subsets of human peripheral CD8+ T-cells (138.18)
Authors: Reilly Brian , Ketron Summer , Hempstead Shannon , Atkinson John
Published: January 2023

There is now considerable evidence that CD8+ T cell reactions can promote or suppress autoreactivity. The type of response is not only dictated by the TCR but also by other cellular receptors. Also known, is that natural deficiencies of certain complement components are associated with autoimmune responses. The potential link between complement and CD8+ T-cells was explored by examining several subsets for the expression of the complement receptor CD35. Mononuclear cells were purified from the peripheral blood of ten donors and further enriched for CD8+ T-cells by either positive or negative selection using immunomagnetic beads, and analyzed for the presence of CD35 by flow cytometry. Upon inspection, 6-12% expressed CD35 and were negative for CD56. Of the CD35+ cells, approximately 65% were found in the naïve subset (CCR7+CD45RA+), 15% in the CD45RA effector memory subset (CCR7-CD45RA+), and 10% in the central memory (CCR7+CD45RA-) and effector memory (CCR7-CD45RA-) subsets. Next, using plate-bound antibodies against CD3 and CD35, in vitro activation experiments were done followed by analysis of several T-cell responses. The results showed a substantial increase in cytokine secretion and intracellular granzyme production with minimal effect on cellular proliferation, compared to TCR cross-linking alone. These data imply that the CD8+ T-cells, which express CD35, modulate T-cell responsiveness following co-engagement of the TCR and CD35.