bluuebunny/crossref_metadata_2025 huggingface dataset:
Clostridium difficile Toxin B blocks effector T cell proliferation by inhibiting PLD signaling. (49.19)
Authors: Chandrasekaran Uma , Iwashima Makio
Published: January 2023

Balance between effector and regulatory T cell subsets play significant role in regulating immune responses. Alterations in this balance would result in suppressed or hyper-immune responses and could lead to disorders such as autoimmunity or infections. Our previous work showed that Phospholipase D (PLD) signaling is a factor that can affect the balance between regulatory T cells (Tregs) and non-Tregs. Phospholipase D is a phosphodiesterase that catalyzes the conversion of phosphatidyl choline (PC) to phosphatidic acid (PA). The data demonstrated that inhibition of PLD signaling preferentially blocks growth of non-regulatory T-cells while allowing Tregs to proliferate in response to T cell receptor (TCR) stimulation. Various endogenous and external factors could modulate PLD signaling in T cells and thus alter the host immune responses. Toxin B secreted by Clostridium difficile (TcdB) is a well documented factor that blocks PLD signaling. Here, we show that TcdB produced by Clostridium difficile selectively blocks expansion of effector T cells. Like PLD signal inhibition, TcdB caused inhibition of CD25 expression by activated non-Tregs but allowed expression of CD25 expression by Tregs. Thus, TcdB mediated T cell growth inhibition is likely caused by PLD signaling inhibition in host T cells and may enable C.difficile to modify host immune response, facilitating its long term survival that leads to disease conditions like pseudomembranous colitis.
AMPA receptors are present on mouse lymphocytes and AMPA receptor antagonist, GYKI-52466, inhibits lymphocyte proliferation. (50.31)
Authors: Manivannan Sivabalan , Griffin Diane
Published: January 2023

AMPA receptors are glutamate-gated ion channels canonically found on mammalian neurons for excitatory neurotransmission. AMPA receptors are composed of a tetrameric combination of subunits, GluR1-4, in the form of a homo or hetero dimers. The presence of a post-transcriptionally edited GluR2 subunit confers Ca++ impermeability to the ion channel. Because treatment of mice with viral encephalitis with AMPA receptor antagonists inhibits inflammation and protects from fatal paralysis, we studied the presence and function of AMPA receptors on lymphocytes. Using RT-PCR and restriction digest assays of mRNAs obtained from mouse spleen cells, we showed the presence of unedited mRNA for Ca++-permeable GluR2 and for variants with alternative splicing patterns. Using an in vitro CFSE proliferation assay with plate bound anti-CD3/CD28 we showed that the noncompetitive AMPA receptor antagonist, GYKI-52466, suppresses T-cell receptor CD3+ lymphocyte proliferation in a dose dependent manner, with the greatest effect on CD4+ T-cells. Decreased proliferation correlated with decreased production of IL-2. Lymphocytes treated with GYKI-52466 did not show an increase in cell death compared to controls. These studies show that AMPA receptors are present on lymphocytes and participate in the proliferative response to signaling through the T-cell receptor.
Regulation of type-III interferons in airway epithelial cells. (51.13)
Authors: Gallagher Grant , Siegel Rachael , Eskdale Joyce
Published: January 2023

The type-III interferons (IFNs) are the most recently discovered IFN family, comprising three protein ligands: IFN-λ1, IFN-λ2 and IFN-λ3. Their expression is induced by viruses and they share many anti-viral characteristics with the type-I IFNs (IFN-α and IFN-β), but type-III IFNs have an important specific functional niche at the immune-epithelial interface. However, their expression is diminished in bronchial epithelial cells of rhinovirus-infected asthmatic individuals. We set out to understand type-III IFN regulation. Here, we identify critical elements for IFN-λ1 promoter regulation in bronchial epithelial cells and define key activators (c-REL/p65 NF-κB heterodimer and IRF-1) and repressors (p50 NF-κB homodimer, BLIMP-1 and ZEB1). We show that IRF-1 and BLIMP-1 may occupy an overlapping promoter element. Following poly I:C stimulation, IFN-λ1 expression peaks at 3 hours. This is accompanied by loss of repression by p50 NF-κB homodimers at the promoter. Two important activators are recruited to the promoter: the c-REL/p65 NF-κB heterodimer and IRF-1. Post-induction repression is mediated by p50 NF-κB homodimers, ZEB1 and BLIMP-1. We show that ZEB1 does not regulate type-I IFN expression; thus, it is the first specific regulator of the type-III IFNs. This study represents the first characterization of any type-III IFN promoter in its native context and conformation, and can now be applied to understanding pathogenic dysregulation of IFN-λ1.
Role of reactive oxygen species in gender-associated autoimmunity (83.15)
Authors: Gangaplara Arunakumar , Massilamany Chandirasegaran , Waldner Hanspeter , Reddy Jay
Published: January 2023

Gender-bias exists in proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in that both male and female SJL mice develop disease but only females show chronic relapses and their underlying mechanisms are unknown. It has been shown that the reactive oxygen species (ROS) play a role in the mediation of autoimmune diseases, but the recent evidence suggests that ROS can suppress autoimmune responses. We hypothesize that T cell-derived ROS are critical to the maintenance of self-tolerance and favor developing resistance to autoimmunity in male mice. To address this hypothesis, we used PLP 139-151-specific T cell receptor (5B6) transgenic (tg) mice and measured ROS production in activated 5B6 T cells by flow cytometry using CM-H2DCFDA as an ROS indicator. Unexpectedly, we found that T cells from female mice produced two fold more ROS when compared to males. We verified these differences by evaluating mRNA expression of oxidative and anti-oxidative defense genes in 5B6 tg T cells and PLP 139-151-specific T cells sorted from immunized wild type (wt) SJL mice by using PLP 139-151 tetramers. TaqMan PCR analysis revealed that interleukin-22 mRNA, a cytokine implicated in ROS metabolism and EAE pathogenesis was increased by three fold in T cells obtained from tg or wt female mice. The data suggests that sex steroids and T cell-derived cytokines have a role in the maintenance of redox homeostasis in autoreactive T cells and T cell tolerance.
Inability of Monoclonal Antibodies to Clear Red Blood Cells during Incompatible Transfusions is not a Property of the Whole Cell (84.19)
Authors: Liepkalns Justine , Cadwell Chantel , Hod Eldad , Spitalnik Steven , Zimring James
Published: January 2023

During an incompatible transfusion (Tx), antibodies (Abs) against some blood group antigens (Ags) will lead to hemolysis while others will allow for continued red blood cell (RBC) circulation despite IgG binding. We tested the nature of RBC clearance using transgenic mice expressing well-defined RBC Ags and monoclonal Abs. Tx of RBCs, expressing human glycophorin A (hGPA), into mice treated with anti-hGPA IgG1 monoclonal Abs led to partial clearance of those RBCs. A population of RBCs persisted and continued to circulate in a 2nd animal also passively immunized against hGPA, indicating that survival was not due to splenic saturation or consumption of the monoclonal Abs. We therefore concluded that these hGPA RBCs were resistant to clearance as they were also positive for hGPA and bound Abs. These studies were also performed using RBCs expressing the Duffy Ag and anti-Duffy IgG2a monoclonal Abs. To test if resistance comprises a whole cell protection from Ab-induced lysis, we transfused blood expressing both hGPA and Duffy Ags into mice passively immunized against hGPA. The resistant RBCs were then transfused into mice passively immunized against Duffy. Although these RBCs were able to resist a 2nd Tx against the same anti-hGPA Ab as the one used during the 1st Tx, these did not resist a 2nd Tx during which anti-Duffy was used. We, therefore, conclude that resistance occurs with different Ags and is not a property of the cell but rather is at the Ag-Ab level.
Isolation and Expansion of Antigen-Specific Mouse T cells using Dynabeads® (85.9)
Authors: Schjetne Karoline , Reed Berit
Published: January 2023

Background: CD134 (OX-40) and CD137 (4-1BB), both members of the TNFR-family, functions as costimulatory molecules promoting proliferation and survival of activated T cells. CD134 and CD137 also identifies recently activated CD8+ and CD4+ T cell and represent a promising approach for the isolation and expansion of viable T cells of a defined specificity. Results: Dynabeads® Mouse CD134 isolates activated antigen-specific T cells by use of an agonistic anti-CD134 antibody conjugated to Dynabeads®. The bead-bound cells are separated on the magnet promoting a prolonged activating signal to the T cells during culture. Dynabeads® Mouse T-Activator CD3/CD28/CD137 enables an easy expansion of the isolated T cells to be used for downstream analysis. Discussion: Dynabeads® Mouse CD134 allows for specific isolation of T cell with defined antigen specificity directly from a crude sample (e.g. splenocytes or tissue from immunized mice) in an easy-to-use in vitro procedure. Its use in combination with Dynabeads® Mouse T-Activator CD3/CD28/CD137 enables easy and reliable isolation and expansion of T cells of a defined specificity. With Dynabeads® Technology, pure, viable T cells can be conveniently isolated for downstream analysis.
Minimum Information About a Genotyping Experiment (MIGen) (88.8)
Authors: Huang Jie , Marthandan Nishanth , Pertsemlidis Alexander , Ding LiangHao , Kozlitina Julia , Maher Joseph , Olsen Nancy , Rios Jonathan , Story Michael , Xing Chao , Scheuermann Richard
Published: January 2023

Genotyping experiments are widely used in clinical and basic research laboratories to identify associations between genes and normal/abnormal phenotypes. Genotyping assay techniques vary from PCR reactions in a test tube with gel electrophoresis detection to high throughput chip or bead array assay employing various technologies for automatic genotype calls. Depending on the study purpose and design, the resulting genotype data could involve millions of markers for thousands of individuals, requiring substantial data analysis using various statistical, modeling or other methodologies. To date, there is no standard for reporting genotyping experiments. Here we present the Minimum Information About a Genotyping Experiment (MIGen) standard. MIGen recommends the standard information required to report a genotyping experiment, covering: study and experiment design, subject information, sample collection and processing, genotyping procedure, and data analysis methods, if applicable. The goal of MIGen is to set a reporting standard for adoption by the research community to facilitate consistent data interpretation and independent validation/reproduction, and to serve as guidance for database design for storing genotyping experiment data. MIGen is being developed as a collaborative project involving international domain experts and is a registered project under MIBBI: Minimum Information for Biological and Biomedical Investigations. Supported by NIH N01AI40076.
ATP and lipoteichoic acid act cooperatively to induce a DC phenotype that promotes Th17 in an IL-23- and IL-1B-dependent manner (89.28)
Authors: Koski Gary , Paustian Christopher , Johnson Terrence
Published: January 2023

We investigated whether pairing TLR ligands with an “endogenous” danger signal, extracellular adenosine triphosphate (ATP), would act in synergy to induce in monocytes unique DC-like properties. The TLR2 ligand lipoteichoic acid (LTA) and ATP were efficient partners for cooperatively enhancing expression of IL-23 and IL-1b when monocytes were cultured in GM-CSF, but not when IL-4 was included. These cells showed DC-like properties including activated DC surface phenotype and enhanced migration towards CCR7 ligands. They also preferentially skewed allogenic CD4pos T lymphocyte responses toward the IL-22hi, IL-17hi/IFN-glo Th17 phenotype, a capacity inhibited by neutralization of either IL-23 or IL-1b, but not IL-6. Although pharmacological activation of either ionotropic or cAMP-dependent pathways acted in together with LTA to enhance IL-23, only inhibition of the cAMP-dependent pathway antagonized enhancement of cytokine production by ATP. In addition, ATP plus atypical lipopolysaccharide from P. gingivalis (signaling through TLR2) was superior to E. coli-derived LPS (TLR4 ligand) for promoting the high IL-23-secreting DC-like phenotype, but considerably inferior for inducing IL-12 p70 production when paired with IFN-g. Collectively, our data suggests TLR2 ligands encountered by innate immune cells in an environment rich in extracellular ATP can induce a distinct activation state that supports an IL-23- and IL-1b-dependent Th17 type response.
Identification of Critical Residues in the Natural Killer (NK) Cell Receptor KIR3DS1 that Control HLA (Human Leukocyte Antigen) Binding (89.55)
Authors: O'Connor Geraldine , Yamada Eriko , Rampersaud Andy , McVicar Daniel
Published: January 2023

The Killer cell Immunoglobulin-like Receptor (KIR) family of NK receptors contains both activating (e.g. 3DS1) and inhibitory (e.g. 3DL1) members. Unlike 3DL1, for which binding to HLA-Bw4 has been characterized, direct binding of HLA to 3DS1 has not been observed. However both genetic studies and in vitro viral replication assays suggest that this interaction can occur and is important in the context of viral infection. 3DL1 and 3DS1 are very similar in the region predicted to interact with HLA, suggesting the subtle differences translate into dramatic functional consequences. Modeling suggests four key differences in the D1 and D2 domains likely to affect interactions with Bw4. Single substitutions in this region dramatically reduced binding by 3DL1, suggesting that any of these changes would result in a lack of binding by 3DS1. In the reciprocal experiment however, we found that reversion of single amino acid residue - position 138 (W to G) was sufficient to confer HLA-Bw4 binding to 3DS1. The HLA-Bw4 reactivity of this mutant was dependent on the residue present at other variable positions, with loss of HLA-Bw4 binding seen with double mutants carrying the 3DL1 residue at both position 138 (G) and position 199 (P). These data provide evidence for a key role for position 138 in determining the HLA binding ability and thus ligand specificity of 3DS1, while suggesting a complex interaction of the residues in this region to determine receptor reactivity and specificity.
Decreased antigen-specific responses and increased lung pathology in MRL-lpr mice following influenza infection. (92.13)
Authors: Crowe Sherry , James Judith , Anderson Jourdan , Edwards Erica
Published: January 2023

Influenza virus infection result in significant morbidity and mortality, especially in individuals with impaired immune systems, such as those with systemic lupus erythematosus. It is unclear, however, whether this increased morbidity is due to intrinsic immune dysfunction or immunosuppressive therapy. To address these questions we used the MRL-lpr mouse model of lupus-like disease. Following infection, MRL-lpr mice had increased weight loss and lung pathology compared to controls. These signs of increased susceptibility were not due to increased viral replication or delayed viral clearance. Splenocytes from MRL-lpr mice did, however, make significantly less TNFα and more IFNγ which might account for the increased weight loss and pathology. Both lupus-prone and control mice developed high levels of anti-influenza antibodies, but the MRL-lpr mice had significantly less antigen-specific antibodies at each time-point. MRL-lpr mice also had decreased numbers of influenza-specific CD8+ T cells. This decreased antigen-specific response and lung pathology was not due solely to the absence of the fas gene since B6-lpr mice were able to mount an immune response equivalent to that seen in B6 mice. This model will provide a unique system to dissect the immune-pathogen interaction in an autoimmune disease setting and provide valuable information on how autoimmunity impacts the development of antigen specific immunity.
Direct discovery of an influenza B peptide displayed by HLA-B*0702. (92.15)
Authors: Fogle Matthew , Wahl Angela , Bardet Wilfried , Schafer Fredda , Buchli Rico , Hildebrand William
Published: January 2023

Annual influenza epidemics caused by influenza A and B viruses substantially impact global public health. The proportion of infections due to influenza B fluctuates from season to season, but on average accounts for 20% of influenza cases each year. We used a direct discovery method to screen for influenza B epitopes presented by HLA-B*0702. We identified epitope TIRLVTEEL (TL9) that lies within the influenza B non-structural protein 2 (NS2) also known as nuclear export protein (NEP). The TIRLVTEEL sequence is found in 86% of influenza B strains (38 out of 44 sequences) and it is a high-affinity binder to HLA-B*0702. To test the immunogenicity of this peptide, we isolated splenocytes from influenza B-infected HLA-B*0702 transgenic mice and found that TIRLVTEEL generated a strong IFN-gamma production by ELISPOT. As three variants of this epitope occur in nature, each differing from TIRLVTEEL by a single amino acid, we tested each variant for B*0702 binding and for cross-recognition by ELISPOT. Each variant is a high affinity HLA-B*0702 binder and each stimulates IFN-gamma production. In summary, we discovered a conserved epitope within the influenza B non-structural protein 2 (NS2) or nuclear export protein (NEP) which may be a candidate for future CTL-based therapies directed towards the influenza B virus.
Influenza A infection enhances cross-priming of CD8+ T cells to cell-associated antigens in a TLR7- and type I interferon-dependent fashion (92.6)
Authors: Chen Weisan , Wei Joe , Waithman Jason , Lata Roleen , Mifsud Nicole , Cebon Jonathan , Kay Thomas , Smyth Mark , Sadler Anthony
Published: January 2023

The initiation of anti-tumor immunity relies on DCs to cross-present cell-associated tumor antigen to CD8+ T cells due to a lack of co-stimulatory molecules on tumor cells. Innate danger signals have been demonstrated to enhance cross-priming of TCD8+ to soluble as well as virally encoded Ags, however their effect on enhancing TCD8+ cross-priming to cell-genome encoded Ags remains unknown. Furthermore, Influenza A virus has not been shown to enhance anti-tumor immunity. Using influenza-infected allogeneic cell lines, we show here that TCD8+ responses to cell-associated Ags can be dramatically enhanced due to enhanced TCD8+ expansion. This enhanced cross-priming in part involves TLR7-, but not TLR3-, mediated sensing of IAV and is entirely dependent on MyD88 and IFN signaling pathways. We also showed that the inflammasome and IFN-r did not play a role in enhancing cross-priming in our system. We further demonstrated in our ex vivo system that CD8+ DCs are the only antigen-presenting cells able to prime TCR transgenic TCD8+. Importantly, pDCs and CD8- DCs were all able to enhance such priming when provided in co-culture. These observations suggest that IAV-infection of tumor cells may facilitate improved cross-presentation of tumor Ags and may be used to augment clinical vaccine efficacy.
Experimental autoimmune uveitis in mice with defective Th1 and Th17 effector responses (93.17)
Authors: Damsker Jesse , Luger Dror , Silver Phyllis , Chan Chi-Chao , Iwakura Yoichiro , Caspi Rachel
Published: January 2023

Experimental autoimmune uveitis (EAU) induced in mice by immunization with the retinal antigen IRBP is a model for autoimmune uveitis in humans. Recently, we showed that Th1 and Th17 responses are redundant for induction of EAU and can substitute for each other. This study examines the effect of deficiency in both the Th1 and the Th17 signature cytokines, IFN-γ and IL-17, on susceptibility to EAU by using IL-17/IFN-double knockout (DKO) mice. Despite lack of both IFN-γ and IL-17, the DKO mice developed full-blown EAU. They exhibited enhanced Th2 responses and undiminished production of IL-22 and proinflammatory cytokines such as IL-1 and TNFα at the systemic level. At the local level, DKO eyes showed strongly elevated Th2 transcription factors, GATA-3 and c-Maf, Th2 cytokines such as IL-13 and IL-5, and eosinophil/granulocyte attracting chemokines. In keeping with this, histological analysis of diseased eyes uncovered a granulocytic/eosinophilic infiltrate in the DKO mice, rather than a lymphocytic/monocytic infiltrate typical of wild type mice. Similarly to WT mice, EAU in DKO mice was inhibited by treatment with a neutralizing Ab to TNF-alpha. Together, these findings suggest the presence of a pathogenic effector response with Th2-like characteristics that develops in the concurrent absence of IFN-γ and IL-17, in which TNF-α remains a pathogenic cytokine. Further studies using neutralizing Abs to other expressed cytokines will address their role in pathogenesis.
Antibodies elicited in response to EBNA-1 may cross-react with dsDNA. (93.29)
Authors: Yadav Pragya , Ebegbe Roland , Gottlieb Paul , Mumbey-Wafula Alice , Kaplan Atira , Spatz Linda
Published: January 2023

Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the EBNA-1 protein, developed antibodies to double stranded DNA (dsDNA). The present study was undertaken to determine the basis for this anti-dsDNA response. We now demonstrate that BALB/c mice immunized with recombinant EBNA-1 protein develop antibodies to EBNA-1 that cross-react with dsDNA. Close examination of two monoclonal antibodies to EBNA-1 has demonstrated their reactivity with dsDNA but not unrelated proteins and absorption on dsDNA cellulose columns removes their reactivity to both dsDNA and EBNA-1. In addition, these monoclonal antibodies recognize different regions of the EBNA-1 protein suggesting that more than one epitope in EBNA-1 may be the target of antibodies that cross-react with dsDNA. These results support the association of SLE with EBV and emphasize the need to prevent EBV infection or to mask EBNA-1 protein from the immune system following EBV exposure, in order to minimize the risk of autoimmunity.
The early inflammatory response to bites of Leishmania major infected Phlebotomus duboscqi sand flies in naïve and pre-exposed mice. (94.15)
Authors: Teixeira Clarissa , Oliveira Luiz , Gomes Regis , Meneses Claudio , Elnaiem Dia-Eldim , Kamhawi Shaden , Valenzuela Jesus
Published: January 2023

Pre-exposure to uninfected sand fly bites (USFB) protects against Leishmania major infection. Little is known about the initial immune response responsible for this protection. During blood feeding, the insect sand fly inoculates Leishmania parasites into the skin alongside saliva. Here, we explore the early inflammatory response in the skin of pre-exposed (PE) mice challenged with L. major-infected P. duboscqi flies. The relative expression of cytokines and chemokines by skin cells were investigated 2- 48h post-infection using a macroarray (Oligo GEArray®). There is a rapid and strong early expression of inflammatory genes in PE compared to naive mice following challenge. Two to twelve hours post-infection, naïve mice showed an increased expression of MCP-1, MCP-3 and eotaxin that attract macrophages and granulocytes while PE mice upregulated TCA-3, BRAK and I-TAC related to migration of NK cells, macrophages, granulocytes and activated T cells. Characterization of leukocytes by FACS 24-48h post-infection showed a significantly increased recruitment of neutrophils, NK cells, macrophages and CD4+ cells in PE mice. PE mice also presented a higher frequency of IFN-γ producing CD4+ and NK cells 24h post-infection. This suggests that induced cytokines and chemokines are altered by pre-exposure to USFB. Thus, protection from Leishmania infection observed in PE mice may result from Th1 priming by an anti-saliva immunity that acts in concert with a modulated innate immune response.
Repercussions of Brucella metabolic activity on protection against virulent Brucella melitensis challenge in mice. (94.17)
Authors: Magnani Diogo , Harms Jerome , Durward Marina , Splitter Gary
Published: January 2023

Brucella spp are highly infective human intracellular pathogens that cause the most prevalent worldwide zoonotic disease, with over 500,000 human infections yearly. No human vaccine exists. Since killed and subunit vaccines present limited efficacy and live attenuated strains retain unacceptable levels of virulence, optimal design for a brucellosis vaccine requires a non-living vaccine that confers effective immunity against Brucella infected cells. Because host cell infection requires synthesis and secretion of new antigens, we hypothesized that bacterial metabolic activity generates the antigenic and adjuvant environment required for efficient triggering of protective responses. Here, we demonstrate that inactivation of B. melitensis by γ-irradiation inhibited its replication capability, yet retained live-Brucella protective features. Irradiated Brucella possessed transcriptional and oxidation-reduction activity, persisted in macrophages, generated antigen-specific cytotoxic T cells and protected mice against virulent bacterial challenge, without signs of residual virulence. In conclusion, pathogen metabolic activity has a positive role in shaping protective responses, and the generation of inactivated yet metabolically active microbes is a promising strategy for safely vaccinating against intracellular organisms like B. melitensis.
Role of hypoxia induced autophagy in the resistance of tumor cells to CTL-mediated specific lysis. (95.11)
Authors: Noman Muhammad , Janji Bassam , Moer Kris , Pierson Sandrine , Buart Stéphanie , Berchem Guy , Mami-Chouaib Fathia , Chouaib Salem
Published: January 2023

Hypoxia is a hallmark of solid tumors. We have recently demonstrated that exposure of tumour targets to hypoxia inhibits CTL induced autologous target cell lysis. Such inhibition is correlated to the cooperative induction of pSTAT3 and HIF-1α. Recent studies show that autophagy plays an important role in protecting cells against hypoxic stress, but the relationship between hypoxic stress and autophagy remains unclear. Here, we provide evidence that hypoxia-dependent impairment of tumor susceptibility to CTL-mediated cell lysis is associated with the induction of autophagy in tumor cells. Interestingly, inhibition of autophagy by targeting Beclin-1 or ATG-5 was sufficient to restore hypoxic tumor susceptibility to CTL and to inhibit the hypoxia-dependent phosphorylation of STAT3 on tyrosine-705 residue (pSTAT3). Furthermore, we show that the inhibition of pSTAT3 in hypoxic Atg5 or beclin-1-targeted tumor cells involves the degradation or the inhibition of the Src family protein kinase by a mechanism that involves the Ubiquitin Proteasome System (UPS). Overall, our data identify a new mechanism of pSTAT3 degradation by cooperate regulation of autophagy and the UPS under hypoxic stress. To our knowledge, this study establishes for the first time a novel functional link between hypoxia induced autophagy and tumour susceptibility to CTL-mediated lysis and highlight the use of autophagy inhibitors in immunotherapy for modulating tumor cell resistance to CTL-mediated lysis.
Simvastatin induces ER stress and p53-mediated apoptosis via eIF2α (95.12)
Authors: Lee Sang Kyu , Lim Ho Yong , Kim Young Sang
Published: January 2023

In addition to cholesterol lowering effect, statins have pleiotropic effects including cytotoxicity to cancer cells. In this study, we investigated the signaling pathways leading to apoptosis induced by simvastatin. Simvastatin induced apoptosis was accompanied by stabilization and translocation of p53 and Bax to mitochondria. The stabilization and the translocation of Bax were alleviated in p53 knockdown MethA and p53-/- HCT116 cells, suggesting p53-dependence of Bax translocation. Simvastatin also induced cardinal features of ER stress; phosphorylation of PERK, eIF2α, and JNK, up-regulation of CHOP and Grp78, and activation of caspase-12. Salubrinal, an inhibitor of dephosphorylation of eIF2α, sustained phosphorylation of eIF2α, suppressed the simvastatin-induced apoptosis, and reduced the stabilization and translocation of p53 and Bax. SP600125, a JNK kinase inhibitor, did not affect the apoptosis and the translocation of p53 and Bax in the condition. Furthermore, PERK knockdown or over-expression of a mutant form of eIF2α (Ser51/Ala) sensitized MethA and HCT116 cells to the simvastatin-induced apoptosis. Over-expression of the eIF2α-mt also enhanced the stabilization of p53 and Bax in response to simvastatin. These results indicate that the PERK-eIF2α axis is involved in stabilization and translocation of p53 and Bax to mitochondria, and eIF2α plays an essential role in induction of ER stress-mediated apoptosis by simvastatin.
Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells (MDSC) (95.16)
Authors: Liu Yuelong , Xiang Xiaoyu , Zhuang Xiaoying , Zhang Shuangyin , Liu Cunren , Cheng Ziqiang , Michalek Sue , Grizzle William , Zhang Huang-Ge
Published: January 2023

Tumor cells released membrane microvesicles - exosomes, can stimulate inflammation through MDSC, and then promote tumor growth by activating immunosuppressive networks. MyD88 is a cytoplasmic adaptor molecule essential for inducing inflammation via the TLR receptors. Very little is known about whether tumor exosome-mediated induction MDSCs and tumor growth is regulated through MyD88. In this study, we observed that mice pretreated with tumor exosomes had a significant acceleration of tumor metastasis in the lung. Tumor metastasis correlated significantly with an increase in recruitment of more MDSCs in the lung of B6 mice pretreated with tumor exosomes. These effects were blunted when MyD88 KO mice were pretreated with tumor exosomes. MDSCs induced by tumor exosomes and isolated from WT B6 mice are also more potent in inhibition of T cell activation and induction of IL-6 and TNF-α than MDSCs isolated from the lung of MyD88 KO mice via activation of the Stat3 pathway. Our in vitro data further support that addition of tumor exosomes to bone marrow derived CD11b+Gr-1+ cells isolated from WT B6 mice results in producing more cytokines, including TNF-α, IL-6 and the chemokine CCL2, than CD11b+Gr-1+ cells isolated from MyD88 KO mice. Less CCL2 in lung was observed in MyD88 KO mice pretreated with tumor exosomes than that in WT mice. Together these data demonstrate a pivotal role for MyD88 in tumor exosome-mediated induction of IL-6, TNF-α, expansion of MDSCs and tumor metastasis.
Murine AIDS and the role of PD-1, PD-L1 and IL-10 in the development of LP-BM5 retrovirus induced CD11b+GR-1+ suppressor cells. (98.26)
Authors: Green Kathy , Green William
Published: January 2023

Pathology due to the immune system’s response to viral infections often represents a delicate balance between inhibition of viral pathogenesis and regulation of protective immunity. In susceptible C57BL/6 mice the murine retroviral isolate LP-BM5 induces splenomegaly, hypergammaglobulinemia, B-and T-cell immunodeficiency, increased susceptibility to opportunistic pathogens and terminal B-cell lymphomas. We have reported that B6.PD-1 and B6.IL-10 knockout(k.o.) mice are more susceptible to LP-BM5-induced disease than wild-type (w.t.) C57BL/6 mice. By reciprocal reconstitution experiments, comparing w.t. vs. PD-1 k.o. sources of the requisite cells for LP-BM5 pathogenesis - CD4 T and B cells, PD-1+ B cells appear crucial in the normal limitation of LP-BM5 induced disease in B6 mice. Recent studies have shown: 1) LP-BM5 infected PD-L1 k.o. mice also develop more severe splenomegaly, hypergammaglobulinemia, and immunodeficiency than infected B6 mice 2) CD11b+GR-1+ spleen cell subsets, from LP-BM5 infected w.t., IL-10 k.o., PD-1 k.o. and PD-L1 k.o. mice are all suppressive in in vitro LPS proliferation assays with suppression most complete for IL-10 k.o. and PD-1 k.o. derived cells. Experiments in progress include: 1) adoptive transfer to define the crucial cell subset required for PD-L1 normal limitation of LP-BM5 induced disease and 2) phenotypic and mechanistic further characterizing CD11b+GR-1+ LP-BM5 induced suppressor cells.
A novel MHC class Ib-mediated immune surveillance mechanism detects the absence of ERAAP (100.16)
Authors: Nagarajan Niranjana , Gonzalez Federico , Shastri Nilabh
Published: January 2023

Immune surveillance mechanisms detect failures in vital parts of the host immune system, such as in the antigen processing pathway. The ER aminopeptidase associated with antigen processing, ERAAP, is an essential component of the antigen processing pathway, and is required to generate the final peptides presented by MHC class I molecules. It has been unclear how the immune system detects deficiencies in ERAAP function, and here we describe a potential mechanism to do so. Non-classical MHC class I, or MHC class Ib, molecules present novel peptides in ERAAP-deficient cells, and elicit potent CD8 T cell responses in wild-type mice. Using these CD8 T cells as a probe, we identified a novel peptide presented by Qa-1b. FL9-Qa-1b-specific CD8 T cells can be readily detected in naïve mice and constitute a substantial fraction of the anti-ERAAP-/- immune response in wild-type mice. Additionally, WT CD8 T cells detect and eliminate MHC class Ib-expressing ERAAP-deficient cells. Therefore, we suggest that presentation of the FL9 peptide by Qa-1b molecules is a surveillance mechanism for detecting inhibition of ERAAP function.
GILT regulates CD4+ T cell tolerance (100.28)
Authors: Rausch Matthew , Metzger Todd , Anderson Mark , Hastings Karen
Published: January 2023

Regulating the balance between T cell activation and tolerance is essential for stimulating an anti-tumor immune response and in controlling autoimmunity. We have shown that gamma-interferon-inducible lysosomal thiol reductase (GILT) is required for efficient MHC class II-restricted processing of an epitope from the melanoma antigen tyrosinase-related protein 1 (TRP1). Using CD4+ TRP1-specific T cell receptor transgenic mice, here we demonstrate a novel function for GILT in the regulation of TRP1-specific T cell tolerance. TRP1-specific T cells are centrally deleted in mice expressing both GILT and TRP1 on the RAG-/- background. In contrast, CD4+ T cells develop in the absence of either GILT or TRP1. GILT’s role in central tolerance is supported by GILT and TRP1 expression in thymic stromal cells. Although TRP1-specific CD4+ T cells escape thymic deletion in the absence of GILT, they are tolerant to TRP1 and do not induce vitiligo. TRP1-specific T cells from GILT-deficient mice initially divide in response to antigen, but fail to produce substantial levels of IFN-γ and IL-2. Tolerance is partially mediated by CD25+Foxp3+ regulatory T (Treg) cells. There is an increased percentage of TRP1-specific Treg cells in GILT-deficient compared to TRP1-deficient mice, and Treg cell depletion allows GILT-deficient TRP1-specific CD4+ T cells to induce vitiligo. These studies highlight a critical role for GILT in shaping the CD4+ T cell repertoire.
S100A4 is dispensable for T cell development and steady state lymph node entry and migration (102.15)
Authors: Chen Yi-Chun , Krummel Matthew
Published: January 2023

T cell activation involves delicate coordination of different signaling pathways. Recent studies of one small calcium binding protein, S100A4, indicate that S100A4 is a promising molecular target for controlling metastasis, possibly through regulating T cell response. The level of S100A4 presence inside and outside of the cells has long been used as a prognosis marker in different human cancers. However, the mechanism(s) of how S100A4 functions intracellularly (from inside of cells) and/or extracellularly (from outside of cells) remains obscure. In S100A4 knockout (KO) mice, the number of thymocytes, splenocytes, and lymphocytes are comparable to wild type (WT) mice, and the developmental profile of S100A4 KO thymocytes are also normal. However, upon stimulation by anti-CD3 and anti-CD28 antibodies, KO thymocytes have higher proliferation rate compared to wt cells, especially at the lower signaling strength. This indicates that S100A4 may be involved in thymocyte survival and selection signaling pathways. In addition, in the presence of high concentration of ICAM, S100A4 KO T cell blasts showed more constrained motility comparing to wt blasts in vitro. However, even the homing of naïve KO T cells is impaired, their motility was normal in vivo. These indicate that S100A4 may play a more important role in T cell infiltration, rather than the steady state motility.
The VCAM-1 cytoplasmic domain is essential for endothelial cell signaling and leukocyte trafficking. (102.17)
Authors: Marchese Michelle , Cook-Mills Joan
Published: January 2023

Under inflammatory conditions, leukocytes migrate into the tissue in a process regulated by adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1). We have reported that leukocyte ligation to VCAM-1 initiates an endothelial cell signaling cascade that is necessary for VCAM-1-dependent leukocyte transendothelial migration. It is unknown whether the cytoplasmic domain of VCAM-1 is involved in initiating the VCAM-1 signaling cascade to enable leukocyte transendothelial migration. Notably, the 19 amino acid VCAM-1 cytoplasmic domain is identical in most mammalian species, including human, mouse and common shrew. This interspecies conservation suggests that the VCAM-1 cytoplasmic domain is important for VCAM-1 signaling function. To test this hypothesis, we generated stably transfected endothelial cells with VCAM-1 constructs containing mutations in the three serines and/or the one tyrosine of the cytoplasmic domain. Using this model, we examined the ability of these mutants to signal via activation of the downstream mediator, Rac 1. We also examined the mutants’ ability to support VCAM-1-dependent leukocyte transendothelial migration under conditions of physiological laminar flow. Mutating the four putative phosphorylation sites resulted in a loss of signaling and a loss of leukocyte transendothelial migration as compared to wildtype VCAM-1. This data suggests that the cytoplasmic domain of VCAM-1 is critical to VCAM-1 signaling and function.
Thioredoxin and lactoferrin differentially regulate Langerhans’ cell migration (102.4)
Authors: Dearman Rebecca , Almond Rachael , Cumberbatch Marie , del Val Greg , Kimber Ian
Published: January 2023

Epidermal LC play a critical role in the initiation and regulation of skin immune and allergic responses. LC migration, including that stimulated by allergen, is dependent upon cytokine signals, provided by tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. We have examined the ability of two human recombinant proteins with immunomodulatory properties to influence LC migration : thioredoxin (Trx), a (12kDa) redox-active protein and lactoferrin (LF), a (79.5kDa) iron-binding protein, produced in E. coli and rice, respectively. LC migration in response to chemical contact allergen (oxazolone; Ox) or to murine TNF-α or IL-1β was assessed in BALB/c mice. Mice were treated by simple topical exposure to TRX or LF 2 h prior to treatment with the migratory stimuli. LC densities were assessed in epidermal sheets by staining for MHC class II or Langerin. Topical application of Trx inhibited Ox- and TNF-α-induced migration whereas LF prevented LC mobilisation stimulated by Ox and IL-1β only. Inhibition of LC migration by TRX was selective for IL-1β-dependent stimuli (Ox and TNF-α) whereas TNF-α-dependent migration (that provoked by Ox and IL-1β) was inhibited by LF. These data suggest that TRX modulates IL-1β production/signaling whereas LF inhibits the activity of TNF-α. The bioactivity of these molecules following topical exposure shows that these proteins can access the viable epidermis and may represent an opportunity for the development of novel anti-inflammatory reagents.
Regulatory T cell mobilization following an acute bout of exercise in human and murine models. (102.5)
Authors: Lowder Thomas , Simpson Richard , Morrison Mark , Bigley John , Rector Jerrald , Kunz Hawley
Published: January 2023

Our lab is interested in how exercise affects regulatory T cells (Tregs). While we have shown that exercise training significantly increases Treg frequency and function in mice, the effects of exercise on human Tregs is less characterized. In the present study we sought to determine if exercise alters Treg frequency in humans following acute exercise. Adult males (n=14) performed a single bout of moderate exercise. Blood was sampled pre-, immediately post-, and 1hr post-exercise and examined for changes in Treg frequency. While a significant increase in CD4+ T cells was seen post-exercise, this was not due to an increase in Tregs. We found a modest increase (~5%) in peripheral Tregs that was due to an increase in total lymphocytes trafficked to the periphery. On a per cell basis, Tregs did not increase following exercise while effector T cells increased significantly (p < 0.05). Only one study has examined Treg responses to acute exercise. In that study, increases in Tregs were seen in elite-level adolescent swimmers. It was hypothesized that during exercise Tregs migrated to interstitial lung tissue. To determine this, we performed an acute bout of exercise in mice. Exercised mice had significantly greater Tregs in their lungs (p < 0.05) when compared to sedentary mice. While exercise-induced leukocytosis is well-documented in human and animal models, this does not appear to be true with Tregs. Tregs do not appear to be mobilized in the same manner as are other leukocytes.
Memory CD4 T cells induce selective expression of IL-27 in CD8+ dendritic cells and maintain homeostatic control of naïve T cell proliferation (104.8)
Authors: Min Booki , Do Jeong-su
Published: January 2023

Naïve CD4 T cells undergo robust proliferation in lymphopenic conditions, while they remain quiescent in steady-state conditions. Mechanisms involved in the induction of the proliferation have been extensively examined. However, a mechanism of how naïve T cells are kept from proliferating under steady-state conditions remains unclear. Here we demonstrate that memory CD4 T cells directly inhibit naïve T cell proliferation within lymphopenic hosts by modulating stimulatory functions of DCs. The inhibition occurred only when both memory and naïve CD4 T cells interact with the same DCs. Thus, when naïve CD4 T cells were introduced into a recipient where a portion of DCs do not interact with memory CD4 T cells, the inhibition was lost. Moreover, the inhibition was mediated by DC-derived IL-27, which was directly induced in CD8+ DC subsets following CD4 T cell-DC interaction. IL-27 appeared to be the major mediator of inhibition as naïve T cells deficient in IL-27R were resistant to memory CD4 T cell mediated inhibition. Finally, IL-27-mediated regulation of memory T cell homeostasis was also observed in steady-state conditions. Taken together, we propose a new model for maintaining peripheral T cell homeostasis via memory CD4 T cells and CD8+ DC-derived IL-27 in vivo.
Caspase-9 deficiency enhances type I interferon production and confers resistance to viral infection (105.1)
Authors: Rongvaux Anthony , Yordy Brian , Iwasaki Akiko , Flavell Richard
Published: January 2023

Type I interferons (IFN) are antiviral cytokines that constitute the first line of defense against infection. Recent progress identified the intracellular sensors that recognize viral nucleic acids as well as the downstream signaling pathways leading to type I IFN production. As the unchecked production of type I IFN can be deleterious for the host, these pathways are under tight control by several negative regulatory mechanisms. Here, we identify caspase-9 as a novel negative regulator of type I IFN production, independently of its well described function in the induction of apoptosis. Caspase-9 deficiency leads to the constitutive production of type I IFNs, the activation of the IFN response and to increased resistance to viral infections. Interestingly, this effect is independent of IRF3 and IRF7, two major transcription factors required for IFN expression. The regulation of IFN production by caspase-9 requires its enzymatic activity as well as the presence of Apaf-1 and the downstream effector caspases. Furthermore, we show that genetic inactivation or pharmacological inhibition of caspases activates the IFN response in vivo and confers increased resistance in models of viral infections in vivo. Thus, caspase inhibition represents a new therapeutic approach to activate the IFN response, with potential applications for the treatment and prevention of viral infections responsible for human diseases.
Human plasmacytoid dendritic cells sequentially up-regulate and co-express functional receptors and IFN-α in response to HIV and HSV stimulation. (105.18)
Authors: Awoyomi Temitayo , Fitzgerald-Bocarsly Patricia
Published: January 2023

Human plasmacytoid dendritic cells undergo functional and phenotypic changes upon viral stimulation. These changes lead to IFN-α production and a coordinated modulation of functional receptors and cytokine production crucial for antigen presentation, T cell activation and trafficking. We investigated whether the same pDC that produce IFN-α express receptors involved in antigen presentation or whether these are functions of distinct subsets. Using flow cytometry and SPICE® analysis, we observed not only time- and stimulus-dependent activation profiles in these activated pDC, but also their ability to concurrently express multiple receptors important for their function as APCs on HIV-1 and HSV-1 stimulation. Kinetics of pDC activation were slightly slower with HIV than with HSV stimulation. CDs 40, 80, 83, 86, PD-L1, MHCII, CCR5 and CCR7 were up-regulated concurrently with IFN-α and TNF-α in a pDC subpopulation, CXCR4 was recycled and CD62L was shed in this same subpopulation. We found subpopulations of pDC that expressed IFN-α and activation markers either singly or together. From the phenotypic progress over time, we hypothesize that these represent pDC that start producing IFN-α, then co-express IFN/activation markers, then go on to become fully mature. Further studies will tease apart interactions between these subsets and other cells of the immune system, especially in disease manifestations like HIV that specialize in suppressing and altering pDC function and phenotype.
Role of MHC class II transactivator isoform 1 in immune system function (109.10)
Authors: Zinzow-Kramer Wendy , Long Alyssa , Rosenthal Kristen , Butler Royce , Evavold Brian , Boss Jeremy
Published: January 2023

Antigen presenting cells (APCs) constitutively express major histocompatibility complex class II (MHC-II) molecules, a crucial component of an organism’s adaptive immune response. Mutations in the master regulator of MHC-II (CIITA) are found in a subset of patients with bare lymphocyte syndrome and result in severe immune response defects. Four promoters control expression of CIITA in a cell type specific manner. Each promoter encodes a unique isoform. It is not known whether the unique isoforms have distinct roles. Isoform 1, expressed by dendritic cells and macrophages induced with IFNγ, encodes a domain with homology to a caspase recruitment domain (CARD). Isoform 1 bears the strongest similarity, of all CIITA isoforms, to the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family. Members of this family have roles in inflammation and pathogen recognition. A gene-targeted mouse model has been designed to determine whether isoform 1 CIITA has a unique role(s) in immune system function. Experiments suggest that isoform 1 CIITA is not required for proper immune system function. Notably, T-cell development and activation appear to be normal, all APCs examined express class II at levels comparable to wild-type, and lacking isoform 1 does not make an organism more susceptible to particular pathogens. These experiments suggest that isoforms 3 and 4 are sufficient for normal immune system development and function.