bluuebunny/crossref_metadata_2025 huggingface dataset:
Severity of autoimmune myocarditis is influenced by Connexin43 expression (171.23)
Authors: Glass Aaron , Coombs Wanda , Taffet Steven
Published: January 2023

Myocarditis, or inflammation of the heart muscle, is associated with severe morbidity and mortality, even in young, otherwise healthy individuals. The disease is initiated by exposure to viruses, parasites, and toxins, but can develop into a chronic autoimmune disease which progresses to severe fibrosis and dilated cardiomyopathy. Work in our lab using experimental autoimmune myocarditis (EAM) as a model, has demonstrated alterations in the sub-cellular localization of the cardiac gap junction protein connexin43 (Cx43) in heart tissue adjacent to myocarditis lesions, a potentially arrythmogenic effect. In the present study, we found the converse to be true; a reduction in Cx43 expression altered the course of EAM, resulting in a more pronounced disease. To generate animals with reduced Cx43, we backcrossed CX43+/- mice on the EAM-resistant C57BL/6 background to the EAM-susceptible BALB/c background. After 8 generations, we initiated EAM by two doses of α myosin heavy chain antigenic peptide at days 0 and 7. Hearts were analyzed 21 days after EAM induction for both the area and intensity of immune infiltrate. We determined that mice heterozygous for Cx43 develop more severe disease than wild type littermates. Closer analysis of infiltrating immune cells by immunofluorescence microscopy revealed differences in myeloperoxidase positive and CD3+ cells, with Cx43 heterozygotes having more of both. These results suggest that Cx43 can alter the course of EAM.
Complement component C1q regulates macrophage expression of Mer tyrosine kinase to promote clearance of apoptotic cells (171.31)
Authors: Galvan Manuel , Foreman Deborah , Bohlson Suzanne
Published: January 2023

Failure to efficiently clear apoptotic cells is linked to defects in development and autoimmunity. Complement component C1q is required for efficient clearance of apoptotic cells, and C1q deficiency is the strongest known susceptibility factor for autoimmunity associated with lupus. Despite the importance of C1q in apoptotic cell clearance, the mechanism by which C1q enhances apoptotic cell clearance is poorly understood. Here we describe a novel C1q-dependent engulfment mechanism for apoptotic cells. We found that macrophage activation with C1q resulted in cycloheximide-sensitive enhanced engulfment, indicating a requirement for de novo protein synthesis. To investigate the cycloheximide-sensitive pathway, C1q-elicited macrophage transcripts were identified by microarray. C1q triggered the expression of Mer tyrosine kinase and Gas 6: a receptor-ligand pair that mediates clearance of apoptotic cells. This novel pathway is specific to C1q since MBL, a related collectin, failed to upregulate Mer expression and function. Soluble Mer-Fc fusion protein inhibited C1q-dependent engulfment of apoptotic cells. In addition, Mer-/- macrophages failed to respond to C1q with enhanced engulfment of apoptotic cells, indicating a requirement for Mer. Our results suggest that C1q elicits a macrophage phenotype specifically tailored for apoptotic cell clearance, and these data are consistent with the established requirement for C1q in prevention of autoimmunity.
S100A4 is expressed in myeloid cells invading inflamed pancreatic islets and supports development of Type 1 diabetes (171.33)
Authors: Kendall Peggy , Pedchenko Tetyana , Case James , Sullivan Allison , Kendall Peggy
Published: January 2023

Type 1 diabetes (T1D) results from adaptive autoimmune destruction of insulin-producing beta cells in pancreatic islets. Macrophages and dendritic cells (DCs) have also been implicated in the disease process, although their roles are less well-studied than those of T cells. S100A4 is a small calcium-activated molecule that contributes to cancer metastases and fibrosis, and has recently been linked to autoimmunity. We have discovered S100A4 among inflammatory infiltrates in pancreatic islets in a T1D model. Cells expressing S100A4 bear surface markers associated with antigen-presentation, such as MHCII and CD86. These cells include CD11c+ DCs, CD11b+ macrophages, and CD11b+/CD11c+ myeloid populations. Neither plasmacytoid DCs (PDCA1+/Ly6C+/B220+) nor lymphoid DCs (CD8+) express S100A4. S100A4-deficiency was engineered by GFP-targeting and introgressed onto the nonobese diabetic (NOD) mouse model of T1D for >10 generations. S100A4-/-/NOD mice are protected from the development of T1D, with a concomitant reduction in insulitis. However, GFP-expression in S100A4-deficient cells indicates that these cell populations remain in the inflamed islets. When compared to S100A4-sufficient counterparts, S100A4-deficient bone marrow-derived DCs have decreased ability to upregulate CD86, or to present antigen and activate T cells in vitro. Thus, S100A4 may contribute to the development of T1D by supporting myeloid cell antigen-presentation to autoreactive T cells.
Interleukin-7 receptor α contributes to experimental autoimmune encephalomyelitis (171.42)
Authors: Jopek Ashbaugh Jessica , Brambilla Roberta , Karmally Shaffiat , Cabello Cecilia , Malek Thomas , Bethea John
Published: January 2023

Multiple sclerosis (MS) is a neurodegenerative disease characterized by inflammation, demyelination, and axonal damage. Recently, a mutation in the interleukin-7 receptor alpha (IL7Rα) locus has been identified as a risk factor for MS. IL7Rα has well-documented roles in lymphocyte development, function, and homeostasis, but its involvement in disease settings is largely understudied. Here we show that IL7Rα defective mice (IL7RTgIL7R-/-) display a less severe form of experimental autoimmune encephalomyelitis (EAE), a model for MS. Compared to wild type (WT), IL7RTgIL7R-/- exhibit reduced levels of paralysis, myelin damage, and inflammation. Furthermore, diseased WT treated with neutralizing anti-IL7Rα show significant recovery in EAE clinical scores and reduced lymphocyte infiltration into the CNS. A series of chimeric mice were then generated by bone marrow (BM) transplantation to identify IL7Rα+ cellular compartments contributing to EAE. Rag-/- mice engrafted with IL7RTgIL7R-/- or WT BM display equivalent disease severity, suggesting that IL7Rα on non-hematopoietic cells contributes to EAE. Lymphodeplete IL7R-/- mice reconstituted with WT or IL7RTgIL7R-/- BM showed dramatic reductions in EAE only with IL7RTgIL7R-/- engraftment and not WT, suggesting that IL7Rα signaling on lymphocytes contributes to disease. Taken together, IL7Rα signaling blockade in multiple cell types is necessary for marked reductions in EAE, and should be considered as a new therapeutic target for MS.
Immunization method influences EAE relapse rate in SJL mice (171.6)
Authors: Marusic Suzana , Olson Katrina , Delp Timothy , Tobin Casey , Lindbergh David
Published: January 2023

We have discovered that administration of pertussis toxin as an adjuvant during induction of experimental autoimmune encephalomyelitis (EAE) in SJL mice results in a more severe first EAE wave, but fewer, milder and later relapses than if pertussis toxin is not used. This finding is inconsistent with the widely-accepted epitope spreading hypothesis, which predicts more severe relapses resulting from more severe tissue damage in the first wave. EAE in SJL mice immunized with PLP139-151/CFA is the mouse model which most closely resembles human remitting/relapsing multiple sclerosis. EAE develops in 90-100% of immunized mice 10-15 days after immunization. The first wave of EAE lasts several days and most mice recover from it. After a disease-free period from one day to several months, most mice relapse. 50-75% of mice develop a relapse during the first 6 weeks after immunization. It is not clear why relapses develop. It is widely accepted that relapses result from epitope spreading - new immune responses to epitopes of self antigens other than the PLP139-151. It is believed that tissue damage from the initial immune response allows “presentation” of new epitopes to auto-reactive T cells. Our findings appear to conflict with this hypothesis. Potential mechanisms of relapse suppression by pertussis toxin will be discussed.
U1-snRNP activates the NLRP3 inflammasome in human monocytes (171.7)
Authors: Shin Min Sun , Kang Youna , Lee Naeun , Kang Ki Soo , Lazova Rossitza , Kang Insoo
Published: January 2023

The NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome is a caspase-1-containing cytosolic protein complex that is essential for processing and secretion of IL-1β. The U1-small nuclear ribonucleoprotein (U1-snRNP) which includes U1-small nuclear RNA (U1-snRNA) is a highly conserved intranuclear molecular complex involved in splicing pre-mRNA. Antibodies against this self nuclear molecule are characteristically found in autoimmune diseases like systemic lupus erythematosus (SLE), suggesting a potential role of U1-snRNP in autoimmunity. Although endogenous DNA and microbial nucleic acids are known to activate the inflammasomes, it is unknown whether endogenous RNA-containing U1-snRNP could activate this molecular complex. Here we show that U1-snRNP activates the NLRP3 inflammasome in CD14+ human monocytes dependently of anti-U1-snRNP antibodies, leading to IL-1β production. Reactive oxygen species (ROS) and K+ efflux were responsible for this activation. Knocking down the NLRP3 or inhibiting caspase-1 or TLR 7/8 pathway decreased IL-1β production from monocytes treated with U1-snRNP in the presence of anti-U1-snRNP antibodies. Plus, the expression of caspase-1 and IL-1β by CD14+ cells was found in cutaneous lupus tissue. Our findings indicate that endogenous RNA-containing U1-snRNP could be a signal that activates the NLRP3 inflammasome in autoimmune diseases like SLE where anti-U1-snRNP antibodies are present.
Slamf8 is a negative regulator of Nox2 activity and antigen presentation by macrophages and dendritic cells. (172.16)
Authors: Wang Guoxing , Abadía-Molina Ana , Berger Scott , Romero Xavier , Liao Gongxian , O’Keeffe Michael , Detre Cynthia , Terhorst Cox
Published: January 2023

Slamf8 (CD353) is a novel cell surface receptor that is expressed upon activation of resident peritoneal macrophages by interferon-gamma or bacteria. In studying innate immune responses of Slamf8-/- macrophages and dendritic cells, we found a very high activity of the Nox2 enzyme, not only in response to E.coli or S.aureus, but also to phorbol myristate acetate. Thus, Slamf8 acts as a negative regulator of Nox2 activity, which was confirmed by a reduction of the enzyme activity after transfection of the receptor into Slamf8-deficient primary or RAW 264.7 macrophages. The elevated Nox2 activity in Slamf8-/- macrophages partially reverted lowering of the pH in E.coli or S.aureus phagosomes. Consequently, in Slamf8-/- macrophages and dendritic cells MHC Class II antigen presentation to CD4 T+ cells is dramatically increased compared to wt cells. Taken together, the data show that upon response to inflammation-associated stimuli the inducible receptor Slamf8 negatively regulates antigen presentation.
Cys106 Oxidation of High Mobility Group Box 1 (HMGB1) Promotes Self-Oligomerization and Cytokine Release (172.22)
Authors: Kwak Man Sup , Jie Wu , Youn Ju Ho , Kwon Min Kyung , Shin Jeon-Soo
Published: January 2023

Oxidative stress can induce covalent disulfide bonds to form between protein-protein thiol groups. HMGB1, a DNA chaperon and damage associated molecular pattern molecule, is integral to oxidative stress. As a redox-sensitive protein, HMGB1 contains three cysteines: Cys23, Cys45, and Cys106. In this study, we focused on the oxidative oligomerization and pro-inflammatory cytokine induction by Cys106. HMGB1 oligomerization is positively modulated by copper and hydrogen peroxide. Mutagenesis of the Cys106 residue within HMGB1 blocks the formation of such oligomers. Addition of copper and hydrogen peroxide in HEK293T cell enhanced the oxidative self-oligomerization of HMGB1, while inhibiting it for mutant HMGB1 C106A. Furthermore, we used a bimolecular fluorescence complementation assay to directly visualize the Cys106 oxidation-induced HMGB1 oligomerization in live cells under oxidative stress conditions. And we were able to reproduce the oligomerization effect of HMGB1 using FRET analysis. Interestingly, the oligomerized-HMGB1 binds to TLR4/MD2 complex with higher affinity than monomer HMGB1. Oligomerized HMGB1 transferred nuclear factor-κB signaling at significant levels using HEK293/TLR4 cells, and the cytokine production by oligomerized-HMGB1 is now under investigation. In conclusion, the oligomerized-HMGB1 most likely plays a regulatory role in the secretion of pro-inflammatory cytokines under physiological conditions.
The Radioprotective 105/MD-1 Complex Contributes to Diet-induced Obesity and Adipose Tissue Inflammation (172.23)
Authors: Watanabe Yasuharu , Nagai Yoshinori , Nakamura Tomoya , Akira Shizuo , Miyake Kensuke , Takatsu Kiyoshi
Published: January 2023

Accumulating evidence suggest that innate immunity is associated with obesity-induced chronic inflammation and metabolic disorders. For example, the TLR4/MD-2 complex recognizes free fatty acids (FAAs) derived from adipose tissue and promotes adipose tissue inflammation and insulin resistance. The RP105/MD-1 complex is a homolog of the TLR4/MD-2 complex. However, the roles of RP105/MD-1 complex in adipose tissue inflammation have remained elusive. We now report that the RP105/MD-1 complex contributes to high-fat diet (HFD)-induced obesity, adipose tissue inflammation, and insulin resistance independent of TLR4 signaling. RP105 mRNA expression is dramatically increased by HFD in stromal vascular fraction of epididymal white adipose tissue (eWAT) in wild-type (WT) mice. In human, RP105 mRNA expression is also increased in the visceral adipose tissue of obese subjects. The RP105/MD-1 complex is expressed by most adipose tissue macrophages (ATMs). HFD increases RP105/MD-1 expression on the M1 ATMs in eWAT. Increased RP105 and MD-1 mRNA expression in macrophages is induced by co-culture with 3T3L-1 adipocytes. RP105 KO and MD-1 KO mice have less HFD-induced adipose tissue inflammation, hepatic steatosis and insulin resistance compared to these in WT and TLR4 KO mice. Finally, the saturated FAAs palmitic and stearic acids activate TLR4/MD-2 but not RP105/MD-1. Thus, the RP105/MD-1 complex is a major mediator of adipose tissue inflammation independent of TLR4 signaling.
Interaction of High Mobility Group Box-1 (HMGB1) with α-synuclein and its aggregation (172.28)
Authors: Ko Eun Ae , Min Hyun Jin , Shin Jeon-Soo
Published: January 2023

HMGB1, also known as amphoterin, is an abundant non-histone nuclear protein and is involved in neurite outgrowth and neuronal migration in CNS. The accumulation of misfolded proteins and neuronal loss are common features in many neurodegenerative diseases. The aggregation of α-synuclein is implicated as a pathologic hallmark of Parkinson’s disease. In this study, we demonstrated that HMGB1, which was translocated to the cytoplasm by the treatment of lysosomal protease inhibitor of MG-132, was co-localized with α-synuclein in SH-SY5Y neuroblastoma cells. HMGB1 showed much more binding to α-synuclein E46K mutant, which is the site of one of familial mutations that accelerate oligomerization, suggesting that HMGB1 could enhance the aggregation of α-synuclein mutant. The binding of HMGB1 and α-synuclein isolated from brain tissue that is originated from Parkinson’s disease patient is under investigation.
Glutathione reductase facilitates host defense by sustaining phagocytic oxidative burst and promoting the development of neutrophil extracellular traps (172.3)
Authors: Liu Yusen , Yan Jing , Meng Xiaomei , Wancket Lyn , Lintner Katherine , Nelin Leif , Chen Bernadette , Francis Kevin , Smith Charles , Rogers Lynette
Published: January 2023

Glutathione reductase (Gsr) catalyzes the reduction of glutathione disulfide to glutathione. Since Gsr has been implicated in the oxidative burst in human PMN, we hypothesized that Gsr-null mice would exhibit marked defects during the immune response against bacterial challenge. We report here that Gsr-null mice exhibited enhanced susceptibility to E. coli challenge, indicated by dramatically increased bacterial burden, cytokine storm, striking histological abnormalities, and substantially elevated mortality. Examination of the bactericidal functions of the neutrophils from Gsr-null mice in vitro revealed impaired phagocytosis and defective bacterial killing activities. Although Gsr catalyzes the regeneration of glutathione, a major cellular antioxidant, Gsr-null neutrophils paradoxically produced far less reactive oxygen species both ex vivo and in vivo. Unlike wildtype neutrophils that exhibited a sustained oxidative burst upon activation, Gsr-null neutrophils displayed a very transient oxidative burst associated with compromised hexose monophosphate shunt. Likewise, Gsr-null neutrophils also exhibited an attenuated oxidative burst upon encountering E. coli. Moreover, Gsr-null neutrophils displayed a marked impairment in the formation of neutrophil extracellular traps, a bactericidal mechanism which operates after neutrophil death. Thus, Gsr-mediated redox regulation is crucial for bacterial clearance during host defense against massive bacterial challenge.
The bone marrow neutrophil reserve is regulated by serpinB1 and cathepsin G through a cell autonomous and caspase-independent death pathway (172.32)
Authors: Benarafa Charaf , Baumann Mathias
Published: January 2023

The bone marrow (BM) neutrophil reserve is a large pool of mature neutrophils that can be rapidly mobilized to the circulation and recruited to tissues in response to danger signals. We have previously shown that serpinB1-/- mature neutrophils have a survival defect that leads to a considerable reduction of the BM reserve and failure to clear bacterial infection. SerpinB1 is one of the best inhibitors of neutrophil serine proteases including elastase (NE) and cathepsin G (CG). Here, BM chimera showed that serpinB1 deficiency in hematopoietic cells was necessary and sufficient to reproduce the bone marrow neutropenia of serpinB1-/- mice. We found that CG, but not NE, was responsible for the BM neutropenia of serpinB1-/- mice. Mice deficient in both CG and serpinB1 had a normal neutrophil reserve, whereas NE-deficiency did not rescue the BM defect of serpinB1-/- mice. Neutrophil survival was increased by pan-caspase inhibitor Q-VD-OPh in both wild-type and serpinB1-/- neutrophils in vitro compared to untreated cells. However, survival was significantly lower in serpinB1-/- neutrophils (35%) vs. wild type (80%) after 72hr. This survival defect in vitro was rescued by CG deficiency. Furthermore, neither transgenic over expression of anti-apoptotic factor BCL-2 nor G-CSF treatment of serpinB1-/- mice rescued CG-mediated neutrophil death. We conclude that CG and serpinB1 regulate neutrophil survival through a proteolytic process distinct from classical apoptotic pathways.
Cross-talk of Nod1 and Nod2 signaling pathways with IFNγ (172.33)
Authors: Duerr Claudia , Fritz Jörg
Published: January 2023

The importance of Nod1- and Nod2-mediated PGN-recognition for the elicitation of antimicrobial immunity has been highlighted by observations demonstrating that animals deficient in Nod1 and Nod2 are more susceptible to several bacterial infections. Moreover, we recently revealed that Nod1-/- and Nod2-/- animals exert altered microbe-specific T cells producing IFNγ. However, sole Nod1 and Nod2 stimulation fails to elicit the Ag-specific type 1 immune response suggesting that PGN recognition by Nod1 and Nod2 acts in concert with (an)other signal(s) for the priming of protective immunity. Since IFNγ provides an innate mechanism of resistance to many intracellular pathogens, we hypothesize that the synergistic activity of Nod1 and Nod2 with IFNγ exerts critical antimicrobial activity through the enhanced priming of CXCR3+ Ag-specific CD4+ and CD8+T cells. Following our hypothesis we acquired preliminary data demonstrating that stimulation of murine and human macrophages and DC by a combination of IFNγ and Nod1- or Nod2 agonists, induces the synergistic release of the chemokines RANTES, MIG and IP-10, known to chemoattract Th1 and NK cells as well as the upregulation of co-stimulatory molecules such as MHC class II, CD40 CD80 and CD86. These finding demonstrate that the synergistic stimulation of IFNγ with Nod1- or Nod2-agonists leads to the potent induction of CXCR3 and CCL5 ligating chemokines and the subsequent polarization of protective antimicrobial type 1 immunity in vivo.
Use of novel small molecule inhibitors to investigate the role of endogenous RIP2 kinase activity in modulating NOD2/RIP2-dependent cytokine signaling (172.4)
Authors: Desai Biva , Haile Pamela , Casillas Linda , Gough Peter , Bertin John , Votta Bart
Published: January 2023

RIP2 is a Ser/Thr protein kinase, which undergoes autophosphorylation and activates NF-κB and MAPKs following stimulation of the upstream NOD1/NOD2 receptors resulting in proinflammatory cytokine production. Previous studies using transient overexpression systems have suggested that RIP2 kinase activity mediates NOD1/2-dependent signaling by regulating cellular RIP2 protein levels. To interrogate whether this is also the case for endogenous RIP2 kinase, we used novel, potent and highly-selective small-molecule RIP2 kinase inhibitors to study MDP-stimulated signaling in both primary human monocytes and human colonic epithelial (HCT116) cells. We demonstrated that inhibition of RIP2 kinase activity blocks proinflammatory cytokine production in monocytes in the absence of detectable changes in total RIP2 protein levels. Using TNFα-primed HCT116 cells, we confirmed these observations and demonstrated a direct correlation between the inhibition of endogenous RIP2 kinase activity (measured as a decrease in pS176-RIPK2 levels) and a reduction in proinflammatory cytokine signaling in response to MDP-stimulation. Our data confirm the importance of endogenous RIP2 kinase activity in modulating proinflammatory cytokine release from both human monocytes and intestinal epithelial cells in response to bacterial ligands. Furthermore, our results suggest that RIP2 kinase inhibition may represent a novel therapeutic strategy for the treatment of intestinal inflammation.
Regulation of Innate Immune Responses by Receptor Interacting Protein Kinases (172.43)
Authors: Chan Francis , McQuade Thomas , Moquin David
Published: January 2023

The Tumor Necrosis Factor (TNF) family of cytokines are crucial regulators of host defense and immunity against pathogens. The Receptor Interacting Protein (RIP) family of serine/threonine kinases play critical roles in the signal transduction pathway of many TNF family receptors. The archetype member of this kinase family, RIPK1, promotes cell survival by activating the latent transcription factor NF-κB. Paradoxically, RIPK1 also promotes apoptosis in the presence of IAP antagonist. When caspases are inhibited, RIPK1 induces programmed necrosis though interacting with another RIP family RIPK3. We found that the switch between cell survival and cell death involves regulation of caspase mediated cleavage, protein ubiquitination and phosphorylation of the RIP kinases. Mice deficient in RIPK3 are highly susceptible to poxvirus infection. In addition, inactivation of the apoptosis mediators Fadd or caspase 8 led to extensive programmed necrosis of lymphocytes that severely impairs antigen-specific lymphocyte expansion. We will discuss the molecular events that regulate RIP kinase dependent necrosis and its role in the immune system.
Lung surfactant protein A attenuates alveolar macrophage classical activation induced by LPS + IFN-γ through binding to IFN-γ and blocking LPS-signaling (172.5)
Authors: Minutti Carlos , García-Fojeda Belén , Casals Cristina
Published: January 2023

There is evidence that lung surfactant protein A (SP-A) plays an important role in modulating inflammation during lung infection. However, it has been reported that SP-A either stimulates or inhibits the inflammatory activity of IFN-γ on alveolar macrophages (aMϕ). The aim of this study was to determine the effect of human SP-A on alveolar macrophages stimulated with IFN-γ in the presence or absence of LPS. To accomplish this, purified rat aMϕ were cultured in the presence or absence of IFN-γ (10 ng/ml), LPS (1 ng/ml), SP-A (5, 12.5, 25 μg/mL), and combinations thereof. We measured iNOS synthesis, TNF-α secretion, as well as STAT-1, ERK, and Akt phosphorylation by rat aMϕ. We found that SP-A inhibited STAT1 phosphorylation activated by IFN-γ. In addition, SP-A diminished LPS-induced ERK and Akt phosphorylation. Finally, SP-A inhibited [LPS+IFN-γ]-induced TNF-α and iNOS production. Therefore, the inhibitory effect of SP-A on [IFN-γ+LPS]-elicited macrophage activation is due to SP-A attenuation of both inflamatory agents. To determine the mechanism by which SP-A abrogates IFN-γ effects, we studied the potential interaction between SP-A and IFN-γ. SP-A specifically bound to IFN-γ, with Kd = 28, 2 ± 8 nM. These data support the biological function of SP-A in host defense, revealing a role of this alveolar protein in limiting classical activation of aMϕ elicited by IFN-γ + LPS.
The cytolytic effects of lamprey variable lymphocyte receptors (172.7)
Authors: Li Qingwei , Wu Fenfang , Chen Liyong , Qiao Xu
Published: January 2023

Lamprey uses variable lymphocyte receptors (VLR), a leucine-rich-repeat (LRR)-based receptors, for antigen recognition instead of the immunoglobulin (Ig)-based receptors used in vertebrates. We report herein the cytolytic effects of antigen stimulated sera in Japanese lamprey (Lampetra japonica) for bacteria and tumor cells. We demonstrated that the antigen-specific VLRB antibodies mediates cytotoxicity, and the humoral immunity based cell lysis involved deposition of VLRB on the surface of target cells with formation of pore-like structures that disrupt cell wall integrity. Our finding provides evidence of role of VLRB in lamprey humoral immunity, and offers new insight into the evolution of adaptive immunity.
Identification of matrix metalloproteinases involved in the activation induced CD16 down-modulation by primary NK cells (172.9)
Authors: Peruzzi Giovanna , Femnou Laurette , Borrego Francisco , Krzewski Konrad , Coligan John
Published: January 2023

CD16 (FcgammaRIIIa), the low affinity receptor for IgG is a potent activating receptor expressed by the majority of human natural killer (NK) cells that functions to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Since the bound IgG can be exchanged, the target cell recognition through CD16 gives NK cells flexibility in receptor specificity that can be manipulated for therapeutic purposes. ADCC dysfunction has been linked to cancer progression, poor prognosis and chronic infections; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. It is well established that upon activation NK cells enter into a refractory period in which CD16 expression is dramatically down-modulated, mainly due to the action of matrix metalloproteinases (MMPs). The aim of this project is to identify the MMP(s) responsible for CD16 cell surface down-modulation on activated NK cells and to determine if this down-modulation can be inhibited. Our data indicate that CD16 is down-modulated upon cell activation in primary NK cells, the released CD16 can be detected in the supernatant of the activated cells and a membrane-type MMP is involved in the process of CD16 shedding.
Inflammation and decreased immunoregulation characterize the leptin receptor deficient (db/db) mouse model of type II diabetes. (173.12)
Authors: Hardie Virginia , Wheeler Matthew , Harms Robert , Boyer Craig , Thiessen Kevin , George Nick , Targy Natalie , Lorenzo Kristina , Mercer David , Sarvetnick Nora
Published: January 2023

Type II diabetes is an inflammatory disease marked by chronic immune activation. Our goal is to describe the fundamental distinctions in the immune system of the db/db mouse. Serum was collected for a 58-factor immunoassay. Splenic leukocytes were isolated for six panels of flow cytometry to examine immune subsets and activation states. Db/db mice exhibit higher levels of proinflammatory factors such as C-reactive protein, granulocyte chemotactic protein-2, monocyte chemotactic protein-1, macrophage inflammatory protein-2, IL1α, and T-RANTES and lower levels in regulatory IL10. Flow cytometry data revealed upregulation of the adhesion molecule CD11b on neutrophils, monocytes and macrophages. Macrophages also expressed increased levels of costimulatory molecule CD80. Inflammatory DC were increased while regulatory DC were decreased. Pre-germinal center B cells were decreased. Plasma and memory B cells were increased with an increase in CD22 maturity marker. FoxP3+ T regs were decreased with a reciprocal increase in CD127. Total CD8 T cells were increased with greater naive populations. Unactivated NK cells were increased, and all NK populations had decreased Ly49H and KLRG1. Above results were significant with p<0.05. Baseline immune activation in db/db mice is marked by increased circulating proinflammatory factors, decreased circulating regulatory factors, increased activation of innate and humoral immunity, and suppressed regulatory and cellular immunity.
The role of sphingosine-1-phosphate receptor-1 in antigen-specific CD8 T cell migration (173.14)
Authors: Bénéchet Alexandre , Maher Leigh , Hla Timothy , Khanna Kamal
Published: January 2023

Our previous studies have helped illuminate the landscape in which the endogenous CD8 T cell immune response occurs. However, the factors that regulate the anatomical program that antigen-specific CD8 T follow after infection are not well understood. Although, it is known that sphingosine-1-phosphate receptor 1 (S1P1) controls naïve T cell trafficking, our understanding of how S1P1 regulates the migration and differentiation of CD8 T cells after infection remains poor. Here we investigated the role of S1P1 in the migration and localization of antigen-specific CD8 T cells in the lymph node (LN) after viral infection. We utilized pharmacological modulation of S1P1 and cell-specific S1P1 knock out mouse models to reveal a central role of S1P1 in controlling effector CD8 T cell migration from the draining LN. In the absence of S1P1 receptor signaling, antigen-specific CD8 T cells failed to utilize the lymphatic sinuses to egress from the draining LN. Furthermore, we used a novel mouse model that contains a mutated native S1P1 protein that cannot be efficiently internalized. Using this mouse model we show that receptor internalization can regulate effector CD8 T cell response early after infection. Therefore, our studies demonstrate that S1P1 receptor signaling is the dominant factor that regulates endogenous effector CD8 T cell migration in secondary lymphoid organs.
Dendritic cells act through IL-6 to enhance naïve T cell trafficking across high endothelial venules during adaptive immunity (173.18)
Authors: Fisher Daniel , Muhitch Jason , Vardam Trupti , Evans Sharon
Published: January 2023

Dendritic cells (DC) are as master regulators of the adaptive immune response, providing both cognate antigen and costimulation required for T cell activation as well as cytokines (e.g., IL-12, TNF, and IL-6) that drive T cell expansion and differentiation. While the role of DCs in T cell activation is well known, here we show that DCs play an unexpected role in shaping the T cell repertoire within inflamed lymph nodes (LN) by promoting trafficking across vascular gateways termed high endothelial venules (HEV). Immunization of mice with bone marrow-derived DCs matured ex vivo by TLR4 and 9 agonists (LPS and ODN-CpG, respectively), but not a TLR2 agonist (Pam3CSK4), increased the intravascular density of two hallmark adhesion molecules, peripheral lymph node addressin (PNAd) and intercellular adhesion molecule-1 (ICAM-1), selectively in HEVs of inflamed LNs. Increased PNAd and ICAM-1 display occurred rapidly after immunization (within 12 h) and coincided with improved trafficking of naïve and central memory T cells as well as increased LN size. PNAd and ICAM-1 expression reverted to homeostatic levels 24-48 h post immunization. Remarkably, increased adhesion molecule expression was abrogated by loss of a single cytokine, IL-6, from immunizing DCs. These findings suggest a model whereby IL-6-producing DC actively promote trafficking in HEVs, thereby increasing the probability that naïve T cells come into contact with cognate antigen-loaded DCs in inflamed LN.
Cognate antigen is necessary and sufficient for the migration of effector and memory T cells to vascularized allografts (173.19)
Authors: Walch Jeffrey , Zeng Qiang , Hoffman Rosemary , Oberbarnscheidt Martin , Camirand Geoffrey , Lakkis Fadi
Published: January 2023

A key step in rejection is the transendothelial migration (TEM) of Ag-specific effector and memory (E&M) T cells into the graft. It is assumed that this step is mediated by Gαi-coupled chemokine receptor signaling. This migratory paradigm was revisited by treating Ag(OVA)-specific(OT-I) and non-specific(P14) CD8 E&M T cells with pertussis toxin (PTx) to block Gαi signaling or not and co-transferring to B6 recipients of B6 Act-OVA or wt vascularized allografts to investigate the roles of cognate Ag and Gαi signaling in T cell TEM. Flow cytometry quantification demonstrated that OVA-specific(OT-I) E&M T cells migrated only to heart grafts that expressed cognate Ag(OVA) and their migration was not inhibited by PTx. Non-specific(P14) T cells, on the other hand, did not migrate to OVA grafts unless OT-I T cells were present and their migration was abrogated by PTx. Two-photon intravital imaging of transplanted OVA kidneys established that PTx only inhibited the firm adhesion and TEM of non-specific(P14) E&M T cells and not the Ag(OVA)-specific(OT-I) E&M T cells. The migration of Ag-specific E&M T cells to vascularized allografts requires cognate Ag and is independent of chemokine signaling. Entry of non-specific E&M T cells, on the other hand, requires chemokines but occurs only if Ag-specific T cells are present. These findings underscore a critical role for cognate Ag in directing the entry of primed T cells to non-lymphoid tissues and suggest novel mechanisms for T cell TEM.
Acute cigarette smoke exposure modulates the inflammatory response in pdmH1N1 and H9N2 infection (P6190)
Authors: Han Yan , Ling Manto , Mao Huawei , Tu Wenwei , Lau Yulung
Published: January 2023

Although the health risks of cigarette smoke are widely reported, the impact and mechanism of short-term cigarette smoke exposure on inflammatory diseases are still unclear. Until now, there is no study about the role of cigarette smoke in 2009 pandemic H1N1 and avian influenza A H9N2 infection. Based on our previous study, which investigated the mouse mortality, weight changes, lung virus loads and inflammatory cytokines and chemokines’ expression, we further analyzed the status of inflammatory responses before the virus infection, pulmonary histological analysis and the lung cell profiles after virus infection. Mild inflammatory responses were shown before influenza A virus infection after cigarette smoke exposure. For pdmH1N1 infection, although there was a trend that smoke exposure group had a lower lung inflammatory score and less neutrophils infiltrated into the lung than the air exposure group, the difference was not significant. However, after H9N2 virus infection, the mice in smoke exposure group displayed a significantly milder disease, as evidenced by a lower lung inflammatory score, as well as less macrophages, neutrophils, CD4+ T cells and CD8+ T cells infiltrated into the lung compared to the air exposure group. Our study provided the first in vivo evidence that acute cigarette smoke exposure may modulate the effect of influenza A virus infection. The underlying mechanism is under investigation.
The N-terminal region of the guanine nucleotide exchange factor Vav1 plays a distinguished role in T cell receptor mediated calcium signaling (P1158)
Authors: Cao Youjia , Li Shiyang , Du Ming-Juan , Wan Ya-Juan , Lan Bei , Liu Yao-Hui , Yang Yin , Zhang Cui-zhu
Published: January 2023

Vav1 is a guanine nucleotide exchange factor specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All the three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor mediated signal transduction. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal region of Vav1 in calponin homology domain to be essential for its interaction with Calmodulin, that leads to TCR-induced calcium mobilization. Substitution of the region of Vav1 with the homologous region of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells activation and IL-2 production. This study highlights the importance of the N-terminal region of Vav1 for calmodulin binding, and that attributes irreplaceable role of Vav1 in T cell activation and signal transduction.
Ca2+ regulates T cell receptor activation by neutralizing the charge of phospholipids (P1164)
Authors: Xu Chenqi
Published: January 2023

The inner-leaflet of the plasma membrane is enriched of anionic phospholipids, which interact with many positively charged proteins and regulate their function. The cytoplasmic domains of CD3ϵ and ζ chains in TCR-CD3 complex are enriched of basic residues and they interact with the inner-leaflet of the plasma membrane in quiescent T cells. Crucial tyrosine residues in ITAM insert into the hydrophobic core of the membrane, preventing the spontaneous tyrosine phosphorylation by Lck. An obvious follow-up question is what would dislodge ITAM from the membrane after TCR engagement. Ligation of TCR with agonist peptide-MHC can induce receptor clustering, which can disrupt ITAM-membrane binding and trigger the initial TCR phosphorylation and downstream signaling events including Ca2+ influx. As a divalent cation, Ca2+ directly binds to the phosphate group of phospholipids, which can disrupt the ionic interaction between CD3ϵ/ζ cytoplasmic domains and the plasma membrane and facilitate ITAM phosphorylation. This regulation can help the phosphorylation of bystander TCRs and amplify the weak initial ligand-induced TCR signaling. When compared with wild-type T cells, T cells deficient of CRAC channel have substantially lower TCR phosphorylation level after triggering. Our study thus demonstrates a positive feedback regulation of Ca2+ to TCR phosphorylation involving lipid manipulation, which helps explain the unique nature of T cell’s hypersensitivity to even a single antigen molecule.
Transcription factor Ascl2 limits Tfh cell differentiation and function (IRC8P.493)
Authors: Bertino Sarah , Dong Xuemei , Laidlaw Brian , Craft Joe
Published: January 2023

Follicular helper T (Tfh) cells promote germinal center (GC) formation and subsequent B cell maturation through the secretion of cytokines such as IL-21 and IL-4. Achaete-scute complex homologue 2 (Ascl2), a transcription factor previously shown to have a role in stem cell maintenance, is highly upregulated in the Tfh-cell subset of CD4+ T cells. However, its role in Tfh-cell development and function has not been established. We generated mice with conditional deletion of Ascl2 in the CD4 T cell compartment to assess whether this protein has a functional role in Tfh cells. Tfh cells deficient in Ascl2 promote a more robust germinal center response, with larger germinal centers and an increased percentage of GC B cells after immunization. This phenotype was coupled with augmented class-switched antibody production in the Ascl2-deficient mice. The expansion of GCs and class-switched antibody production is likely a result of increased IL-4 production by Tfh cells in the Ascl2-deficient mice. These data suggest that Ascl2 may play a role in limiting the functionality of Tfh cells by preventing their secretion of IL-4 and repressing their potential as cell helpers.
In vivo monitoring of the immune response to mIgG conjugated to iron oxide nanoparticles using the pulp (dermis) of growing feathers as a test-tissue in chickens, (IRC8P.495)
Authors: Alaamri Olfat , Byrne Kristen , Falcon Daniel , Erf Gisela
Published: January 2023

Biomedical applications of nanoparticles (NPs) are growing. Data on the biological activities of NPs are, however, limited. We are evaluating the growing feather (GF) in chickens as a test-site (US-Patent 8,216,551; GF Erf) to monitor in vivo activities of NPs. In this study, the immune response to mouse (m) IgG initiated with different antigen-adjuvant combinations was examined. Specifically, 8-week-old male chickens were immunized i.m. with 0.1 mL of mIgG alone, a mixture of Alum adjuvant & mIgG, or mIgG conjugated to iron oxide NP (IONP-mIgG, 10 nm) (n=6 birds/treatment). To monitor the immune response, 0.01 mL mIgG was injected into the pulp (dermis) of 20 GF/bird ten days post-immunization. GF were collected before (0 h) and at 0.25, 1, 2, 3, 5, and 7 d post-GF-injection for flow cytometry, mRNA and histological analysis. Flow-cytometric cell-population analysis of pulp cell suspensions revealed higher levels (%, P<0.001) of lymphocyte infiltration [due to B cells (P<0.001), alpha-beta TCR+ T cells (P<0.005) and CD4+ T cells (P<0.05)] in birds immunized with IONP-mIgG compared to Alum & mIgG or mIgG. These data demonstrate the effectiveness of GF as a test-site to monitor qualitative and quantitative aspects of immune activities in vivo and the usefulness of this minimally invasive procedure as a window into biological activities of NP. Moreover, the results suggest effective adjuvant activities of IONP in stimulating a primary immune response to protein antigen.
Tim-3 enhances CD8 T cell responses to acute listeria monocytogenes infection (IRC9P.711)
Authors: Gorman Jacob , Starbeck-Miller Gabriel , Pham Nhat-Long , Rothman Paul , Traver Geri , Harty John , Colgan John
Published: January 2023

Tim-3 is a cell surface molecule expressed on multiple immune cell populations that can mediate both stimulatory and inhibitory effects. Tim-3 has been previously shown to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute infection remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes (LM) infection. Analysis of wild-type (WT) mice infected with LM showed that Tim-3 was transiently expressed by activated CD8 T cells and was associated with acquisition of an effector CD8 T cell phenotype. Comparison of responses to LM by WT and Tim-3 KO mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells. To address the T cell-intrinsic role of Tim-3, we analyzed responses to LM infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the generation of cytokine-producing cells and proliferation by Tim-3 KO cells were significantly reduced, demonstrating that Tim-3 has a direct effect on CD8 T cell function. Our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses during an acute infection.
T Cell Factor-1 and β-catenin specifically and critically control the generation and function of T helper 2-type natural killer T cells (LYM7P.725)
Authors: Berga Bolanos Rosa , Sharma Archna , Sultana Dil , Chen Qinghua , Heller Nicola , Sen Jyoti
Published: January 2023

Phenotypically and functionally heterogeneous natural killer T (NKT) cells develop from common thymocyte precursors. Transcriptional programs that regulate the development of different subsets of NKT cells remain to be delineated. In this report we demonstrate that transcription factors T cell factor (TCF)-1 and β-catenin direct the development of CD4+ T helper (Th)2-type, IL-4-producing NKT2 cells in the thymus without influencing the development of Th1-type NKT1 cells. NKT2 cells are significantly diminished in TCF1-deficient and β-catenin-deficient mice and are increased in mice with enforced expression of β-catenin. Increased NKT2 cells in mice with enforced expression of β-catenin are functional and produce increased cytokines that exacerbate the development of IL-25-dependent airway hypersensitivity.
FoxP3+Helios+, but not FoxP3+Helios-, Tregs express GARP and LAP, representing the expanded subpopulation in pancreatic cancer patients (TUM2P.911)
Authors: Elkord Eyad , Al Samid Abd Al Samid , Khaled Yazan , Ammori Basil
Published: January 2023

Recent evidences show that Tregs negatively impact anti-tumour immunity and cancer immunotherapy. We, and others, reported that Tregs are expanded in peripheral blood and tumour microenvironment of cancer patients, correlating with poor prognosis and reduced survival. Our work has further established that Treg infiltration of tumors is correlated with a lack of patients’ responsiveness to therapy. Recent observations indicate that Tregs could be phenotypically and functionally different in cancer settings. However, molecules contributing to their enhanced immunosuppressive activity are yet to be elucidated. We recently found that the majority of peripheral and tumor-infiltrating FoxP3+ Tregs expresses Helios, an Ikaros family transcription factor. Interestingly, FoxP3+Helios+ Treg subset is significantly higher in cancer patients, compared to healthy individuals. Herein we show that markers of activated Tregs [latency associated peptide (LAP) and glycoprotein A repetitions predominant (GARP, or LRRC32)] are mainly expressed on FoxP3+Helios+ Treg subset, but not on FoxP3+Helios- Treg subset. Additionally, FoxP3+Helios+GARP+LAP+ activated Tregs represent the expanded subset in pancreatic cancer patients, compared to controls. Taken together, these observations confirm that the expanded FoxP3+Helios+ Tregs in cancer patients represent the activated Treg subset, with more suppressive characteristics, that could be targeted by specific protocols.
CTLA-4+ regulatory T cells are increased in cetuximab treated head and neck cancer patients, suppress NK cell cytotoxicity and correlate with poor prognosis (TUM2P.916)
Authors: Jie Hyun-Bae , Schuler Patrick , Lee Steve , Srivastava Raghvendra , Argiris Athanassios , Ferrone Soldano , Whiteside Theresa , Ferris Robert
Published: January 2023

The EGFR-targeted antibody, cetuximab, is clinically effective against head and neck cancer (HNC) but only in 15 - 20% of patients. We hypothesized that regulatory T cells may have functional and clinical impact antitumor immunity induced in cetuximab-treated patients. Indeed, Treg frequency and expression of CTLA-4, CD39, and surface TGF-β were significantly increased after cetuximab-based therapy. These Treg were inversely associated with functional activity of intratumoral NK cells and with clinical outcome in cetuximab-treated HNC patients. Importantly, NK cells selectively eliminated intratumoral Treg in the presence of anti-CTLA mAb (ipilimumab), which overcame the Treg suppressive activity on cetuximab-mediated ADCC. These results suggest that differences in Treg-mediated suppression may play a role in the clinical response to cetuximab therapy, and that efficacy of cetuximab therapy could be enhanced by adding ipilimumab to eliminate Treg, thus promoting anti-tumor immunity.