bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of HSH2 adaptor protein expression in the B lineage regulates the qualitative and quantitative nature of the humoral response (109.2)
Authors: Justement Louis , King R.
Published: January 2023

Endogenous expression of the adaptor protein HSH2 is regulated in a dynamic manner during B cell maturation and differentiation. Developing B cells lack detectable HSH2, whereas T1 and T2 B cells in the periphery exhibit increasing levels of expression. Mature follicular B cells exhibit decreased expression of HSH2 compared to T2 cells and expression is further downregulated in germinal center B cells. In contrast, marginal zone B cells and B1a/b B cells exhibit high-level HSH2 expression. Regulation of HSH2 expression plays a critical role in determining the outcome of the humoral immune response as demonstrated using HSH2 transgenic mice. Constitutive expression of HSH2 in B cells results in decreased serum Ig titers for all subclasses with the exception of IgA. HSH2 Tg mice immunized with T-dependent or T-independent antigens exhibit a moderate decrease in the production of antigen-specific IgM, whereas class switched isotypes are decreased by approximately 80-90% compared to control mice. In contrast, HSH2 hypomorphic mice exhibit accelerated and enhanced class switching in response to challenge with T-dependent antigen. Analysis of HSH2 Tg B cell activation in vitro demonstrated that HSH2 selectively regulates the B cell response to TNF family receptors (i.e. CD40 and BAFF-R), but not BCR- or TLR-dependent signals. These data demonstrate that changes in HSH2 expression have profound effects on the humoral immune response.
Sumoylation-dependent cSMAC localization of PKCθ is critical for T cell activation (109.25)
Authors: Gong Yu , Gong Bei-Ni , Wang Xu-Dong , Li Shi-Qing , Xu Anlong , Altman Amnon , Li Yingqiu
Published: January 2023

Small ubiquitin-like modifier (SUMO) modification is an important regulatory mechanism underlying regulation of transcription factors in the immune system. However, whether it is involved in T cell receptor (TCR) proximal signaling is not known yet. Protein Kinase C-θ (PKCθ) plays an essential role in TCR/CD28-induced T cell activation. Here, we report that sumoylation is required for physiological activation of PKCθ in T cells. There are two sumoylation sites on PKCθ. Expressing a desumoylation mutant form of PKCθ(2KR) significantly impairs TCR/CD28-induced signaling and localization of PKCθ to the central supramolecular activation cluster (cSMAC), and results in inhibition of IL-2 production and T cell activation. Collectively, these results uncover a mechanism involved in TCR/CD28-triggered PKCθ activation and cSMAC localization, indicating the crosstalk between sumoylation and TCR proximal signaling pathways.
Divergent role of heme oxygenase inhibition in the pathogenesis of sepsis (111.7)
Authors: De freitas Andressa , Alves-Filho Jose C , Cellone Trevelin Silvia , Spiller Fernando , Moreira Suavinha Marina , Nascimento Daniele , Pestana Cezar , Dal Secco Daniela , Sonego Fabiane , Curti Carlos , Barja-Fidalgo Christina , Cunha Fernando
Published: January 2023

The reduction of neutrophil migration to an infectious focus is associated with high mortality in severe sepsis. The present study was designed to determine the role of heme oxygenase (HO) in sepsis induced by cecal ligation and puncture (CLP) model. We demonstrated that pre-treatment, but not the combination of pre- plus post-treatment with ZnPP IX, a HO inhibitor, prevented the reduction of CXCR2 on circulating neutrophils and the failure of intraperitoneal neutrophil migration to site of infection. Consequently, bacterial dissemination, systemic inflammatory response and organ injury were prevented. In addition, pre-treatment with the HO inhibitor avoided hypotension and consequently increased survival. Moreover, in mice subjected to Severe-CLP, the pre-treatment, but not the combination of pre- plus post-treatment with ZnPP IX, prevented the increase of plasmatic free heme observed in non-treated Severe-CLP. The administration of exogenous hemin to mice subjected to moderate sepsis consistently increased the mortality rate. Furthermore, hemin resulted in a reduction of neutrophil migration both in vivo and in vitro. Altogether, our results demonstrated that pre-treatment with the HO inhibitor prevents the pathological findings in Severe-CLP. However, the combination of pre- plus post-treatment with ZnPP IX enhances sepsis severity due to an increase in circulating levels of heme, which is deleterious to the host tissues and also inhibits neutrophil migration.
Paxillin associates with the microtubule cytoskeleton and the immunological synapse of CTL through its LD domains and contributes to microtubule organizing center reorientation (112.24)
Authors: Ostergaard Hanne , Robertson Leslie
Published: January 2023

The cytoskeletal adaptor protein paxillin localizes to the microtubule organizing center (MTOC) in T cells and upon target cell binding is recruited to the supramolecular activation complex (SMAC). We mapped the region of paxillin that associates with both the MTOC and SMAC to the leucine-aspartic acid (LD) domains and showed that a protein segment containing LD2-4 was sufficient for MTOC and SMAC recruitment. Paxillin localizes to the peripheral area of the SMAC along with LFA-1 suggesting that LFA-1 may contribute to its recruitment. To test this possibility, beads coated with anti-CD3 were used alongside non-antigen bearing target cells, thus physically separating adhesion and TCR signals. Paxillin preferentially localized to sites of integrin engagement rather than to sites of TCR engagement, further suggesting that LFA-1 contributes to paxillin recruitment. These sites of antigen-independent integrin binding were not sufficient to induce MTOC reorientation. Paxillin has been shown to be phosphorylated downstream of ERK, but when we generated a mutation that abolished detectable phosphorylation we found that paxillin still bound to the MTOC and was recruited to the SMAC. Finally, expression of the LD2-4 region of paxillin reduced MTOC reorientation. These studies demonstrate that paxillin is recruited, through its LD domains, to sites of integrin engagement and may contribute to CTL adhesion and subsequent MTOC reorientation required for directional degranulation.
Mechanisms for TNF-α-mediated down-regulation of murine B cell responses with age (113.13)
Authors: Blomberg Bonnie , Romero Maria , Diaz Alain , Landin Ana , Riley Richard , Frasca Daniela
Published: January 2023

In humans, inflammatory cytokines increase with age and have deleterious effects on the immune, cardiac and nervous systems. In this study, we investigated whether this increase also occurs in mice and whether B cells contribute to this by secreting pro-inflammatory cytokines, such as TNF-α. Our hypothesis is that the following changes occur in B cells from old mice: ↑TNF-α → ↑TTP → ↓E47 and TNF-α → ↓AID, CSR Briefly, the transcription factor E47 and AID (activation-induced cytidine deaminase), both crucial for class switch recombination, are decreased with age. We show that unstimulated (ex vivo) B cells from old mice have significantly higher levels of TNF-α than young mice and pre-incubation of young B cells with additional TNF-α before stimulation with LPS decreases both young and old B cell responses. B cell function was restored by adding anti-TNF-α antibody before LPS in young and more significantly in old cultured B cells. To address a molecular mechanism, we found that B cells pre-incubated with TNF-α, before LPS stimulation, induce tristetraprolin (TTP), a negative regulator of mRNA stability of the transcription factor E47and TNF-α. These results reveal new molecular mechanisms which may contribute to reduced antibody responses in aging and potentially other conditions of increased chronic inflammation.
Tracking in vivo IL-17A production during infection and autoimmunity (114.11)
Authors: Price April , Liang Hong-Erh , Reinhardt R. Lee , Locksley Richard
Published: January 2023

Interleukin (IL)-17A plays a crucial role in host defense against a variety of pathogens and also contributes to the pathogenesis of autoimmune diseases. Various studies have suggested that IL-17A can be produced by multiple cell types, including CD4+ T cells, CD8+ T cells, γδ T cells, NKT cells, dendritic cells, macrophages and granulocytes. However, since most studies rely on ex-vivo restimulation to assay IL-17A production, it is unclear which cell types are the predominant sources of IL-17A during the actual course of an immune response in vivo. To address this issue, we generated IL-17A reporter mice in which IL-17A-expressing cells are marked by the presence of a surface marker. We verified the ability of these reporter mice to mark the activation of IL-17A-producing Th17 cells in vitro. We then used these mice to gain a better understanding of the temporal and cell-type specific patterns of IL-17A expression during infections with Citrobacter rodentium and Klebsiella pneumoniae and in the mouse model of experimental autoimmune encephalomyelitis (EAE). In all of these models, we found that a significant portion of γδ T cells produced IL-17A, while smaller fractions of CD4+ T cells and NKT cells produced this cytokine. These mice should prove to be a valuable tool for understanding the in vivo biology of IL-17A.
FOXP3 levels in T cells negatively correlate with cardiovascular risk factors in healthy controls and patients with SLE (115.27)
Authors: Okereke Chetachi , McMahon Maureen , Skaggs Brian , Philips Rachel , Singh Ram Raj , Divekar Anagha
Published: January 2023

Autoimmune diseases such as SLE manifest with increased risk for atherosclerosis. The role of regulatory T cells (Tregs) in atherosclerosis associated with autoimmune diseases is not clear. Here, we correlated T cell FOXP3 mRNA levels with clinical and laboratory risk markers of atherosclerosis in 36 patients with SLE and 33 healthy control subjects enrolled in the UCLA Biomarkers of Atherosclerosis in SLE study. Subjects underwent a carotid ultrasound to assess plaque and an evaluation for SLE disease activity and Framingham cardiovascular risk factors. PBMCs were obtained by Ficoll gradient and the T-cell fraction was isolated using AutoMACS. RNA from T cells was isolated and used for estimating FOXP3 mRNA using real-time qPCR. We found that FOXP3 was lower in patients than in controls. Interestingly, FOXP3 levels correlated with total cholesterol (r=-0.49), LDL cholesterol (r=-0.42), and homocysteine (r=-0.37) in healthy subjects. In patients with SLE, FOXP3 levels correlated with HDL cholesterol (r=-0.42), apoJ (r=-0.45), and adiponectin (r=-0.49). Thus, FOXP3 correlates with molecules associated with atherosclerosis risk in healthy and SLE subjects. Ongoing studies will determine the significance of these correlations.
Bruton's Tyrosine Kinase and Nrf2 mediate TLR-induced activation of Heme oxygenase-1 gene expression in macrophages (116.16)
Authors: Vijayan Vijith , Baumgart-Vogt Eveline , Naidu Srivatsava , Qian Guofeng , Immenschuh Stephan
Published: January 2023

The inducible isoform of heme oxygenase (HO)-1, is involved in heme degradation producing CO and biliverdin, mediating cytoprotection against oxidative stress. Recently, HO-1 has been shown to evoke potent anti-inflammatory and immunomodulatory effects in macrophages. Here we demonstrate that Bruton's tyrosine kinase (Btk), the gene of which is mutated in human immunodeficiency X-linked agammaglobulinemia, is involved in toll-like receptor (TLR)-induced gene expression of HO-1 in macrophages. The Btk inhibitor LFM-A13 blocked the induction of HO-1 by LPS (TLR4 ligand) in RAW264.7 macrophages and primary mouse alveolar macrophages. Furthermore, LPS-stimulated up-regulation of HO-1 gene expression was abrogated in alveolar macrophages from Btk-/- mice. In addition, inhibition of Btk with LFM-A13 attenuated the LPS-induced luciferase activity of the HO-1 promoter in transfection studies in RAW264.7 cells. This effect was mediated by the transcription factor Nrf2, which is a master regulator of the cellular antioxidant defense. Immunofluorescence studies revealed that the nuclear translocation of Nrf2 in LPS-treated macrophages was reduced by Btk inhibition. Moreover, the generation of reactive oxygen species (ROS), but not that of NO, was involved in this regulatory pathway. Btk-dependent induction of HO-1 gene expression via Nrf2 was also observed upon stimulation by TLR7 and TLR9 ligands, suggesting Btk to be generally involved in TLR-mediated HO-1 gene activation.
Enhanced interleukin-33 expression and mast cell activation in pancreas and lung tissues of pancreatic duct ligation-induced acute pancreatitis in rodents (117.16)
Authors: Kempuraj Duraisamy , Yuan Zuobiao , Theoharides Theoharis , Samuel Isaac
Published: January 2023

IL-33 is a newly identified member of the IL-1 cytokine family mainly expressed by epithelial cells, endothelial cells, fibroblasts, macrophages and mast cells. IL-33 release is known to increase in tissues of inflammation and is a potent activator of immune cells including mast cells. Elevation of IL-1β and TNF-α in acute pancreatitis has been already reported, and these cytokines upregulate IL-33 expression in inflammation. Activation of mast cells leads to the release of preformed mediators (eg., histamine) and proinflammatory cytokines (eg., IL-1β, TNF-α) and chemokines. Here we investigated mast cell activation and IL-33 expression following 5-48 hr of distal pancreatic duct ligation (PD)-induced acute pancreatitis in rats and mice. PD showed mast cell degranulation in the pancreas and lung, compared to sham controls. Rat plasma showed increased histamine levels with 48 hr of PD than sham controls. Immunohistochemistry, ELISA and immunoblotting results showed increased IL-33 expression in the rat and mouse exocrine pancreas and lung with 5-48 hr of PD. Murine bronchoalveolar lavage fluid showed higher IL-33 level in 48 hr of PD. Mast cell derived mediators and enhanced IL-33 expression may contribute to the progression of pancreatic and pulmonary inflammation. These results will enhance our understanding of acute pancreatitis pathogenesis and could indicate new therapeutic targets.
Chronic and acute inflammatory conditions determine MEK1 or MEK2 usage as regulators of IL-1β and sIL-1ra expression (117.18)
Authors: Burger Danielle , Carpintero Rakel , Gruaz Lyssia , Brandt Karim
Published: January 2023

Deregulation of the production of IL-1β and its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra), plays an important role in chronic inflammatory diseases such as multiple sclerosis. Relevant to this condition direct cellular contact with stimulated T cells potently triggers cytokine production in human monocytes. In this study we investigated the implication of MEK1 and MEK2 in the control of IL-1β and sIL-1Ra production by human monocytes upon two different stimuli: (i) soluble extracts of plasma membranes from stimulated T cells (CEsHUT), mimicking cellular contact with T cells that is relevant to chronic/sterile inflammation; and (ii) LPS that is relevant to acute/infectious inflammation. By using MEK1/2 (U0126) and MEK1 (PD98059) inhibitors and siRNA specific to MEK1 and MEK2 we demonstrate that MEK1 and MEK2 are differentially involved in the induction of IL-1β production upon chronic/sterile and acute/infectious inflammatory conditions, MEK1 being dispensable to IL-1β induction in monocytes activated by LPS but required in CEsHUT-activated monocytes. Together with the fact that immunomodulatory treatments of multiple sclerosis - IFNβ and glatiramer acetate - are using MEK2 to induce sIL-1Ra production in human monocytes, MEK1 represents a potential therapeutic target which could participate in the restoration of IL-1β/sIL-1Ra balance in chronic/sterile inflammation without affecting normal response to infectious agents.
TGF-β renders in vivo regulatory T cell function independent of TNF-α. (117.3)
Authors: Housley William , Adams Catherine , Nichols Frank , Puddington Lynn , Lingenheld Elizabeth , Zhu Li , Ragan Thiruchandurai , Clark Robert
Published: January 2023

TNF-α plays a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti-TNF-α therapy. Recent studies have suggested that TNF-α may modulate autoimmunity partially via effects on regulatory T cells (Tregs) mediated through the type II TNF receptor (TNFR2). We have now investigated the requirement for TNFR2 in both murine natural Treg (nTreg) and induced Treg (iTreg) suppression of autoimmunity. Surprisingly, we find that TNFR2 is required for nTreg but not iTreg suppression of colitis. Abnormal TNFR2-/- nTreg function was not associated with a decrease in accumulation, stability or phenotypic markers known to be relevant in Treg function. As iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2-/- nTregs. While pre-activation alone did not restore suppressive function of TNFR2-/- nTregs, pre-activation in the presence of TGF-β did. These results suggest for the first time that nTregs require TNF-α as a critical factor for activation, while iTregs replace this TNF-α requirement with TGF-β. Our findings thus not only expand on previous reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes, but also lend themselves to a new mechanistic understanding of the enigma of dichotomous responses to anti-TNF-α therapy in human autoimmune diseases.
Plasmacytoid dendritic cell depletion augments CD8 T cell IFN-γ associated atherogenic responses in a mouse model of atherosclerosis. (147.18)
Authors: Daissormont Isabelle , Christ Anette , Seijkens Tom , Sampedro Stefan , Rousch Mat , Boon Louis , Poggi Marjorie , Rademakers Timo , Halvorsen Bente , Aukrust Pal , Biessen Erik
Published: January 2023

Plasmacytoid dendritic cells (pDC) have been shown to play a pivotal immunogenic role in viral immune responses by releasing high IFN-alpha levels upon TLR9 activation. However, evidence is culminating that pDC also play a major role in inducing immune tolerance in chronic low grade inflammation. In this study we set out to address effects of pDC depletion on atherosclerosis development. Carotid artery lesions were induced in LDLr-/- mice by perivascular collar placement. To study effects of pDC depletion on atherogenesis, a pDC depleting antibody, 120G8, was administered daily for 4 weeks. GL113 mAb was used as isotype control. pDC depletion resulted in exacerbated atherosclerosis, characterized by an increased lesion CD3+ T cell infiltration. FACS analysis showed an increase in CD3+ T cell numbers in spleen and lymph nodes with a concurrent shift towards CD8+ T cells. This was paralleled by strongly elevated plasma IFN-γ levels. Moreover, immature but not mature pDC from spleen of atherosclerotic mice expressed increased levels of IDO and PD-L1 compared to pDC from non-atherosclerotic mice. Finally, we found that pDC isolated from human UAP/SAP patients displayed reduced IFNalpha expression, while intralesional expression of IFNalpha was unaltered at later stages of disease progression. In conclusion, our data demonstrate that immature pDC act atheroprotective by dampening CD8+ T cell responses and keeping Th2/Th1 responses in check.
TGFβ1 promotes adaptive immune response by modulating skin dendritic cell function and migration (147.24)
Authors: Mohammed Javed , Gunderson Andrew , Koubek Richard , Glick Adam
Published: January 2023

TGFβ1 is required for the development of Langerhans cell (LC) in the skin as TGFβ1 null mice are devoid of any LC’s. While autocrine or paracrine effects of TGFβ1 have been studied in cell-specific TGFβ1 or TGFβ1RII knockout mouse models, little information is available on effects of TGFβ1 overexpression in LC function. We overexpressed active TGFβ1 in keratinocytes and monitored the paracrine effects on skin antigen presenting cell (APC) migration in vitro and in vivo. There was an increase in migration of MHC II+CD11c+ cells from ear skin following TGFβ1 overexpression by keratinocytes and a significant increase in APC migration following treatment of mouse ear skin explant with exogenous TGFβ1. Analysis of epidermal sheets following 5 days of TGFβ1 overexpression in vivo suggested an activated phenotype of APC with loss of typical dendritic morphology and increase in MHC II staining. There was enhancement in migration of skin APC’s to the skin draining lymph nodes following application of a hapten to TGFβ1 overexpressing skin. Induction of TGFβ1 in the skin 24 hrs prior to sensitization with 0.5% Dinitro-fluorobenzene resulted in increased inflammation following challenge. These results suggest that paracrine effects of TGFβ1 promote activation and migration of skin APC’s thereby enhancing skin adaptive immune response.
Naïve T cells sense the cysteine protease allergen papain and propel basophil-mediated T helper type 2 immunity (149.13)
Authors: Liang Genqing , Xie Zhihui , Charles Nicolas , Rivera Juan , Druey Kirk
Published: January 2023

Sensitization to protease allergens, such as papain, or helminth infection is associated with basophil recruitment to draining lymph nodes. Basophils have the capacity to present antigen to naïve T cells and promote TH2 differentiation. How papain induces basophil migration is unknown. Here, we show that papain immunization induces production of chemokines including CCL2, CCL17 and CCL22 in lymph nodes preceding basophil accumulation. Papain-evoked basophil trafficking and TH2 differentiation were diminished by the absence of CCR2 or CCR4. Papain binds and activates antigen-inexperienced T cells directly to produce CCL17 and CCL22, and T cells appear to be the major source of chemokine following papain immunization. Papain-elicited accumulation of basophils and production of CCL17 and CCL22 in lymph nodes were abolished by T cell deficiency. These results support a previously unanticipated innate function for naïve T cells in the recognition of a cysteine protease allergen, which lies upstream of basophil-evoked TH2 effector cell development.
Eosinophils regulate dendritic cell-induced Th2 polarization following allergen challenge in a mouse model of asthma (151.10)
Authors: Jacobsen Elizabeth , Zellner Katie , Colbert Dana , Lee Nancy , Lee James
Published: January 2023

Eosinophil recruitment and accumulation in the lungs of asthmatics is a hallmark feature of asthma. The causative relationship between eosinophils and induced Th2 pulmonary pathologies remains unclear. Our data suggest a novel role for eosinophils as immune modulators that drive Th2-mediated pulmonary pathologies through modulation of dendritic cell function leading to the suppression of Th1/Th17 immune responses. Methods: Eosinophils and ovalbumin (OVA)-pulsed myeloid dendritic cells were adoptively transferred into IL-5-/- (eosinophil-low) or PHIL mice (eosinophil-null) at the time of allergen challenge using an established acute OVA protocol. Allergen-induced pulmonary changes were assessed as changes in lung and lung draining lymph node (LDLN) cells, activation/polarization of T cells, and induced lung histopathologies. Conclusions: We demonstrate that a threshold level of eosinophils must migrate to the lung draining lymph node to promote dendritic cell recruitment and thus activation of antigen-specific memory T cells. Expression of MHC II or CCR7 on eosinophils is not necessary for proliferation of T cells in the LDLN. These lymphatic eosinophils are necessary to suppress Th1/Th17 neutrophilic polarized immune responses initiated by OVA-pulsed dendritic cells during allergen challenge. These results imply an important function for eosinophils in modulating the balance between Th2 and Th1/Th17 pathways in the development of allergic respiratory inflammation.
Fullerene derivatives as a novel approach to controlling asthma via induction of the dual-specificity Phophatase-1 gene in mast cells (151.8)
Authors: Dellinger Anthony , Zhou Zhiguo , MacFarland Darren , Kepley Christopher
Published: January 2023

Fullerenes are a class of novel carbon allotropes being investigated potential therapeutic agents; here we report a biological function for C70-based fullerenes derivatized with inositol (C70-I). Human Mast Cells (MC) were harvested from tissue and incubated with C70-based fullerenes derivatives. Next, mediator release (cytokine and beta-hexosamindase) or protein levels (Western blot or immunoprecipitation) was measured. This derivative induced significant increases in the gene and protein expression of human mast cell dual-specificity phosphate one (DUSP1); master regulator of the corticosteroid response in asthma. This increase in FCϵRI-induced DUSP1 expression resulted in the dephosphorylation of mitogen-activated protein kinases and acted synergistically with corticosteroids to diminish mediator release from MC. The increase in DUSP1 protein expression was due in part on the ability of C70-I to prevent the ubiquitination and degradation of DUSP1. These findings identify yet another potential application of functionalized fullerenes as a way to increase the efficacy of corticosteroids or use as an anti-inflammatory through the control of DUSP1.
Two distinct Th9 subpopulations are generated by activating naïve CD4 cells with either antigen/APC, or anti-CD3/CD28 antibodies (152.12)
Authors: Tan Cuiyan , Vistica Barbara , Nugent Lindsey , Zureick Andrew , Shi Guangpu , Gery Igal
Published: January 2023

Recent studies identified and partially characterized the Th9 lineage, using anti- CD3/CD28 Abs for activation during polarization. Using TCR transgenic mice we showed, however, that Th9 are also generated by activation with the specific Ag, hen egg lysozyme (HEL) (J. Imm., 185:6795, 2010). Here, we compared Th9 lines generated by either HEL, or anti-CD3/CD28 Abs, but similarly polarized by IL-4/TGF-β. Main observations: (i) IL-9 and IL-10 are similarly secreted in the supernatant by HEL or Ab-activated cultures. Intracellular IL-9 is expressed earlier in the Ab-activated cells, but IL-10 is similarly produced by the two subsets. (ii) Both subsets induced ocular inflammation when adoptively transferred into mice expressing HEL in their eyes, but pathogenicity of Ag-stimulated Th9 was restricted to day 3 in culture, whereas Ab-stimulated Th9 induced disease when collected after 3-5 days in culture. (iii) The two Th9 subsets expressed similarly the majority of tested major surface Ags, but differed in expression of CD45RB, CD69, α4β7 and αEβ7. (iv) The two Th9 subsets differed remarkably in their expression of several chemokine and chemokine receptor transcripts, with patterns strikingly similar to those reported for the two Th17 subsets. Thus, different modes of activation of naïve CD4 cells during polarization yield two distinct subpopulations of Th9.
The expression of the abundant secondary granule proteins MBP-1 and EPO is required for the maturation of eosinophil-lineage committed progenitor cells in the hematopoietic compartments of mice (153.27)
Authors: Doyle Alfred , Jacobsen Elizabeth , Ochkur Sergei , McGarry Michael , Shim Kevin , Nguyen David , Protheroe Cheryl , Colbert Dana , Dyer Kimberly , Rosenberg Helene , Lee Nancy , Lee James
Published: January 2023

Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.
Id3 is a critical regulator of γδ-T cell development (153.41)
Authors: Lee Sang Yun , Zuniga-Pflucker Juan-Carlos , Wiest David
Published: January 2023

αβ and γδ T cells are thought to arise from a common precursor during development in the thymus. Previously, we have shown that αβ/γδ lineage choice is dictated by differences in T cell receptor (TCR) signal strength defined by differential activation of the ERK-Egr-Id3 pathway. In particular, we found that Id3 is required for strong TCR signals to both promote adoption of the γδ-fate and oppose the αβ-fate outcome. Importantly, the effects of Id3-deficiency on γδ T cell development differ depending on the γδ subset. While Id3-deficiency impairs the development of the Vγ2-expressing and Vγ3 dendritic epidermal T cell (DETC) subsets, it results in the dramatic expansion of Vγ1.1Vδ6.3 T cells which are thought to be innate type NK γδ T cells. Fetal thymic organ culture analysis indicates that Id3-deficiency blocks DETC development by interfering with selection in the thymus, not by blocking expression of the canonical DETC TCR complex. Conversely, single-cell PCR analysis has revealed that the junctional diversity of CDR3s of the Vγ1.1Vδ6.3 expressing cells that are expanded in Id3-deficient mice exhibit evidence of ligand-mediated selection. These results are consistent with the notion that development of these cells is normally restricted by ligand-engagement, but this restriction is disabled in the absence of Id3. Altogether, these results indicate that Id3 plays a critical role not only in regulating fate selection, but also in shaping the γδ TCR repertoire.
Immunisation analysis and animal protection experiments of a recombinant multi-epitope assembly peptide from Herpes Simplex Virus Type 2 (155.35)
Authors: Yuexi Li , Xingsheng Wang , Guangyan Xie , Jianming Liao , Dengke Yin , Wenyan Guan , Mingjie Pan , Jingnian Li
Published: January 2023

Human herpes simplex virus 2 (HSV2) has been the main cause for genital herpes, causes a significant health problem worldwide, and no effective vaccine is available. Multi-epitope assembly peptides vaccination is a promising mean to achieve protective immunity and to avoid immunopathology. A recombinant Multi-Epitope Assembly Peptide (MEAP) including 12 antigen epitopies from HSV2 was expressed and purified by genetic engineering, its immunity and protection efficacy against HSV2 infection were identified in mice. The twelve epitopies contained six B cell epitopies from six envelope glycoproteins, B, C, D, E, G and I of HSV2 respectively, four CD4+ T cell epitopes from B and D, and two CD8+ T cell epitopes from D. they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. all above epitopes were inserted into the extracellular fragment of HSV-2 glycoprotein D to construct multi-epitope assembly peptides by replacing some non-epitope amino acid sequences. The MEAP could elicit high titer neutralizing antibodies and cell-mediated immune responses in mice and rabbits. The mice immunized with the MEAP were completely protected against HSV-2 infection death at a lethal dose, and the virus shedding, inflammation severity in the mice were reduced significantly compared with the control mice, which indicates that it might be a potential candidate vaccine.
A novel HER-2-specific exosome-targeted T cell-based vaccine: immunotherapy of established lung metastasis in transgenic HLA-A2/HER-2 mice (155.37)
Authors: Wang Lu , Xie Yufeng , Wei Yangdou , Mulligan Sean , Xiang Jim
Published: January 2023

CD4+ T cell vaccine has less been studied. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of ovalbumin (OVA)-pulsed dendritic cell (DCova)-derived exosome (EXOova) express exosomal peptide/major histocompetibility complex (pMHC) I and costimulatory molecules. EXOova-uptaken (targeted) CD4+ aT (aTexo) cells can stimulate central memory CD8+ cytotoxic T lymphocyte (CTL) responses, more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than DCova, and break the self immune tolerance in two transgenic mouse models leading to OVA- and neu-specific CTL-mediated diabetes and antitumor, respectively. We further elucidated that aTEXO’s stimulatory effect is mediated via its IL-2 secretion and acquired CD80 costimulation, and specifically targeted to CD8+ T cells in vivo via acquired pMHC I complexes. Therefore, EXO-targeted CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases. Therefore, EXO-targeted CD4+ T cell vaccine may represent a novel effective vaccine strategy for therapeutic treatment of patients with early stage of HER-2-positive or trastuzumab-resistant HER-2-positive breast cancers, and for adjuvant treatment of patients with potential risk of metastasis after surgical treatment.
LIGHT expression in prostate cancer inhibits tumor growth and induces prostate antigen-specific immunity (156.2)
Authors: Yan Lisa , Da Silva Diane , Kanodia Shreya , Gray Andrew , Kast W. Martin
Published: January 2023

An immunosuppressive tumor microenvironment has always been a hurdle for successful immunotherapy even in the presence of induced tumor-specific T cells. Regulatory T cells (Tregs) appear to be key regulators in local immune suppression. LIGHT, a ligand for lymphotoxin-β receptor (LTβR), is predominantly expressed on activated immune cells, signaling via LTβR is required for the formation of organized lymphoid tissues. Forced expression of LIGHT recruits naive T cells into tumors and is capable of establishing tumor specific immunity. However this has never been tested in prostate cancer models where tolerance to self-antigen likely exists. Here we test the hypothesis that forced expression of LIGHT in prostate tumors induces prostate cancer-specific immunity and results in tumor regression by altering the suppressive activity of Tregs and consequently enhancing a more persistent proinflammatory microenvironment. Our data show that intratumoral expression of LIGHT via adenovirus delivery in TRAMP-C2 tumor challenged mice develop de novo CD8+ IFNγ-secreting prostate antigen-specific T cells and display increased survival compared to control treated mice. LIGHT-treated mice also display an increase in ratio of tumor infiltrating lymphocytes to Tregs as well as decrease in Treg suppression activity. Our data suggest that LIGHT treatment can alter the microenvironment such that natural and vaccine-induced prostate tumor antigen specific T cells mediate tumor regression.
mTOR signaling pathway regulates the IL-12/IL-10 axis in Leishmania donovani infection (158.17)
Authors: Cheekatla Satyanarayana , Aggarwal Amita , Naik Sita
Published: January 2023

Macrophage stimulation with TLR ligands and concurrent infection with L. donovani promastigotes results in decreased IL-12 and increased IL-10 production compared to stimulation with TLR agonists. Because mTOR deficiency has been associated with increased IL-12, we hypothesized that host mTOR is central to the evasive effect of parasite on TLR-induced IL-12 inhibition. Here we demonstrate that L. donovani activate mTOR via phosphoinositide 3-kinase/Akt. Parasite enhanced phosphorylation of P70S6K (a correlate of mTOR activity), was completely blocked by rapamycin (mTOR inhibitor). This inhibition resulted in reversal of IL-12, IL-10 production and iNOS expression modulated by the parasite. These observations suggest that mTOR dependent pathway is operative in the modulation of IL-12/IL-10 cytokine axis by the parasite. Parasite internalization by macrophages was not affected by rapamycin treatment, suggesting cytokine reversal mediated by rapamycin is independent of parasite entry. Further blocking TLR2 resulted in the reversal of leishmania-induced IL-10 production. Collectively, these findings provide new insight into the mechanism controlling modulation of IL-12 & IL-10 cytokines by leishmania involving the intracellular network of mTOR pathway in a TLR2-dependent manner. mTOR inhibitors may be an attractive molecule to reverse cytokine modulation and control the infection.
Degeneracy analysis of T cell receptors in mice. (160.17)
Authors: Buntzman Adam , Vincent Benjamin , Krovi S.Harsha , Steele Shaun , Walsh Jesse , Kepler Thomas , Frelinger Jeffrey
Published: January 2023

The potential T cell receptor (TCR) diversity in mice is presumed to be vast. Each mouse contains a small sampling of the possible TCR variants that can be generated, therefore the likelihood that multiple mice generate the same TCR is low. Yet TCR clones have been observed in the memory and naive T cell populations in multiple mice. A global analysis of the inter-mouse TCR repertoire overlap may shed light on a mechanism for the synthesis and retention of shared TCRs. One current model of TCR sharing between mice is based on the hypothesis that degeneracy exists in the TCR recombination machinery at multiple levels. Degeneracy can exist at the recombination level, at the codon usage and amino acid level, at the TCR nucleotide level and at the TCR protein level. This degeneracy has been proposed to drive the generation of TCRs at a higher frequency in each mouse, thus leading to a higher probability of being shared between mice. To globally address whether degeneracy and relative TCR frequency are correlated with the extent of TCR sharing, we have sequenced hundreds of thousands of TCR beta chains from the cytotoxic T cell population of multiple mice and have analyzed the multi-level degeneracy and frequency profile of the shared and unshared pools of TCRs. Our approach provides the first analysis of TCR sharing between mice in whole repertoire.
Chronic differentiation of human mucosal IgA-secreting cells during B cell depletion therapy with rituximab. (161.3)
Authors: Mei Henrik , Frölich Daniela , Giesecke Claudia , Loddenkemper Christoph , Reiter Karin , Schmidt Stefanie , Feist Eugen , Daridon Capucine , Tony Hans-Peter , Dörner Thomas
Published: January 2023

During mucosal immune responses, IgA-secreting plasmablasts are generated from mucosal B cells. We previously demonstrated that at steady-state, most human peripheral blood antibody-secreting cells produce IgA and express mucosal homing receptors, and suggested their generation in mucosal immune responses. In the current study, we analyzed the blood of patients who received B cell depletion therapy with rituximab (anti-CD20) for treatment of rheumatoid arthritis before and at 2-9months after rituximab infusion by flow cytometry. While CD20+ B cells were reduced to <0.02% of their intial numbers before therapy, CD19+/CD27hi/CD20- antibody-secreting cells remained detectable in the blood at 26-119% of their initial numbers. These circulating antibody-secreting cells expressed IgA that bound to bacterial antigens, beta7 integrin and CCR10 before and during B cell depletion, suggesting their mucosal origin. High expression of HLA-DR and Ki-67 and in vitro migration towards CCL28 and CXCL12 qualified these cells as recently activated plasmablasts, reflecting the survival of functional B cells during rituximab treatment. Consistently, IgA+ plasmablasts and plasma cells were identified in the lamina propria during B cell depletion. Our data implicate that a population of mucosal B cells is resistant to rituximab and self-sufficient in adult humans and not replenished by CD20+ B cells immigrating from blood, lymphoid tissue, or bone marrow, that is, B cells depleted by rituximab.
The role of Gr1int interstitial cells in lung host defense against gram-negative bacterial infection. (162.8)
Authors: Poe Stephanie , Arora Meenakshi , Oriss Timothy , Krishnamoorthy Nandini , Yarlagadda Manohar , Ray Anuradha , Ray Prabir
Published: January 2023

We have recently identified a CD11b+Gr1intF4/80+ (Gr1int) regulatory, non-migratory, cell type in the lung that is able to suppress a Th2 effector response via secretion of IL-10, arginase 1 and nitric oxide (NO). These regulatory cells also secrete IL-6 and GM-CSF, but low levels of IL-12. Our studies show that although Gr1int cells are present in the lung of naïve mice, their frequency increases following LPS exposure in a TLR4/MyD88 dependent fashion. The rapid appearance of these cells in response to a TLR ligand, combined with their non-migratory nature and their anatomical location in the lung interstitium, suggests a unique function during host defense against bacterial infections. Much is already known about the role of alveolar macrophages and neutrophils in host defense against Klebsiella Pneumoniae, however little is known about host defense mechanisms in the lung tissue/interstitium. For the first time, we have examined the role of Gr1int interstitial cells in defense against K. Pneumoniae. We show that the Gr1int cell population increases following exposure to K. Pneumoniae, are highly phagocytic, and co-localize with K. Pneumoniae, suggesting phagocytic uptake in vivo. Furthermore, Gr1int secrete high levels of NO, previously shown to be critical for effective innate immunity against K. Pneumoniae.
The balance between autoimmune thyroiditis and cancer immunotherapy (164.6)
Authors: Kari Suresh , Flynn Jeffrey , Zulfiqar Muhammad , Snower Daniel , Kong Yi-chi
Published: January 2023

Clinical trials of immunotherapeutic agents for both autoimmune diseases and cancer often lead to opportunistic autoimmune disorders, where highly prevalent autoimmune thyroid disease is prominent. To simulate cancer patients undergoing immunotherapy, we established a tumor protection model in experimental autoimmune thyroiditis (EAT)-susceptible CBA/J (H2k) mice. Strong tumor immunity required both regulatory T cell (Treg) depletion and immunization with irradiated tumor cells from spontaneous CBA mammary adenocarcinoma line (A22E-j). All mice withstood initial and repeat lethal tumor challenge; protection was mediated by CD4 & CD8 T cells. In EAT, Treg depletion also increases the incidence and severity when immunized with mouse thyroglobulin (mTg), 16x over 4 wks, without adjuvant; soluble mTg mimics physiologic release of circulatory Tg. When combined, EAT was enhanced in mice protected from lethal tumor challenge. To simulate tumor patients with pre-existing autoimmunity undergoing immunotherapy, we induced EAT with mTg + IL-1β (d -29,-22), followed by Treg depletion (d -14,-10), tumor vaccination (d -7,0), lethal challenge (d 0) and soluble mTg (d 0-24, 16x). This regimen had no effect on established EAT and ~75% mice withstood tumor challenge (d 35). The data suggest that ongoing EAT is not affected by induction of tumor immunity, yet tumor immunity may be tempered. Whether subclinical autoimmune disease could be exacerbated by such therapy remains to be determined.
Rewiring of the PKCβII and the Bcr/Abl signal transduction pathways (165.37)
Authors: Hoekstra David , Carlson Louise , Louise Kelvin
Published: January 2023

Chronic myeloid leukemia (CML) accounts for 20% of adult leukemias, and is characterized by the presence of the constitutively active tyrosine kinase Bcr-Abl. Its presence is essential for CML blast survival even in advanced disease, and underlies the success that the Bcr-Abl kinase inhibitor imatinib has had. However, resistance to imatinib occurs in a significant number of accelerated phase or blast crisis patients. Instead of trying to inhibit Bcr-Abl signaling, we propose a previously unexplored approach to rewire signaling pathways to activate proteins that would not normally be activated by Bcr-Abl. We have previously shown that activation of Protein kinase C isoform βII (PKCβII) drives dendritic cell differentiation in normal and leukemic cells. We hypothesized that inserting the Abl kinase motif into a specific domain of PKCβII, would allow Bcr-Abl to activate PKCβII. Using molecular modeling techniques, we have shown that the modified PKCβII pseudosubstrate sequence is largely unchanged as a result of this mutation and still able to respond to traditional activating signals. Additionally, our modified PKCβII is activated in Bcr-Abl+ but not in Bcr-Abl- cells resulting in a decrease in viability and increase in apoptosis. Future experiments plan to characterize the immunogenic potential of these converted leukemic cells, and to fuse the protein transduction domain from HIV onto our modified PKCβII to test the efficacy of this modified protein in a murine model of CML.
Involvement of Axl receptor tyrosine kinase in the pathogenesis of DSS-induced IBD (166.14)
Authors: Ji Kon-Young , Kim Dong-Hern , Kang Hyung-Sik
Published: January 2023

To elucidate the role of Axl receptor tyrosine kinase on the pathogenesis of inflammatory bowel disease (IBD), IBD-associated prognostic factors were examined in dextran sulfate sodium (DSS)-treated wild type and Axl-/- mice. We found that body weight and colon length were decreased in DSS-treated Axl-/- mice compared to those in DSS-treated WT mice. In addition, the reduced number of regulatory T cells was observed in the large intestine, mesenteric lymph node, and peritoneal cavity of DSS-treated Axl-/- mice. Expression of the proinflammatory cytokines was overexpressed in the large intestine of DSS-treated Axl-/- mice. These results suggest that the overexpression of proinflammatory cytokines may be due to the reduced number of regulatory T cells by Axl deficiency. Taken together, it suggests that Axl may be involved in the pathogenesis of IBD, and the regulation of its signaling may be helpful to ameliorate the progression of IBD.
Genes encoding Th2 cytokines and other proteins of immunological interest in the Oryctolagus cuniculus (rabbit) whole genome sequence and ENCODE (170.20)
Authors: Mage Rose , Schäffer Alejandro , Gertz E. , Agarwala Richa
Published: January 2023

Rabbits, a valuable resource for diagnostic and therapeutic antibodies, are becoming increasingly important for vaccine development, and are used as animal models of human autoimmune, infectious and other diseases. The unique characteristics of their immune system, resulting in antibodies of high affinity and specificity, make them a major source of polyclonal and monoclonal antibodies. Whole genome sequencing (WGS) at 7x coverage and assembly of a high quality draft rabbit genome sequence was recently completed at Broad Institute. The Thorbecke Rabbit (TR) chosen for sequencing was less heterozygous than outbred New Zealand White (NZW), but was heterozygous at the immunoglobulin heavy chain locus. The TR strain was more susceptible to Tuberculosis and had obvious developmental abnormalities. Any follow-up on the biology must use another rabbit strain because the TR strain was lost in a fire. The ENCODE Project, with ~ 1% of rabbit genomic sequence from a different, normal NZW animal, includes genes of immunological interest such as a cluster containing the coordinately regulated Th2 cytokine genes IL5, IL13, and IL4. Cross-species comparative genome analysis revealed conservation of some coding and regulatory sequences within syntenic regions of rabbit and 9 other mammals. A few differences between the coding sequences of one TR and one NZW rabbit, and many differences in regulatory sequences across mammals were found. Other genes of the immune system are now under study.