bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoproteasomes shape the transcriptome and regulate the function of dendritic cells (IRM7P.476)
Authors: Rouette Alexandre , de Verteuil Danielle , Hardy Marie-Pierre , Lavallée Stéphanie , Trofimov Assya , Gaucher Étienne , Perreault Claude
Published: January 2023

Background: By regulating protein degradation, constitutive proteasomes (CPs) regulate practically all fundamental cellular processes. Vertebrates also express immunoproteasomes (IPs), but the only well-established non-redundant role for IPs is their enhanced ability to generate peptides for MHC-I presentation. Results: Here we show that IPs regulate the expression of 8,104 genes in maturing bone marrow-derived dendritic cells (DCs). IPs regulated transcription of many mRNAs and maturation of a subset of them. These genes were separated into 15 different kinetic patterns, and Gene-Ontology analysis revealed enrichment linked to immune functions and housekeeping cellular processes, highlighting the complexity of the transcriptional cascade regulated by IPs. Notably, IPs’ impact on transcription was mediated through the non-redundant regulation of critical immune-related pathways, such as Nf-kB, STATs and IRFs. Furthermore, even when loaded with optimal amounts of SIINFEKL, IP-deficient DCs were inefficient for in vivo priming of OT-1 T cells. Conclusion: Our study shows that the role of IPs is not limited to antigen processing and highlights a new and critical role for IPs in regulation of gene expression. The dramatic impact of IPs on the transcriptional landscape could explain the various immune and non-immune phenotypes observed in vertebrates with IP-deficiency or -mutation.
Conditional STAT3-deficiency augments Flt3 ligand-driven myeloid-derived suppressor cell expansion but limits their suppressor function (IRM7P.487)
Authors: Matta Benjamin , Rosborough Brian , Mathews Lisa , Liu Quan , Raich-Regue Dalia , Angus Thomson , Turnquist Heth
Published: January 2023

Fms-like tyrosine kinase 3 ligand (Flt3L) is a potent hematopoietic growth factor that profoundly expands immunostimulatory dendritic cells (DC). Despite this, the influence of Flt3L on immunoregulatory myeloid-derived suppressor cells (MDSC) has not been described. Since DC and MDSC arise from common myeloid progenitors, and Flt3L-driven DC expansion is STAT3-dependent, we sought to precisely define the role of STAT3 in MDSC downstream of Flt3L signaling. We bred myeloid cell-specific STAT3-deficient mice (LysMCre x STAT3loxP) and administered Flt3L (10 μg/d ip, 10d). Splenic DC (CD11c+) and MDSC (Gr-1+) were isolated by magnetic bead selection. Stimulation and suppression of CD3+ T cell responses was assayed in MLR. As expected, STAT3-deficiency prevented Flt3L-mediated CD11c+ DC expansion. However, we observed an increase in MDSC (CD11b+Gr1+) frequency and absolute numbers in STAT3-deficient mice administered Flt3L. Interestingly, Flt3L-expanded MDSC from STAT3-deficient mice exhibited reduced suppressive capacity against CD3+ T cells in MLR compared to controls. These data identify a previously unappreciated STAT3-independent pathway for MDSC expansion, but reinforce the need for STAT3 signaling for their full suppressive capacity. Likewise, we establish that STAT3 has reciprocal effects on suppressive MDSC and immunostimulatory DC expansion. Together, these findings enhance understanding of the immunomodulatory properties of Flt3L.
Properties of virus-induced NK cells correlating with their ability to kill activated CD4 T cells (IRM7P.489)
Authors: Hatfield Steven , Daniels Keith , Waggoner Stephen , Welsh Raymond
Published: January 2023

Activated natural killer (NK) cells induced during viral infections of mice can regulate adaptive immunity by lysing activated CD4 T cells. This seems to be a general property of interferon-inducing viruses, but the efficiency of this process is in part dependent on the nature of the instigating virus. Here we examined the differential expression of activating and inhibitory receptors on NK cells from lymphocytic choriomeningitis virus (LCMV) and vaccinia virus (VACV)-infected mice, because NK cells from VACV-infected mice were far less efficient at killing activated CD4 T cells than those from LCMV-infected mice. Relative to expression levels on LCMV-activated NK cells, NKG2A expression was higher in VACV-induced NK cells while NKG2D was lower. NK cells activated by infections with LCMV, Pichinde virus (PV), mouse hepatitis virus (MHV), and murine cytomegalovirus (MCMV) displayed similar expression patterns for all of the examined receptors, whereas VACV-induced cells had a distinct expression pattern. Another major difference between VACV-induced NK cells and NK cells activated by the other viruses was in the expression of granzyme B, an essential component of NK cell-mediated cytolysis. VACV-induced NK cells expressed granzyme B at lower levels than NK cells from mice infected with LCMV, PV, MHV A59, or MCMV. These results indicate that the NK cell receptor expression and the cytolytic activity of NK cells can vary with the infecting virus.
TSC1 is critical for iNKT-cell terminal maturation and controls iNKT-1/17 balance (IRM7P.490)
Authors: Zhong Xiaoping , Wu Jinhong
Published: January 2023

iNKT-cells terminal maturation from CD44+NK1.1- stage 2 to CD44+NK1.1+ stage 3 is accompanied by functional acquisition of predominant IFNγ-producing (iNKT-1) but not IL-17-producing (iNKT-17) capability. iNKT-17-cells are rare and restricted to the CD44+NK1.1- subset. The mechanisms that control iNKT terminal maturation and predominance of iNKT-1 over iNKT-17 remain poorly understood. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling. We demonstrate here that deficiency of the TSC1 causes iNKT-cell developmental block at stage 2 and functional alteration from iNKT-1 predominance to iNKT-17 predominance, leading to enhanced airway hypersensitivity. TSC1 promotes T-bet but downregulates ICOS expression in iNKT-cells by inhibiting mTOR complex 1. Decreased T-bet contributes to both terminal maturation defect and iNKT-17 predominance over iNKT-1 while increased ICOS expression is required for iNKT-17 predominance in TSC1-deficient iNKT-cells. Thus, TSC1and its proper control of mTORC1 are crucial for iNKT-cell terminal maturation and effector lineage decision for iNKT-1 predominance over iNKT-17.
Identification of a novel epigenetic code, histone H1 fucosylation, and its roles in macrophage plasticity in rheumatoid arthritis (IRM7P.491)
Authors: Li Jun , Hsu Hui-Chen , Yang PingAr , Wu Qi , Luo Bao , Mobley James , Bridges S. , Mountz John
Published: January 2023

Macrophages (MΦ) are key players in rheumatoid arthritis (RA). High plasticity renders MΦ an attractive tool for studying epigenetic reprogramming. We have observed a highly positive correlation between TNFα and fucosylation catalyzing enzymes, fucosyltransferases (FUTs) 1, 2, 3, 4, 5, 6, 7, 9, 10, 11 (p=0.0001) in human RA synovia; these FUTs are predominantly detected in M1 inflammatory MΦ but not M2 MΦ and T cells from RA synovial fluid. A fucosylation inhibitor, 2-Deoxy-D-galactose (2-D-gal) precluded collagen II-induced arthritis in DBA/1J mice (scores 9.5±1.7 vs 0.5± 0.3, p<0.01) with reduced M1 MΦ in draining LN, decreased TNF-α and anti-CII in the serum (p<0.05).Ulex Europaeus Agglutinin I (UEA 1) affinity chromatography coupled to mass spectrometry indicated that the linker Histone H1, but not core histone H 2-4, is the major protein that modified by a fucose-α-(1-2)-gal moiety in M1 MΦ. Disrupting this moiety by 2-D-gal skewed the differentiation of M1 MΦ toward M2 anti-inflammatory phenotype with a reprogrammed gene expression, including i) robustly upregulated Il10, Arg1, and Megf8 (p<0.01) (M2 markers); ii) downregulated Ifnγ and Il6 (p<0.05) (M1 markers); iii) inhibited Actin and its associated molecules including Ahnak and Cap1, and suppressed the antigen presentation of M1 MΦ phenotype (p<0.01). We uncovered Histone 1 fucosylation as a novel epigenetic code that orchestrates MΦ transcriptome reprogramming and renders it an attractive target for RA therapy.
Effect of 1,25-dihydroxyvitamin D on all-trans retinoic acid metabolism and interleukin-4 signaling in porcine alternatively activated lung macrophages. (IRM7P.493)
Authors: Heibel Sandra , Chen Celine , Urban Joseph , Dawson Harry
Published: January 2023

All-trans retinoic acid (ATRA), the most active vitamin A (VA) metabolite, and 1,25-dihydroxyvitamin D (VD3), the most active form of vitamin D (VD), and interleukin-4 (IL-4) each positively regulate the development of alternatively activated macrophages (AAM) however, their interactions are unknown. We have previously shown that VA, ATRA, and IL-4 have differing mechanisms for sustaining a Th2-type AAM response. Herein, we demonstrate that VD3 regulates the contribution of ATRA and IL-4 inducible signals to this system. While ATRA (10-7 M) increases mRNA expression of retinol producing dehydrogenase/reductase (SDR family) member, 4 fold, VD3 (10-7 M) decreases its expression 3-fold and additively increases expression of retinal producing, Aldehyde dehydrogenase 1 family, member A2, and dehydrogenase/reductase (SDR family) member 9 by 5 fold when present along with ATRA. Conversely, VD3 decreases IL-4 (10 ng/ml) -induced chemokine (C-C motif) ligand 17 by 4 fold, and C-type lectin CD209 antigen by 60 fold. These results suggest that VD3 increases expression of genes involved in ATRA anabolism while decreasing expression of IL-4 responsive genes. In conclusion, VD3 may regulate the development of AAM by increasing levels of ATRA and decreasing the effect of IL-4, thereby supporting an alternate signal for AAM development.
Ezrin tunes the magnitude of humoral immunity. (IRM8P.709)
Authors: Gupta Neetu , Pore Debasis , Parameswaran Neetha , Matsui Ken , Stone Matthew , Saotome Ichiko , McClatchey Andrea , Veatch Sarah
Published: January 2023

Ezrin is a member of the Ezrin-Radixin-Moesin (ERM) family of membrane-actin cytoskeleton crosslinkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice with conditional deletion of ezrin in the B cell lineage to investigate the physiological significance of ezrin’s function in antigen receptor-mediated B cell activation and humoral immunity. B cell development, as well as the proportion and numbers of major B cell subsets in peripheral lymphoid organs were unaffected by the loss of ezrin. Using super resolution imaging methods we show that in the absence of ezrin, BCRs respond to antigen binding by accumulating into larger and more stable signaling microclusters. Loss of ezrin led to delayed BCR capping and accelerated lipid raft coalescence. Although proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling components displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into antibody-secreting cells ex vivo, and stronger T cell-independent and -dependent responses to antigen in vivo. Overall, our data demonstrate that ezrin regulates amplification of BCR signals and tunes the strength of B cell activation and humoral immunity.
Antigen-induced reorientation of the microtubule organizing centre to the B cell immune synapse requires Rap activation and actin dynamics (IRM8P.704)
Authors: Wang Jia , Freeman Spencer , Lee Jeff , Gold Michael
Published: January 2023

B cell activation can be initiated by antigen presenting cells (APC) that display intact antigen (Ag) on their surface. Reorientation of the B cell microtubule-organizing centre (MTOC) towards the APC facilitates Ag internalization at the immune synapse (IS), which is critical for B cells to present Ag to T cells and elicit T cell help. We showed previously that BCR-induced activation of the Rap GTPase, a key regulator of cell polarity and actin dynamics, is required for IS formation. We now show that BCR-induced Rap activation is critical for MTOC polarization towards anti-Ig-coated beads and towards APCs. Activated Rap increases actin dynamics and promotes cytoskeletal reorganization by activating the actin-severing protein cofilin. We found that both disrupting the actin cytoskeleton with latrunculin A and blocking cofilin function prevented MTOC polarization, suggesting that Rap controls MTOC reorientation via its effects on actin dynamics. To assess how actin dynamics regulates the microtubule (MT) network, we tested the role of the actin-MT crosslinking protein IQGAP1 in BCR-induced MTOC polarization. We found that both Rap and cofilin controlled the accumulation of IQGAP1 at the site of Ag contact, and that depleting IQGAP1 prevented MTOC polarization. Thus Rap regulates the polarity of the MT network via its effects on the actin cytoskeleton and this may involve proteins such as IQGAP1 that link the actin and MT cytoskeletons.
Identifying key mechanisms in fibroblast disregulation using a systems biology approach. (IRM9P.727)
Authors: Warsinske Hayley , Linderman Jennifer , Moore Bethany , Kirschner Denise
Published: January 2023

Pulmonary fibrosis, stiffening and scarring of the lung tissue, is a symptom of interstitial lung disease with no available treatments and poor prognosis for patients. It occurs when disregulation of the proliferative and remodeling phases of the wound healing process results in uncontrolled proliferation and activation of fibroblasts, excessive extracellular matrix deposition, and detrimental tissue remodeling. The molecular mechanisms underlying this disregulation are poorly understood providing an untapped pool of potential therapeutic targets. TGFβ is known to be an important factor in fibroblast activation while Pge2 has been shown to counteract TGFβ; however the mechanistic details of this relationship have not been characterized. To this end, we combined computation and experimental techniques to study these cellular and molecular scale events. In vitro experiments are done in parallel with computation experiments and used to build and validate an in silico model. We created a hybrid multi-scale model focusing on the roles of TGFβ receptor ligand signaling dynamics with downstream signaling events leading to fibroblast activation, and fibroblast and epithelial cell co-regulation. Analysis of our model allows evaluation of real-time molecular events in the context of a simultaneous fibroblast/epithelial cell co-culture system. With these tools we have identified key factors driving fibroblast regulation and predict potential therapeutic targets for pulmonary fibrosis.
Tyk2 catalytic activity is required for p40 and interferon-alpha dependent responses (IRM9P.724)
Authors: Strnad Joann , Cheng Lihong , Gillooly Kathleen , McIntyre Kim , Zupa-Fernandez Adriana , Burke James
Published: January 2023

Tyk2 transgenic mice were generated by replacing the Tyk2 wild type allele with a catalytic domain mutation that rendered the enzyme inactive. Characterization of these mice and their WT littermates demonstrate that Tyk2 catalytic activity is required for IL-23, IL-12 and interferon alpha-induced signaling and responses and that these transgenic mice are protected in models of autoimmunity.
The inducible costimulator (ICOS) potentiates TCR-mediated calcium mobilization through Lck (IRM10P.749)
Authors: Suh Woong-Kyung , Leconte Julien
Published: January 2023

It was previously shown that T cell costimulatory receptor ICOS plays an important role in inducing acute graft-versus-host disease (GVHD) in murine models. Although ICOS potentiates TCR-mediated phosphoinositide 3-kinase (PI3K) activation and intracellular calcium mobilization, relative roles of each signaling pathway in GVHD remains to be clarified. Using a knock-in strain of mice in which ICOS selectively lost the ability to activate PI3K, we have shown that T cells utilize PI3K-independent ICOS signaling pathways to induce GVHD. This led us to hypothesize that ICOS-Ca2+signaling pathways play important roles in GVHD. Through site-directed mutagenesis and flow cytometric analysis of calcium fluxing capacities of mutant ICOS proteins, we discovered a membrane proximal triple lysine motif in the ICOS cytoplasmic tail that is essential in inducing Ca2+ flux. We also have genetic and biochemical evidence that the Src family kinase Lck plays a crucial role in ICOS-mediated Ca2+signaling through the activation of ZAP-70 and PLCγ1. Moreover, ICOS appears to utilize different mechanisms than CD28 to potentiate TCR-mediated Ca2+ flux suggesting a synergy between the two related T cell costimulators. Thus our study identified ICOS-Lck-PLCγ1 signaling axis in T cell calcium signaling. Based on these, we aim to define the role of ICOS-Ca2+ signaling in GVHD by generating ICOS mutant mice with selective defect in Ca2+ signaling.
mTORC1-dependent metabolic reprogramming orchestrates T cell activation and fate decisions (IRM10P.750)
Authors: Yang Kai , Shrestha Sharad , Zeng Hu , Karmaus Peer , Neale Geoffrey , Vogel Peter , Chi Hongbo
Published: January 2023

Naïve and activated T cells exhibit distinct bioenergetic and biosynthetic demands. Naïve T cells are in a quiescent state and utilize a catabolic metabolism, whereas antigen-stimulated T cells are highly anabolic. How the metabolic reprogramming is regulated by immune signaling is unclear. In the present study, we reveal that Raptor/mTORC1 orchestrates antigen-induced metabolic reprogramming, and this is a key process to mediate T cell exit from quiescence for ensuing activation and differentiation. Raptor is essential for the integration of T cell receptor and CD28 co-stimulatory signals for sustained mTORC1 activation. Ablation of Raptor in T cells impaired T cell exit from quiescence, indicated by defective cell growth and cell cycle entry. Raptor deficiency abrogated multiple metabolic programs, including glycolysis, lipid synthesis and oxidative phosphorylation, which are required for antigen-triggered exit from quiescence. Aside from the requirement of Raptor in T cell exit from quiescence, Raptor/mTORC1 linked glucose metabolism to the initiation of Th2 cell differentiation through regulating cytokine receptor expression and cytokine responsiveness. Raptor/mTORC1 also impinged upon the differentiation of other T cell lineages including Th1 and Th17 cells. Our studies indicate that Raptor/mTORC1-dependent metabolic reprogramming fuels quiescence exit of naïve T cells, and this in turn coordinates lymphocyte expansion and differentiation in adaptive immune responses.
Sam68 governs T cell activation and proliferation by programming selective NF-κB transcription (IRM10P.751)
Authors: Wan Fengyi
Published: January 2023

The alpha chain of the interleukin-2 receptor (IL2Rα) is expressed in activated T cells and plays a significant role in adaptive immune response; however, the molecular mechanisms that ensure robust and accurate transcription of IL2RA remain poorly understood. We identified the Src-associated substrate during mitosis of 68kDa (Sam68) as a novel component in the nuclear factor-kappaB (NF-κB) complex that specifically confers IL2RA transcription. We also demonstrated that Sam68 plays an essential role in the induction and maintenance of IL2Rα in human T cells derived from both healthy donors and adult T-cell leukemia/lymphoma patients. T cell receptor (TCR) engagement triggered translocation of the inhibitor of NF-κB kinase alpha from the cytoplasm to the nucleus, where it phosphorylates Sam68, causing complex formation with NF-κB. Moreover, TCR engagement-induced activation and proliferation was significantly attenuated in T cells derived from Sam68 deficiency mice, compared to Sam68 proficient controls. Our results reveal an important role of Sam68 in T cell activation and proliferation, and the potential role of Sam68 in T cell-mediated autoimmune disease model will be further discussed.
MicroRNA-21 regulates T cell apoptosis by directly targeting the tumor suppressor gene Tipe2 (IRM10P.755)
Authors: Ruan Qingguo , Wang Peng , Wang Ting , Qi Ji , Wei Monica , Wang Shaowen , Fan Tingting , Johnson Derek , Wan Xiaochun , Shi Weiyun , Sun Honghong , Chen Youhai
Published: January 2023

MicroRNAs (MiRs) are short non-coding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of NF-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly down-regulated while miR-21 expression up-regulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, over-expression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; MiR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.
Flow cytometric analysis of EGR1 expression in human T cells. (IRM10P.740)
Authors: Stepniak Dariusz , Alvarez Miguel , Johnson Ben , Patkar Priya , Kang William , Miranda Cherlyn , Li Shourong , Schrantz Nicolas , Funatake Castle , Peggy Just
Published: January 2023

Early Growth Response 1 (EGR1) is a zinc finger transcription factor that is transiently up-regulated in T and B lymphocytes in response to mitogenic stimuli. It activates transcription of target genes required for cell division and differentiation. In T cells, EGR1 is known to integrate TCR signaling pathways with signaling cascades resulting from co-stimulation and cytokine ligation. In this study, we developed and characterized a novel monoclonal antibody for human EGR1 that can be used for intracellular (nuclear) staining and flow cytometric analysis. Using this antibody we have analyzed stimulatory requirements for the expression of this transcriptional regulator in human peripheral blood T cells.
The orphan nuclear receptor estrogen-related receptor α negatively regulates Toll-like receptor-induced inflammation through A20 induction (IRM11P.759)
Authors: Kim Tae Sung , Yuk Jae-Min , Jo Eun-Kyeong
Published: January 2023

Estrogen-related receptor (ERR) α, an orphan nuclear receptor member, is a key metabolic regulator in various organs and tissues. However, its function in regulating innate immune responses has remained largely unknown. Herein we show that ERRα negatively regulates toll-like receptor (TLR)-induced inflammatory responses through induction of A20. ERRα-deficient (ERRα-/-) mice showed a higher susceptibility to endotoxin-induced septic shock, with a concurrent increase of proinflammatory responses in vivo and in vitro. Global gene expression analysis showed that ERRα regulated TLR-induced inflammation through induction of A20, a deubiquitinating enzyme in TLR signaling. Lack of ERRα in macrophages increased NF-κB signaling and IKK activation. In addition, treatment of macrophages with inverse agonist XCT-790 led to an increase of inflammatory responses induced by TLR4 stimulation. These findings identify a novel function of ERRα as an intrinsic negative regulator of inflammation through induction of A20.
The role of CD99 in T cell activation, signaling, and immunological synapse formation (IRM11P.760)
Authors: Nam Giri , Choi Eun Young
Published: January 2023

Cytotoxic T lymphocytes (CTLs) provides a defense against virus-infected and tumorigenic cells. The recognition of antigen-presenting cells (APCs) for T cells and the association with them are essential in the formation of the immunological synapse (IS) which triggers T cell activation. Although various proteins that stabilize the IS structure for delivering appropriate signals in T cells have been identified, the role of adhesion molecule CD99 in CTL activation and IS formation has not been investigated. Here we found that CD99 in CTLs participates in T cell activation, proliferation, and effector differentiation by inducing of cluster formation between T cells and APCs. Interestingly, it is shown that CD99 is co-localized more at the peripheral supramolecular activating cluster (pSMAC) region than the central supramolecular activating cluster (cSMAC) in the IS, indicating the involvement of CD99 in IS formation. The absence of CD99 in CD8+ T cells resulted in slower formation of T-APC conjugates than WT CTLs. In addition, Akt-mediated signaling, followed by activation, proliferation, and cytokine production, was delayed in CD99KO CTLs upon TCR stimulation. Results presented here demonstrate for the first time that CD99 is recruited to the IS during APC and T cell interaction and plays an important role in subsequent T cell activation.
CD28 induces mitochondrial respiration in long lived plasma cells for reactive oxygen species dependent survival and NFκB-mediated Blimp1 expression. (LYM6P.771)
Authors: Utley Adam , Rozanski Cheryl , Nair Jayakumar , Lee Kelvin
Published: January 2023

Sustained humoral immunity is dependent upon bone marrow resident long-lived plasma cells (LLPCs). We have shown that CD28 signaling in LLPCs is necessary for their survival and antibody production; however, the signaling pathways that define LLPC survival and function are not completely understood. In T cells, CD28 is known to induce glycolysis at the expense of mitochondrial respiration. To our great surprise, CD28 induces mitochondrial respiration-dependent reactive oxygen species (ROS) through PLCγ1 in LLPCs but not splenic short lived plasma cells (SLPCs). Paradoxically, inhibition of ROS kills LLPCs while promoting survival of SLPCs. In LLPCs, CD28-mitochondrial ROS activates NFκB, which in turn binds directly to the Blimp1 promoter, the master regulator of plasma cell identity. Furthermore, CD28 induces Blimp1 expression in LLPCs but not SLPCs. These data suggest a model wherein CD28-induced mitochondrial respiration drives survival of LLPCs through mitochondrial ROS-dependent activation of NFκB and subsequent Blimp1 upregulation. By understanding CD28 regulation of LLPC metabolism, new therapeutic targets arise for antibody-mediated autoimmune disease and vaccine design.
Differential regulation of pro-inflammatory cytokine expression by MAP kinases in macrophages in response to intestinal parasite infection (MPF3P.816)
Authors: Lim Mei Xing , Jiao Huipeng , Png Chin Wen , Tan Shyong Wei Kevin , Zhang Yongliang
Published: January 2023

Blastocystis is a common enteric parasite that is associated with inflammatory bowel disease. However, the pathogenic status of Blastocystis remains unclear. In this study, we found that Blastocystis induced expression of pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α in murine colon and macrophages. We showed that Blastocystis also induced the activation of the three major groups of MAP kinases (ERK, JNK, and p38) in macrophages. The use of MAPK-specific inhibitors in this study demonstrated differential regulation of pro-inflammatory cytokines in macrophages by MAPKs in response to Blastocystis. We found that ERK activation is important for the expression IL-6 and TNF-α at both mRNA and protein levels and the mRNA expression of IL-1β in response to Blastocystis stimulation. Inhibition of JNK significantly suppressed all three pro-inflammatory cytokines at both mRNA and protein levels. p38 inhibition only suppressed IL-6 secretion but not IL-1β and TNF-α. Furthermore, we found that serine proteases from Blastocystis are important for the activation of MAPKs and cytokine expression in macrophages. Our study demonstrated for the first time that Blastocystis could induce the expression of various pro-inflammatory cytokines via activation of MAP kinases, and infection with Blastocystis may contribute to the pathogenesis of inflammatory intestinal diseases through the activation of inflammatory pathways in host immune cells such as macrophages.
Nanogel-based PspA nasal vaccine induces microRNA-associated protective immunity in nonhuman primates (MUC4P.834)
Authors: Fukuyama Yoshiko , Yuki Yoshikazu , Katakai Yuko , Takahashi Haruko , Sawada Shinichi , Joo Sunyi , Park Eun Jeong , Akiyoshi Kazunari , Kiyono Hiroshi
Published: January 2023

We previously demonstrated a cationic type of cholesteryl group-bearing pullulan based nanogel containing pneumococcal surface protein A (PspA-nanogel) induced both protective Th2-mediated Ag-specific systemic and mucosal antibody (Ab) responses and Th17 cell-mediated immunity. In this study, we examined whether PspA-nanogel nasal vaccine could induce PspA-specific protective immunity and cytokines-related miRNA expression in nonhuman primates. When cynomolgus macaques were nasally immunized with 25 μg of PspA-nanogel/dose, increased levels of Th2 cell dependent PspA-specific serum and broncho alveolar lavage fluid (BALF) IgG, and nasal wash (NW) secretory IgA (SIgA) Ab responses with elevated Th17 cell immunity were seen when compared with control macaques nasally immunized with 25 μg of PspA alone or PBS. MicroRNA (miRNA) analysis of serum and nasal tissues from these PspA-nanogel immunized macaques revealed specific elevation of miR-181a and miR-326 which have been shown to corresponding to Th2 and Th17 responses, respectively. These results suggest that these two miRNAs are also associated with non-human primate Th2 and Th17 cell responses after nasal vaccination with PspA-nanogel vaccine which accounted for the induction of protective immunity against pneumonia.
Mechanism by which commensal bacteria limit inflammation (MUC4P.844)
Authors: Paynich Mallory , Jones Sara , Knight Katherine
Published: January 2023

Trillions of bacteria live in homeostasis within the gastrointestinal tract and provide a variety of benefits to the host immune system. One beneficial function of the healthy microbiota is to protect the host from enteric pathogens; however, little is known about the mechanisms by which this occurs. We utilize a mouse model in which a single dose of the commensal bacterium, Bacillus subtilis, protects mice from disease induced by the enteric pathogen Citrobacter rodentium. Our goal is to elucidate the mechanism by which B. subtilis protects from inflammation caused by infection with C. rodentium. We identified exopolysaccharides (EPS) to be the active molecule of B. subtilis, and a single i.p. dose of EPS protects mice from infection. Because protection occurs within a few days, we hypothesized that EPS elicits a protective response by modulating the innate immune system. EPS binds F4/80+CD11b+ peritoneal macrophages, and after i.p. injection of EPS, we identified F4/80+CD11b+ cells that are MHCII+CD206+, indicative of alternatively activated macrophages. We further show that peritoneal macrophages from EPS-treated mice suppress T cell proliferation in vitro, suggesting that these cells contribute to EPS mediated protection from inflammation. We are testing if these cells differentiate in the peritoneum following EPS treatment and whether they mediate protection from C. rodentium induced inflammation.
Immune modulatory mechanisms of Oncostemum bojerianum extracts during Clostridium difficile infection in mice (MUC4P.845)
Authors: Viladomiu Monica , Hontecillas-Magarzo Raquel , Kingston David , Eaton Alex , Zhang Xiaoying , Philipson Casandra , Schiff Eric , Eden Kristin , Lu Pinyi , Carbo Adria , Bassaganya-Riera Josep
Published: January 2023

Activation of Peroxisome Proliferator-Activated Receptor γ has proven beneficial for the treatment of several inflammatory diseases. However, current drugs targeting PPAR result in significant side effects. Therefore, the development of safer anti-inflammatory therapies is important. This project aimed to discover new natural constituents from ethnomedicinal plants that target PPARγ and elucidate the mechanisms of action underlying their anti-inflammatory activities. From a screen of 200 plant extracts, we identified more than 50 that activate PPARγ, with a high predominance in Oncostenum species. The anti-inflammatory properties of two O. bojerianum extracts were further tested in a mouse model of Clostridium difficile infection, which resulted in a detectable improvement in disease activity, weight loss, colonic inflammatory lesions, and increased levels of colonic IL-10-producing CD4+ T cells. Oral treatment with Oncostemum extracts increased PPARγ and PPARα activity and elevated transcriptional expression of their responsive genes. Bioassay-guided fractionation studies in combination with in silico molecular screening, identified the resorcinol family as the PPARγ-activating compounds contained in the extracts. Characterizing the modulation of cellular and molecular signatures by resorcinols at the sites of inflammation will translate natural products discovery into the development of safer and more efficacious therapeutics for infectious and immune-mediated diseases.
Segmented filamentous bacteria induce systemic IgA responses to commensal bacteria (MUC4P.854)
Authors: Wilmore Joel , Sonnenberg Gregory , Artis David , Allman David
Published: January 2023

IgA is a component of the mucosal barrier surface and plays an important part in maintaining separation between the host and the commensal microbiota. Mucosal IgA responses are considered short-lived and restricted to mucosal sites with no systemic dissemination. However, there is little known about the regulation and maintenance of IgA secreting plasma cells (PCs) and how they compare to bone marrow PCs. Interestingly, we find that IgA secreting PCs make up a large proportion of the bone marrow PC pool in mice colonized with segmented filamentous bacteria (SFB), but not in germ-free or conventional SFB free mice. The increase in IgA PCs in the bone marrow correlates with a large increase in serum IgA. The co-housing of SFB free mice with SFB colonized mice results in significantly increased levels of IgA in the serum and higher numbers of IgA+ PCs in the bone marrow. This process is dependent upon T cells, because SFB colonized TCRβ-/-δ-/- mice have similar levels of IgA bone marrow PCs as wild-type SFB free mice. The SFB associated increase of IgA antibody titers coincides with the ability to detect commensal bacteria specific IgA in the serum. These data suggest that the induction of systemic IgA is influenced by the composition of the commensal microbiota. These results highlight the need for greater understanding of the role commensal bacteria play in systemic immunity, which could impact oral vaccine development and autoimmunity.
Phosphoinositide-3-kinase p110δ isoform promotes influenza pathogenicity (MUC5P.858)
Authors: Hancock Aidan , Boesteanu Alina , Chroneos Zissis , Turner Martin , Katsikis Peter
Published: January 2023

We have found that mice deficient in a tissue specific isoform of the signaling enzyme phosphoinositide-3 kinase (PI3K) p110δ are protected from PR8 influenza virus infection and have reduced weight loss and lung viral loads post-infection. Lung inflammation and immune cell infiltration is also decreased after viral challenge. In addition, p110δ KO mice had fewer influenza virus infected alveolar epithelial cells when infected with a GFP-flu virus and GFP expression was measured in lung epithelial cells. Cytokine measurements in bronchoalveolar lavages suggest p110δ KO mice exhibit increased IFN-gamma and MIG post PR8 infection. Slightly increased numbers of NK cells are detected in the lungs of p110δ KO mice as early as day 3 post-infection however activation was decreased within this population as compared to controls, suggesting they are not the primary source of resistance to influenza. In order to determine which cell type was causing the decreased pathogenicity we used two different tissue specific knockout mouse lines. Mice with a myeloid specific KO of p110δ showed similar weight loss and viral loads to C57BL/6 controls, suggesting alveolar macrophages are not primarily responsible for the observed protection in p110δ KO mice. However, mice with alveolar epithelial cell (AEC) specific deletion of p110δ were protected from influenza virus challenge. These data suggest PI3K signaling cascade is important for influenza virus protection within AEC.
Respiratory infection alters archetypal anti-viral CD8 T cell differentiation programs resulting in diminished memory potential (MUC5P.861)
Authors: Shane Hillary , Verbist Katherine , Klonowski Kimberly
Published: January 2023

Our understanding of memory CD8 T cells has been largely derived from acute, systemic infection models. However, memory CD8 T cells generated from mucosal infection exhibit unique properties and, in respiratory infections, are poorly maintained long-term. To better understand how infection route modifies memory differentiation, we compared murine CD8 T cell responses to an identical VSV challenge generated intranasally (IN) or intravenously (IV). IN infection resulted in greater peak expansion of VSV-specific CD8 T cells. However, this numerical advantage was rapidly lost during the contraction phase of the immune response, resulting in numerical memory CD8 T cell deficiencies when compared to IV VSV infection. Interestingly, the anti-viral CD8 T cells generated in response to IN VSV exhibited a biased and sustained proportion of early effector cells (CD127loKLRG1lo) akin to the CD8 developmental program favored after IN influenza infection, suggesting that respiratory infection favors an incomplete memory differentiation program. Correspondingly, IN infection resulted in lower CD122 expression and EOMES levels by VSV-specific CD8 T cells, further indicative of an inferior transition to bona-fide memory. Together these data suggest that the environments encountered in distinct immunization routes are sufficient to modulate both the quality and quantity of anti-viral CD8 T cells in response to an identical pathogen and should be considered in CD8-based vaccine design.
Accurate quantitation of proteins involved in autoimmune disease using simple western (TECH2P.872)
Authors: Tu Andrea , Nguyen Uyen , Kazakova Irina , Ramirez Francisco , Xu Hui , Proctor John , Boge Annegret
Published: January 2023

Why do researchers use Western blots? Since the technique was developed thirty years ago it’s become a trusted method for confirming the presence or absence of a protein but lacks the quantitative rigor for accurate measurement of the amount of protein present. The process requires many steps which introduces multiple variables. While portions of the technique have been automated to improve consistency, until now there has been no major leap in the technology that propels this method of protein analysis from qualitative to quantitative. Simple Western is the modern evolution of traditional immunoassay techniques. Wes, the latest addition to the Simple Western platform, is an easy to use, fully automated system that removes the variability seen with traditional Westerns for more reproducible results run to run and between users. Researchers can now identify their protein and achieve reliable quantitation of their target proteins. To demonstrate the precision and data reliability of Wes we look at proteins indicative of autoimmune disease in human samples from either normal individuals or individuals suffering from the disease. Wes not only reduces the hands on time and the time to results, but generates highly reproducible data that gives researchers a high degree of confidence in the differences detected between the two disease states. Accurate quantitation of these proteins can lead to the identification of predictive biomarkers for either diagnosis or response to treatment.
Anti-murine CD52 therapy provides anti-inflammatory and neuroprotective effects in an EAE mouse model (THER3P.888)
Authors: Turner Michael , Chretien Nathalie , Havari Evis , Huang Jianhua , Pang Petti , LaMorte Michael , Roberts Bruce , Kaplan Johanne , Siders William
Published: January 2023

To better understand the mechanism of action of alemtuzumab in multiple sclerosis (MS), we have investigated the effects of anti-mouse CD52 antibody (anti-muCD52) treatment in an EAE mouse model. Therapeutic administration of anti-muCD52 antibody significantly reduced the severity and progression of disease. Treatment benefit was associated with a reduction in the number of lymphocytes in the spleen and CNS as well as a reduction in MOG35-55 auto-reactive T cells that produce IFNγ or IL-17 following ex vivo restimulation. Neuroprotective effects were also observed following antibody treatment. Specifically, anti-muCD52 treatment reduced the level of white matter damage in the CNS as determined by histological analysis of myelin basic protein and by measurement of serum phosphorylated neurofilament heavy chain. In addition, axonal conductance of the spinal cord was assessed through electrophysiological measurement of motor-evoked potentials. Animals treated with anti-muCD52 demonstrated higher peak amplitude as well as reduced peak latency compared to control animals treated with vehicle. Collectively, these results suggest that the therapeutic benefit of anti-muCD52 treatment in EAE can be attributed in part to a reduction in the number of auto-reactive T cells, decreased CNS inflammation, and protection of CNS integrity.
Generation and characterization of a mouse LIGHT specific inhibitor in a murine model of colitis (THER4P.891)
Authors: Felix Nathan , Kansal Selena , Stetsko Dawn , Yang XiaoXia , Xie Jenny , Suchard Suzanne
Published: January 2023

LIGHT (TNFSF14, CD258) is a member of the TNF superfamily that is expressed on activated T cells, immature DCs, macrophages, and NK cells. LIGHT interacts with its receptors (HVEM and LTβR) and is reported to provide co-stimulatory and pro-inflammatory signals to T cells, B cells, DCs, and macrophages. Numerous studies have implicated the pro-inflammatory effects of LIGHT as an important contributor to disease severity in models of inflammatory bowel disease and fibrosis. However, the inhibitors used in many of these studies (e.g. LTβR-Ig and HVEM-Ig) are not specific for LIGHT, and are likely to impact other pathways such as LTαβ and BTLA. Our focus was to develop domain antibodies (dAbs) that specifically block LIGHT-HVEM and LIGHT-LTβR interactions to better understand the therapeutic potential of LIGHT specific blockade. We were able to develop a mouse LIGHT specific inhibitor that potently blockades LIGHT-HVEM and LIGHT-LTβR interactions in vitro. Notably, this dAb had little effect when used as a monotherapy in a murine model of colitis, but when combined with CD40 blockade showed a high degree of efficacy (equivalent to CTLA4-Ig treatment). These data suggest that LIGHT monotherapy may not be sufficient to impact disease severity, but LIGHT blockade may have potential as part of a combination approach.
VNN2 is a new surface marker for monocytic human myeloid-derived suppressor cells (TUM4P.900)
Authors: Soler David , McCormick Thomas , Cooper Kevin , Young Andrew
Published: January 2023

Studies of myeloid-derived suppressor cells (MDSC) have been hampered by the scarcity and lack of cell-specific markers. Specifically, monocytic MDSC (M-MDSC) are currently characterized as CD14+ HLA-DR-/low and only the absence or low expression of HLA-DR is used as a marker to specifically isolate and purify live M-MDSC. However, identifying cells based on the absence or reduce expression of a protein significantly hampers their study. Reviewing previous studies regarding monocytic markers, we noted Vanin-2 as a protein that has been described in the past to be present in monocytic subsets displaying reduced antigen presentation as well as high reactive oxygen production, hallmarks of MDSC. In order to determine if this marker was present on cells we currently classify as M-MDSC, we used flow cytometry to isolate MDSCs and compared by qPCR CD14+ HLA-DR-/low (M-MDSC) versus CD14+ HLA-DR+ monocytes for VNN2 expression. Interestingly VNN2 showed a 12.43 fold increase in CD14+ HLA-DR-/low M-MDSC compared to CD14+ HLA-DR+ monocytes. Next, we sorted CD14+ VNN2+-expressing monocytes (>10%) and we performed a suppressive assay with autologous bead-stimulated CD8 T-cells. This assay showed CD14+ VNN2+ monocytes suppressive capabilities were comparable to those of traditional CD14+ HLA-DR-/low M-MDSC at 24%, compared to CD8 T-cells alone (90%) or CD14+ VNN2neg monocytes (~70%). These results suggest VNN2 might be a viable surface marker to positively identify live human M-MDSC.
Potentiating dendritic cells to target hypoxic environment of brain tumor (TUM4P.911)
Authors: Ginwala Rashida , Sagar Divya , Karakashev Sergey , Khan Zafar , Reginato Mauricio , Jain Pooja
Published: January 2023

Glioblastoma multiforme (GBM) is the most malignant type of brain tumor with a mean survival time of one year. The most difficult problem to treat tumors is their hypoxic microenvironment that changes the phenotypic characteristic of immune cells. In hypoxic conditions, HIF-1α, a transcription factor, accumulates and stimulates various genes that promote angiogenesis and tumor growth. We studied tumor-mediated adaptations to hypoxia in glioblastoma cells and showed an upregulation of HIF-1α and VEGF expression in a 2- and 3- dimensional culture model. We then confirmed that hypoxic environment increases HIF-1α expression in dendritic cells (DCs), but downregulates the expression of HLA-DR, CD86 and HLA-ABC and affects their antigen processing and presentation capability. First, we reversed the effect of cancer-driven hypoxia on DC functionality in vitro. Then by injecting DCs containing antisense HIF-1α plasmid into mice with GBM, in vivo imaging revealed fluorescently labeled DCs migrating away from the site of injection into the spleen and mediastinal lymph nodes by 54h p.i., indicating chemoattraction of DCs to areas of ongoing inflammation. Finally, these DCs were loaded with glioblastoma tumor antigens via a glycotargeting approach which lead to potentiation of DC functionality and antigen presentation under hypoxic conditions. This translational approach has applicability in eradicating tumors by DC-based vaccination or being adjunct to existing radio- and chemotherapy.
Evaluation of myeloid cell suppression related to cancer cell released products (TUM4P.913)
Authors: Motta Juliana , Sperandio Aline , Castelo-Branco Morgana , Locati Massimo , Rumjanek Vivian
Published: January 2023

Dendritic cells (DCs) and macrophages may recognize tumor antigens and initiate an antitumoral response before and after the formation of tumor mass. However, the secretion of cytokines and factors that inhibit myeloid cells is a mechanism used by tumor cells to escape from immune attack. Our aim was to understand how tumor cells modulate the generation of murine macrophages and human DCs. For this, we collected bone marrow cells from C57Bl/6 mice and differentiated with M-CSF in the presence of 3LL (Lewis lung carcinoma) or MN/MCA (fibrosarcoma) supernatants. Some cells were activated with IFN-γ and LPS to obtain M1 macrophages or IL-4 to M2. Data showed increased expression of arginase-1 mRNA in macrophages differentiated with MN/MCA supernatant and it happened both in M1 and M2. About 20% of cells proliferate in the presence of supernatants, even in the absence of M-CSF and they express higher level of CD16. Moreover, we differentiated human DCs from monocytes with IL-4 and GM-CSF in the presence of K562 (chronic myeloid leukemia) supernatants. TNF-α was added to activate the cells. It was observed higher percentage of cells expressing CD80 when the differentiation was done with K562 supernatant. Higher secretion of IL-10 and lower production of IL-12 were found in cells differentiated with K562 supernatant and later activated. Therefore, the development and function of macrophages and DCs are affected by tumor products and both cells present some suppressive features.