bluuebunny/crossref_metadata_2025 huggingface dataset:
Over-, ectopic-expression of FoxN1 at early life adversely influences lymphopoiesis (HEM3P.280)
Authors: Ruan Linhui , Burnley Preston , Wang Liefeng , Mu Lili , Coder Brandon , Shaw Jennifer , Zhuge Qichuan , Su Dong-Ming
Published: January 2023

FoxN1 expresses in thymic epithelial cell (TEC) and skin epithelium to regulate their development. Prenatal mutation in FoxN1 results in thymic development failure and nude phenotype, while postnatal insufficient FoxN1 expression induces thymic atrophy, resulting in declined T-lymphopoiesis. Although enhancing FoxN1 expression in aged thymus promotes functional rejuvenation, whether FoxN1 over-expression at early age or ectopic expression in the bone marrow is beneficial for lymphopoiesis is unknown. Using our newly generated R26-STOPflox-FoxN1 (gain-of-function) mutant mice, which have an over-expressed FoxN1 after various promoter-driven Cre-mediated deletion of the roadblock STOPflox, we found that K14Cre-mediated prenatal FoxN1 over-expression resulted in a newborn lethal phenotype, displaying abnormal permeability in the skin. Ubiquitous deletion of the STOPflox mediated by uCreERT progressive leak in infant mice influenced TEC development, and T- and B-lymphopoiesis. Although the K5CreERT mediated FoxN1 over-expression in adult mice may be beneficial for postponing thymic aging, early induction of K5CreERT activation in infants induced adverse influences to thymoycte and hair follicle development. Therefore, FoxN1 has a stage and tissue sensitivity. Over- and ectopic-expression of FoxN1 at early life adversely influences immature thymic, T-, and B-cell, and skin epithelial development.
Essential regulatory role for factor H and properdin in the formation of platelet/granulocyte aggregates in human whole blood (HUM1P.301)
Authors: Blatt Adam , Saggu Gurpanna , Cortes Claudio , Ferreira Viviana
Published: January 2023

An increased number of activated platelets and platelet/leukocyte aggregates are found in patients with chronic inflammatory diseases including cardiovascular disease. Platelet/leukocyte aggregates are not only indicative of cardiovascular disease, but also play an important role in the initiation and progression of disease. The role that complement regulatory proteins properdin and factor H play in controlling the formation of platelet/granulocyte aggregates, as well as which factor H domains are essential for the interaction with platelets on which complement is activating remain unknown. Here, we have determined that (a) the C-terminus of factor H is critical for the ability of the protein to protect platelets from the alternative pathway of complement, (b) blocking of properdin function using an inhibitory antibody significantly reduces the formation of platelet/granulocyte aggregates, while adding properdin increases the formation of these aggregates in a dose-dependent manner, and (c) factor H controls the properdin-mediated formation of platelet/granulocyte aggregates. Our data support critical roles for properdin and the C-terminus of factor H in controlling the formation of platelet/granulocyte aggregates. These studies may contribute to the understanding of the pathophysiological mechanisms involved in the interaction between platelets and granulocytes in chronic inflammatory diseases.
Role of immune cell subsets in bone disease associated to chronic kidney disease and haemodialysis patients. (HUM1P.315)
Authors: Gigante Margherita , Cafiero Cesira , Brunetti Giacomina , Grano Maria , Colucci Silvia , Simone Simona , Ranieri Elena , Pertosa Giovanni , Gesualdo Loreto
Published: January 2023

Chronic kidney disease (CKD) is a progressive loss in renal function, which severity is classified in five stages, with stage 1 being the mildest and stage 5 being a severe illness evolving to hemodialysis (HD). Chronic inflammatory state in HD and CKD patients (pts) may contribute to the development of bone disease through the increase of osteoclasts (OCs), the bone resorbing cells. In pathological condition, immune cells and inflammatory cytokines belonging to tumor necrosis factor superfamily, such as LIGHT and RANKL, play a key role in osteoclastogenesis. The aim of our study was to evaluate the osteoclastogenic potential of unfractionated and T cell-depleted PBMCs from CKD and HD pts. We also evaluated the presence of circulating osteoclast precursors (CD14+/CD16+) and characterized the immune cell subsets for LIGHT and RANKL expression through flow cytometry and RT-PCR. PBMCs from HD and CKD pts (stage IV-V) showed a spontaneous osteoclastogenesis in vitro (80±7). Conversely, exogenous cytokines were essential to trigger and sustain osteoclastogenesis in CKD pts (stage I-II) and controls (10±4). It correlated with a significant increase of both LIGHT and RANKL expression on CD4+ T cells as well as CD14+CD16+ monocytes in HD and CKD pts (stage IV-V) compared to controls (p<0.005). LIGHT and RANKL may represent a new link between immune cells activation and bone-associated disease in CKD and HD pts suggesting new potential therapeutic targets in the setting of these pts.
Upregulation of immunomodulatory molecules by matrine treatment in experimental autoimmune encephalomyelitis (HUM1P.316)
Authors: Zhu Lin , Nan Liu
Published: January 2023

Dysfunction of the immunologic mechanism is a primary characteristic of experimental autoimmune encephalomyelitis (EAE), which is a classical experimental model of multiple sclerosis (MS). Matrine (MAT), a quinolizidine alkaloid, has been used for hepatitis B in clinical. Previous reports have suggested that MAT could ameliorate the clinical symptoms of EAE rats, but haven’t clarified the mechanism of action until now. Our present study showed that MAT reduced the neurological scores of EAE rats, suppressed infiltration of inflammatory cells and demyelination in the CNS significantly. Furthermore, we studied the possible immunologic mechanism underlying the treatment of MAT for EAE by measuring the expression of system and molecules related with immunoregulation mechanism. In our study, an administration of MAT significantly increased the expression and production in the CNS of Nrf2 and HO-1, the correlation factors of oxidative stress that confront inflammatory cells into the CNS. Furthermore, the expressions of Interleukin-4, Interleukin-5, Interleukin-10, transforming growth factor-1 and Foxp3, which play an important role in the balance between Th1 and Th2 cells in EAE, were also significantly increased. Our findings reveal that MAT could promote the expressions of factors for inhibiting the inflammation. In conclusion, our study clarifies the possible mechanisms underlying the treatment of MAT for EAE, implies that MAT may be a novel therapeutic option for MS.
Treatment of MS patients with BG-12 results in a decline in circulating memory T and B lymphocytes (HUM1P.317)
Authors: Lundy Steven , Wu Qi , Le Lisa , Mao-Draayer Yang
Published: January 2023

BG-12 (Tecfidera®) is a recently approved oral treatment for multiple sclerosis that has shown remarkable efficacy. The mechanisms by which BG-12 acts on the human immune system are poorly defined. In the current study, we analyzed the effects of BG-12 treatment on subsets of circulating human T and B lymphocytes using multicolor flow cytometry. Patients treated for four months with BG-12 had decreased total numbers of lymphocytes in their blood that did not reach significance, and no changes in their overall percentages of CD4+ T cells, CD8+ T cells, or CD19+ B cells compared to pretreatment levels. Significant differences were observed when subsets of T and B cells were evaluated. BG-12 treatment resulted in a decrease in percentages of circulating CD4+CD45RO+CD45RAneg memory TH cells and CD8+CD45RO+CD45RAneg memory CTL. Treatment with BG-12 also resulted in significant decreases in circulating CD27+CD19+ memory B cells, and a relative increase in the levels of CD24highCD38highCD19+ regulatory B cells. A CD27+CD24lowCD19+ B cell subset was present in the circulation of some MS patients prior to treatment, but returned to the very low levels found in healthy controls following four months of BG-12 treatment. The data suggest that BG-12 has in vivo down regulatory effects on some lymphocyte subsets, particularly memory T and B cells. Ongoing experiments are focused on measuring functional differences in T and B cell cytokine production in BG-12 treated patients.
The effects of microRNAs on regulatory T cells in multiple sclerosis (HUM1P.319)
Authors: Severin Mary , Lovett-Racke Amy
Published: January 2023

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS. The pathophysiology that propagates MS is hypothesized to be an autoimmune reaction mediated by pro-inflammatory T cells activated against myelin. Regulatory T cells (Tregs), protectors against such autoimmune responses, are dysfunctional in MS patients. We investigated which microRNAs, modulators of cellular and disease processes, are regulators of MS pathogenesis and the extent to which these miRNAs regulate genes critical for Treg development. To this end, miRNA profiling studies performed on naïve CD4+ T cells have identified 85 differentially expressed miRNAs in MS patients compared to healthy donors. Of the miRNAs, 23 were predicted to target genes of the transforming growth factor beta (TGFβ) signaling pathway. Given the importance of TGFβ in regards to Treg development and function, we hypothesized that dysregulated miRNAs in the naïve CD4+ T cells of MS patients target the TGFβ-signaling pathway, resulting in defective Tregs and enhanced susceptibility to developing MS. Our data indicate that 1) expression of TGFβ-associated genes is decreased in MS patients, 2) several of the identified miRNAs directly target genes of the TGFβ-signaling pathway, and 3) overexpression of TGFβ-targeting miRNAs causes a decrease in Treg development. Taken together, our data have allowed us to highlight specific miRNAs as being potentially highly relevant in the development of defective Tregs in MS.
Exacerbation of neurodegeneration by an α-synuclein specific CD4+ effector T cell line in a mouse model of Parkinson’s disease (HUM1P.326)
Authors: Wilshusen Rebecca , Anderson Kristi , Szlachetka Adam , Mosley R.
Published: January 2023

We have previously shown in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model that T cell-mediated immunity regulates innate immunity to exacerbate neuroinflammation. Effector T cells that recognize nitrated α-synuclein (N-α-syn) as non-self, increase reactive myeloid microglia and inflammatory mediators with prolonged progression and increased dopaminergic neurodegeneration within the substantia nigra (SN) of MPTP treated mice. This study focuses on the generation of an N-α-syn specific CD4+ T cell line with the ability to increase release of reactive nitrogen species (RNS) from myeloid lineage cells and also intensify the loss of tyrosine hydroxylase (TH) expressing dopaminergic neurons within the SN of MPTP intoxicated mice. Flow cytometric analysis showed the T cell line exhibited very distinct expression of CD3, CD4 and IL-17a, moderate staining for CD146, yet very little expression of CD8 and FoxP3. Co-culture of the T cells with N-α-syn and BV-2 cells increased RNS production of BV-2 cells by 150% compared to controls. Lastly, adoptive transfer of the T cell line to MPTP intoxicated mice exacerbated the loss of TH+ dopaminergic neurons by 63% compared to controls. Together, these results support the notion that effector T cells specific for N-α-syn increase oxidative stress as measured by RNS release from reactive myeloid microglia, increase the loss of TH+ neurons, and thus may play a pivotal role in the tempo and progression of Parkinson’s disease.
The autoimmunity-associated gene CLEC16A controls HLA-II expression by participating in the molecular machinery of late endosomal maturation (HUM2P.337)
Authors: Laman Jon , van Luijn Marvin , Kreft Karim , Jongsma Marlieke , Mes Steven , Wierenga-Wolf Annet , van Meurs Marjan , Melief Marie-Jose , van der Kant Rik , Janssen Lennert , Janssen Hans , Priatel John , Tan Rusung , Neefjes Jacques , Hintzen Rogier
Published: January 2023

In GWA studies, CLEC16A has been identified as a risk gene for several major autoimmune diseases, including multiple sclerosis (MS). We show that CLEC16A is increased in MS and functionally links to HLA-II, and uncovered the molecular mechanism of CLEC16A as key regulator of HLA-II antigen presentation. We used the human melanoma cell line MelJuSo and primary moDC as APC models to silence CLEC16A with different siRNA and lentiviral shRNA. CLEC16A knockdown in MelJuSo cells and moDC resulted in cytoplasmic dispersion and strong accumulation of HLA-II+ late endosomes, suggesting impaired formation of multivesicular late endosomal compartments, i.e. MIIC. Indeed, in CLEC16A shRNA transductants, MIIC contained no or a few abnormally enlarged internal vesicles and showed an increased number of HLA-II molecules as compared to scrambled shRNA transductants of MelJuSo cells. CLEC16A associated with Rab7-interacting lysosomal protein (RILP) and the homotypic fusion and protein sorting (HOPS) complex, two members of the dynein motor complex regulating late endosomal trafficking and maturation. In addition, CLEC16A silencing disrupted RILP-mediated recruitment of HLA-II+ late endosomes to perinuclear regions. We reveal direct involvement of CLEC16A in the machinery regulating late endosomal processing of HLA-II. This novel C-type lectin function supports a pathogenic role of CLEC16A in autoimmune disease by promoting HLA-II antigen presentation via the formation of MIIC.
Alcohol consumption increases ultraviolet radiation induced immune dysfunction. (HUM2P.341)
Authors: Brand Rhonda , Stottlemyer J. Mark , Paglia Melissa , Falo Louis
Published: January 2023

Alcohol ingestion and sun exposure often occur simultaneously. Excessive ultraviolet radiation (UVR) is associated with increased incidence of nonmelanoma skin cancers (NMSCs). More recently, epidemiologic studies have revealed an independent correlation between alcohol use and increased incidence of NMSC. Although multiple factors have been proposed to explain these interactions, the underlying mechanisms responsible for this association have not been determined. Impaired tumor surveillance contributes to the development of skin cancer, and UVR can suppress immune function. Furthermore, the metabolism of alcohol can also suppress immune function. We therefore sought to determine whether UVR mediated immune dysfunction is affected by alcohol consumption. Specifically, we evaluated UVR induced immune suppression in the presence or absence of alcohol ingestion by combining standard mouse models of alcohol consumption and UVR induced immune suppression. In this controlled system, our results demonstrate that mice exposed to both ETOH and UV suffer greater immune suppression than those exposed to either alone. These results suggest that combining alcohol and UV exposure increases immune suppression, and support additional efforts to investigate the interactions between UVR and alcohol relevant to immunosuppression and the development of NMSC.
Activating mutations in p110δ cause human immunodeficiency and reveal important roles of PI3K in T cell senescence and homeostatic cell death (HUM2P.334)
Authors: Lucas Carrie , Lenardo Michael
Published: January 2023

The p110δ subunit of phosphatidylinositol-3-kinase (PI3K) is selectively expressed in leukocytes and plays critical roles in lymphocyte biology. We have identified 14 patients from 7 families who were heterozygous for one of three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ) and suffered from sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia, and viremia due to cytomegalovirus and/or Epstein-Barr virus. Strikingly, hyperactivation of PI3K signaling caused significant skewing of peripheral blood CD8 T cells toward an effector phenotype with exaggerated effector characteristics in vitro. Mechanistically, T cell blasts harboring gain-of-function mutations in p110δ exhibited constitutively hyperactive Akt and mTORC1 signaling with enhanced glucose uptake. These changes promoted marked terminal effector cell differentiation and senescence of CD8 T cells. Notably, treatment of patients with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely ‘rescued’ the in vitro T cell defects, and greatly improved clinical course. In addition to enhanced effector characteristics, in vitro analyses of patient T cell blasts also revealed a greater susceptibility to TCR restimulation-induced cell death. Indeed, in an independent chemical genetics screen, PI3K stood out as a critical pro-death kinase whose downstream signaling connected with caspase activation upon TCR restimulation.
Fluvastatin suppresses IgE-mediated mast cell activation in vitro and in vivo (HYP3P.411)
Authors: Kolawole Elizabeth , Ndaw Victor , Ryan John
Published: January 2023

Fluvastatin, the HMG-CoA reductase inhibitor known for its role in the treatment of hypercholesterolemia and cardiovascular disease, has more recently been shown to play a role in the immune response. Given the critical role that mast cells play in allergy and inflammatory diseases such as asthma, which effects one third of America’s population, we assessed the effect of Fluvastatin on mast cell function. We demonstrate that Fluvastatin downregulated IgE-mediated cytokine production and mast cell migration. Additionally, in vivo studies showed that Fluvastatin suppressed IgE-mediated anaphylaxis. Interestingly, the effect of Fluvastatin may be restricted to mast cells cultured from mice with a Th1-prone background. Characterizing the role of Fluvastatin on mast cells may prove to be therapeutically important.
Suppression of IgE-mediated disease by rapid desensitization with anti-FcεRIα mAb (HYP3P.345)
Authors: Khodoun Marat , Kiselev Artem , Mulloy James , Wunderlich Mark , Morris Suzanne , Strait Richard , Finkelman Fred
Published: January 2023

IgE-mediated mast cell and basophils activation is critical for the pathogenesis of anaphylaxis, drug allergy, food allergy and allergic skin disorders and contributes to the pathogenesis of asthma and allergic rhinitis. We recently demonstrated that IgE-mediated anaphylaxis in mice can be safely blocked by rapid desensitization with ascending doses of an anti-mouse FcεR1α Abs, starting with a dose too small to induce detectable disease and ending with a dose that induces anaphylaxis if injected into previously untreated mice (JACI, 131:1555-64, 2013). We have now modified this approach, which removes IgE from mast cells, to make it human-applicable, faster and safer. To do this, we: (1) generated a chimeric anti-human FcεR1α mAb that has V regions from a mouse anti-human FcεR1α mAb (15.1) and C regions from human IgG1; (2) substituted a mAb that binds FcεR1 regardless of whether it is associated with IgE for a mAb that only binds FcεR1 that is not IgE-associated; and (3) mutated the γ1 chain C region to increase its affinity for the inhibitory receptor, FcγRIIB. The potential usefulness of this approach has been demonstrated in IgE-mediated anaphylaxis models in mice that express human, instead of mouse FcεRIα and in human cord blood-reconstituted NSGS mice, which generate human mast cells, basophils and IgE-secreting B cells. These observations suggest that full development of this approach should provide a way to safely and rapidly suppress IgE-mediated human allergy.
Signaling via neurokinin 1 and 2 receptors exerts opposing effects on mast cell pro-inflammatory and Th2-biasing functions. (HYP3P.347)
Authors: Sumpter Tina , Tkacheva Olga , Shufesky William , Morelli Adrian , Larregina Adriana
Published: January 2023

Following activation via FcεRI, peripheral tissue mast cells (MCs) initiate and sustain atopic diseases, by secreting proinflammatory and Th2 biasing cytokines. MC express neuropeptide receptors and are exposed to neuroinflammatory mediators. However, the role of neuropeptides in FcεRI-activated MCs remains mainly unknown. Two of the most potent pro-inflammatory neuropeptides, substance-P (SP) and hemokinin-1 (HK-1), bind with high affinity to the neurokinin 1 receptor (NK1R) and with lower affinity to the NK2R. We addressed in vitro and in vivo the role of the NK1R and NK2R in MC responses initiated via FcεRI. In vitro, FcεRI activation of bone marrow derived (BM)MCs from wild type (WT) mice increased NK1R and NK2R expression and HK-1 release, but not release of SP. Utilizing NK1R-/- BMMCs, we determined that the NK1R was necessary for Akt-NFκB signaling and for the secretion of the pro-inflammatory TNF-α, IL-6 and Th2 biasing IL-4 and IL-13. Conversely, inhibition of NK2R signaling in WT BMMCs with GR159897 decreased the release of proinflammatory and Th2 cytokines. In vivo, NK1R signaling was necessary for amplifying IgE-Ag mediated cutaneous and systemic anaphylaxis and chronic asthma. We conclude that NK1R and the NK2R display opposing effects on the inflammatory function of FcεRI-activated MCs relevant for the control of MC derived atopic disorders.
Tetraspanin CD151 specifically regulates ERK1/2 signaling to limit FcεRI-mediated production of cytokines by mast cells (HYP3P.349)
Authors: Abdala-Valencia Hiam , Bryce Paul , Wechsler Joshua , Cook-Mills Joan , Hsu Chia-Lin , Berdnikovs Sergejs
Published: January 2023

Mast cells and basophils are granulocytes critical in the pathogenesis of allergic disease due to the release of preformed and newly synthesized mediators. Tetraspanins are emerging as novel modulators of signals for cellular activation, however, mechanistic understanding of their function is currently lacking. We found tetraspanin CD151 to be specifically induced in human and mouse mast cells upon IgE stimulation, suggesting a functional role in activation of these cells. Here, we show that CD151 modulates IgE-induced mast cell activation by exerting selective control over downstream pathways regulating de novo synthesis of cytokines IL-4, IL-13 and TNF-α. Mechanistically, CD151 selectively targets IgE-induced activation of ERK1/2, associated with cytokine production, but has no regulatory effect on PLCγ1 or calcium release necessary for degranulation. Inhibition of ERK1/2 blocks the exaggerated induction of cytokines by CD151 deficient mast cells. In vivo, CD151 deficiency significantly exacerbates IgE-mediated late phase response in a murine model of passive cutaneous anaphylaxis. Collectively, our data indicate that CD151 functions in the late phase of IgE-induced mast cell activation by limiting cytokine synthesis.
Leukotriene enhanced allergic lung inflammation through induction of chemokine production (HYP4P.414)
Authors: Kwon Bo-In , Shin Kihyuk , Lee Seung-Hyo
Published: January 2023

Leukotrienes, which are mainly expressed by mast cells, macrophages and eosinophils, have been shown to act as important mediators in allergic asthma. In addition to leukotrienes, IL-13, a Th2 specific cytokine, has also been considered as the most important effector cytokine to induce allergic asthma, by increasing airway hyper-responsiveness, airway inflammation, eosinophilia and mucus hyper-production. But, the exact mechanisms how leukotrienes elicit allergic lung responses at initial stage and the effect of leukotrienes on allergic asthma through IL-13 remain unclear. To reveal this, we adopted two distinct models of allergic inflammation: intranasal Aspergillus protease allergen and recombinant IL-13 induced Th2 mediated inflammation. Here we measured AHR, bronchoalveolar lavage cytology, glycoprotein secretion, as well as IL-13 related chemokines such as CCL7, CCL11 and CCL17, which directly regulate homing of Th2 cells to lung with 5-lipoxygenase deficient and wild type mice. 5-lipoxygenase that convert arachidonic acid into leukotriene A4 null mice showed attenuated airway inflammation, AHR and bronchoalveolar lavage eosinophilia, as well as diminished chemokines including CCL7 and CCL17. Finally, addition of LTB4 and LTC4 to the airways of 5-lipoxygenase deficient mice restored rIL-13 induced asthma. Thus, our results indicate that LTs augment the effect of IL-13 in allergic asthma, are required for Th2 specific chemokine production.
Allergen-specific lung resident CD4+ Th2 cells in asthma (HYP4P.417)
Authors: Pepper Marion , Hondowicz Brian , Moon James
Published: January 2023

Asthma is an inflammatory disease of the airways that affects more than 300 million people worldwide. Clinically, asthma is characterized by acute, intermittent and recurrent episodes of inflammation that can be induced by a specific allergen. Allergen-specific CD4 T cells contribute to this process by producing cytokines and inducing B lineage production of IgE. Allergen-specific CD4 memory T cells are also thought to persist after resolution of inflammation to rapidly drive asthma-induced pathology. Due to the challenge of tracking small populations of CD4 T cells that express allergen pMHCII-specific TCRs, little is known about what regulates Th2 memory cell differentiation, migration and maintenance. We have therefore produced a MHCII tetramer containing a peptide from the Derp1 protein of the house dust mite (HDM), the most common cause of atopic asthma, to elucidate the underlying mechanisms of Th2 memory cell differentiation during atopic asthma. Tetramer-based enrichment strategies used to isolate Derp1:I-Ab+ cells from the secondary lymphoid organs and lungs have revealed that heterogenous Th2 memory populations form, including a CD69+ Derp1:I-Ab+ population in the lung thats persists post-contraction. Intravascular staining techniques demonstrate that these cells reside in the lung parenchyma and are driven to the lung by early cytokine exposure. These findings have important implications for novel immunotherapeutics in both asthma and allergy.
Peroxiredoxin released in inflammasome condition activates the classical complement pathway (INM2P.427)
Authors: Lee Hyun Sook , Kim Sook Young , Shin Jeon soo
Published: January 2023

Peroxiredoxin (Prx) family proteins are expressed in diverse cellular components and play roles as antioxidant proteins that control ROS level. Recently, it has been reported that Prx was released in ischemic brain. We, therefore, focused on investigating how Prx is released and the role of released Prx. In this study, we show that Prx could be released by LPS stimulation in immune cells. Moreover, the release of Prx was dramatically increased in inflammasome condition through priming LPS followed by ATP treatment. To examine the interaction between released Prx and complement, we performed in vitro binding assay. Recombinant Prx protein binds recombinant C1q protein. Also, we observed membrane attack complex (MAC) in normal human sera, which is a result of activation of the complement pathway. To clarify whether these complement activation by Prx is a C1q-dependent response; we confirmed the formation of MAC using C1q-depleted human sera. The effect of Prx in formation of MAC was significantly reduced in C1q-depleted human sera. Through this, we found that complement activation by Prx is a C1q-dependent response. In conclusion, our results reveal that Prx was released by inflammasome-dependent and -independent pathways in immune cells. Released Prx, then, binds to C1q and activates the classical complement pathway. These findings suggest that released Prx may have synergistic effect with complement in inflammation, which can be a target in septic inflammation therapy.
Decoy FcγR-Ig molecules ameliorate immune-complex induced blood vessel damage by blocking the release of inflammatory mediators from macrophages (INM2P.432)
Authors: Mula Ramanjaneya , Machiah Deepa , Wang Xinyu , Selvaraj Periasmay , Shashidharamurthy Rangaiah
Published: January 2023

Autoimmune vasculitis is an endothelial inflammatory disease that results from deposition of immune-complexes (ICs) in blood vessels. Interaction of FcγRs expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is prerequisite to prevent the blood vessel damage. Herein, we have shown that dimeric FcγR-Ig (CD16A-Ig and CD32A-Ig) molecules are able to block these interactions using in vitro and in vivo vasculitis models. FcγR-Igs could block 70% of RAW 267.4 cells binding to antibody-coated Human umbilical vein endothelial cells (HUVEC). FcγR-Igs significantly inhibited the IC-mediated expression of inducible nitric oxide synthase (iNOS) and Nitric oxide (NO) release in RAW 264.7 cells. We observed that exogenous NO induced the upregulation of pro-apoptotic genes such as Bax, Bak, caspase-3 and caspase-8 in HUVEC cells. Further, in vivo studies revealed that circulating ICs deposits in the capillaries of various vital organs but not in large arteries. Interestingly, dimeric FcγR-Igs are distributed in the areas where ICs are deposited. The co-localization of ICs and FcγR-Igs revealed that dimeric FcγR-Ig molecules bind specifically to ICs and thus prevent the vascular damage. Taken together, these results suggest that IC-induced NO might be a major factor promoting the blood vessel damage, which can effectively be blocked using recombinant dimeric FcγRs molecules during IC mediated vasculitis.
Adaptor protein Crk-like (CrkL) controls natural killer cell function via regulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at immunological synapses (INM2P.433)
Authors: Zheng Peilin , Hanson Celine , Liu Dongfang
Published: January 2023

Very few studies have addressed how CrkL, a ubiquitous adaptor protein, regulates the immune response, especially in NK cells. Previous studies indicate that CrkL is required for NKG2D-mediated cellular cytotoxicity (NC) and CD16 signaling triggered antibody mediated cellular cytotoxicity (ADCC). However, how CrkL protein controls NK cell cytotoxicity remains unclear. Here we used lentiviral inducible shRNA to silence CrkL in human NK cell line and primary NK cells. Inducible knockdown of CrkL significantly reduced NK-target conjugation, degranulation, and cytotoxicity in both NC- and ADCC-mediated immune responses. Further investigation on the function and signaling transduction at the NK cytotoxic immunological synapse (IS) reveals that polarization of lytic granules, and accumulation of both F-actin and phosphorylated Vav-1 at the IS were greatly reduced in CrkL-silenced NK cells. NK cell activation involves in actin-dependent activating receptor clustering. CrkL silencing resulted in reduced accumulation of ligands for CD16 and NKG2D on the lipid bilayer. Similar results were obtained in NK cells from DiGeorge Syndrome patients (CrkL gene deletion). We thus propose a new model for adaptor protein CrkL function in NK cell activation: CrkL controls NK cell cytotoxicity via promoting F-actin accumulation and lytic granule polarization by modulating the accumulation of activating receptors and phosphorylated Vav-1 molecules at the IS.
Characterizing bone marrow-derived mast cell interaction with electrospun bioresorbable vascular grafts (INM2P.436)
Authors: Abebayehu Daniel , Spence Andrew , Sell Scott , Bowlin Gary , Ryan John
Published: January 2023

Upon implantation, the regenerative potential of a vascular graft will be dependent on the body’s innate immune response. Thus, it is imperative to evaluate and regulate how innate immune cells interact with a vascular graft’s structural architecture. Different pore-sizes and fiber diameters of electrospun grafts should modulate mast cell activation and secretion, thus dictating their innate ability for inflammation or angiogenesis and regeneration. Grafts were electrospun with two different polymer concentrations on a solid mandrel, an air-impedance mandrel (increasing scaffold porosity), and altered by compression (decreasing scaffold porosity). Murine bone marrow-derived mast cells (BMMC) were seeded on scaffolds, and supernatants and cell lysates were collected after LPS and IL-33-mediated activation. ELISA analysis demonstrated that BMMC released TNFα and IL-6 at significantly greater concentrations on grafts with decreased fiber diameter and porosity. This was matched by greater NFκB activation, as measured by western blotting. In contrast, significantly greater VEGF secretion was observed on grafts with the largest fiber diameter and porosity. These results indicate that graft architecture regulates mast cell interactions and subsequent potential for healing, with increased fiber diameter and porosity promoting an anti-inflammatory and angiogenic response.
Regulation of IFN-γ by IL-13 dictates the susceptibility to secondary post-influenza MRSA pneumonia (INM3P.439)
Authors: Rynda-Apple Agnieszka , Harmsen Ann , Erickson Anfin , Harmsen Allen
Published: January 2023

Post-IAV superinfections are the primary cause of deaths during IAV pandemics. The superinfection in mice 7 days after IAV infection results in exacerbated bacterial pneumonia mediated by type I and II IFNs. However, the sequelae of cytokine responses early in IAV infection, that eventually determines the later susceptibility is not understood. Here we examined whether the susceptibility to superinfection with MRSA varies during IAV infection. In the first 3 days of IAV infection mice had increased resistance to MRSA superinfection, which required IFNAR and IL-13 signaling to inhibit IFN-γ. IFN-γ-mediated high susceptibility to MRSA pneumonia 7 days after IAV infection was associated with increased IL-13Rα2 production, and was reversible by treatment with mrIL-13 or anti-IL-13Rα2. Thus, early in IAV infection IFNAR signaling maintained IL-13, which increased resistance to MRSA. Then, as IAV infection progressed, the capacity for IL-13 signaling was inhibited, in part by increased IL-13Rα2, resulting in increased IFN-γ levels and susceptibility to MRSA superinfection. Thus, IAV induces a sequence of cytokine responses where early IFNAR-dependent IL-13 signaling suppresses IFN-γ production resulting in enhanced resistance to MRSA challenge. Understanding these cytokine sequelae is critical to development of immunotherapies for IAV-MRSA coinfection since perturbations in the sequence of these events at the wrong time could increase susceptibility to MRSA and/or IAV.
NLRP3 and AIM2 mediate nitric oxide production and macrophage resistance to T. cruzi infection (INM3P.442)
Authors: Silva Grace , Carneiro Natália , Zamboni Dario , Silva João
Published: January 2023

The Toll-like receptors (TLRs), 2, 4 and 9 are important to immunity against Trypanosoma cruzi, etiologic agent of Chagas disease. Intracellular receptors, including NLRP3 and AIM2, associate to ASC forming a complex that active caspase-1, called inflammasome. This complex is required for cleavage of the active forms of the IL-1β and IL-18. Recently, we show that ASC inflammasome is necessary to control T. cruzi infection, however the receptors that participate in this signalizing are not complete clear. Here, we investigated the participation of NLRP3 and AIM2 in the immune response against T. cruzi. First, we found high IL-1β production and caspase-1 activation in BMMs from WT mice, but not from NLRP3-/- and AIM2-/- mice. To understand the role of the inflammasome pathway during T. cruzi infection, WT, caspase-1-/-, ASC-/-, AIM2-/- and NLRP3-/- mice were infected with Y strain of T.cruzi. Our results showed that ASC-/- and caspase-1-/- mice presented higher mortality, whereas WT, AIM3-/- and NLRP3-/- are resistant to infection, suggesting more than one NLR is necessary to confer resistance for T. cruzi infection. In addition, caspase-1-/-, ASC-/-, AIM2-/- and NLRP3 BMMs presented higher parasitism and reduced amount of Nitric oxide (NO) production. Together, these results suggest that AIM2 and NLRP3 via ASC inflammasome participate of the infection against T. cruzi. Financial Support: FAPESP
Leptin upregulates CRAC channel expression in T lymphocytes (IRC2P.444)
Authors: Ebersbacher Charles , Chimote Ameet , Obici Silvana , Conforti Laura
Published: January 2023

Obesity is a major health problem associated with systemic diseases whose pathogenesis has been linked to inflammation, but the underlying mechanisms are poorly understood. Leptin, an adipokine secreted by adipose tissue, is known to augment the antigen-dependent release of pro-inflammatory cytokines by T lymphocytes. Cytokine production is tightly regulated by cytoplasmic Ca2+ (Cac ) which is under the control of ion channels (Kv1.3, KCa3.1, calcium release activated Ca2+channel [CRAC, formed by Orai1 and Stim1]). We tested the hypothesis that ion channels contribute to the pro-inflammatory effects of leptin. CD3+ T cells isolated from healthy human donors were treated with 100-250 ng/ml leptin and ion channels’ gene expression was measured by RT-qPCR. Leptin induced a dose- and time-dependent increase in Orai1 expression up to 1.53 ± 0.21 (100 ng/ml) and 2.04 ± 0.40 folds (250 ng/ml, n=3) at 12 h. There was no significant difference in Stim1, Kv1.3 and KCa3.1 expression. CRAC channels are important for activation-mediated Ca2+ influx in T cells. Thus, we investigated whether Orai1 regulation by leptin affects Cac using a protocol that bypasses the T cell receptor and allows assessment of ion channel-dependent changes in Cac. Incubation with leptin-depleted serum reduced Ca2+ fluxes, while addition of 250 ng/ml of leptin to the leptin-free serum augmented Ca2+ fluxes. These findings suggest that leptin may promote hyperactivity of T cells via upregulation of ion channels.
Leucine-rich repeat containing 8a (LRRC8A) is a novel T cell co-stimulatory receptor for T cell activation (IRC2P.453)
Authors: Chou Janet , Kumar Lalit , Yee Christina , Fraulino David , Hedayat Mona , Geha Raif
Published: January 2023

LRCC8A is a ubiquitously expressed cell surface receptor necessary for lymphocyte development. The role of LRRC8A in peripheral T cell function is unknown. We now demonstrate that LRRC8A ligation enhances TCR-driven T cell proliferation and cytokine secretion. LRRC8A co-capped with the TCR/CD3 complex after anti-CD3 stimulation, and its ligation led to the phosphorylation of AKT through a Src kinase-Zap-70-PI3K pathway. A GST-LRRC8A fusion protein containing the extracellular leucine-rich region of LRRC8A bound to fibroblasts and keratinocytes, but not dendritic cells or B cells, indicating that non-professional APCs express a ligand for LRRC8A. The LRRC8A receptor-ligand interaction was essential for T cell proliferation to antigen presented by non-professional APCs, and for tissue inflammation induced by contact hypersensitivity to hapten. LRRC8A functions as a co-stimulatory receptor for T cell activation by non-professional APCs. Blocking LRRC8A signaling may be useful in the treatment of T cell-driven inflammatory diseases.
Role of SNX17 in receptor recycling and antigen recognition by T cells. (IRC2P.445)
Authors: Osborne Douglas , Billadeau Daniel
Published: January 2023

A key component in T cell activation is the endosomal recycling of receptors to the cell surface allowing for continual integration of signaling and antigen recognition. Multiple early endosomal associated proteins have been found to interact with T cell surface receptors and play a role in the recycling and transport of these receptors to the cell surface. One endosomal protein possibly involved in T cell receptor transport is sorting nexin 17 (SNX17). SNX17 has been found to bind with receptors involved in T cell activation, but its role in receptor recycling and T cell activation is unknown. Using lentivirus shRNA against SNX17, we have found that the knockdown of the SNX17 protein resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared to control T cells. Using immunofluorescence, we have found that SNX17 colocalizes with TCR and localizes to the peripheral-SMAC at the immune synapse in T-APC conjugates. This data suggest that SNX17 plays a synergistic role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse.
Cell-intrinsic TGF-β receptor signaling limits virus-specific CD4 T cell responses and prevents lethal pathology during chronic viral infection. (IRC2P.450)
Authors: Lewis Gavin , Maloveste Sebastian , Streeck Hendrik , Zuniga Elina
Published: January 2023

Persistent viruses create a profound immunosuppressive environment that is conserved in mice and humans. While several pathways suppressing CD8 T cell responses have been identified, much less is known about the immune regulatory molecules restricting the number and helper function of CD4 T cells. By using Lymphocytic Choriomeningitis Virus (LCMV) infection in mice, we observed enhanced signaling of the immunosuppressive cytokine Transforming Growth Factor-β (TGF-β) in virus-specific T cells during chronic infection. Importantly, genetic ablation of TGF-β receptor (R) resulted in rapid mortality upon chronic, but not acute, LCMV infection. While TGF-βR deficient mice showed comparable CD8 T cell responses, CD4 T cell proliferation, terminal differentiation and a cytotoxic program characterized by Granzyme B, Perforin and EOMES expression were significantly enhanced in the absence of TGF-βR signaling. Importantly, EOMES overexpression recapitulated the phenotype of TGF-βR deficient CD4 T cells while EOMES inhibition suppressed it. Finally, preliminary experiments showed TGF-β signaling also suppressed cytotoxic molecules and EOMES in human CD4 T cells. Together these data uncover a novel TGF-βR-dependent axis that restricts EOMES-driven differentiation and cytotoxicity in murine and human CD4 T cells while highlighting a critical role for TGF-βR in limiting anti-viral CD4 T cell responses and mortality upon chronic viral infection.
A new pathway for central B cell tolerance: interaction of AID, MyD88, and BCR (IRC3P.467)
Authors: Kuraoka Masayuki , Snowden Pilar , Kelsoe Garnett
Published: January 2023

Expression of activation-induced cytidine deaminase (AID) in immature and transitional B cells in mice and humans is genetically linked to the first tolerance checkpoint. In the absence of AID, autoreactive immature/transitional-1 (T1) B cells are inefficiently purged and exhibit increased resistant to receptor-induced apoptosis. These substantial effects are surprising given that AID expression in the immature/T1 B cells is only 3% of that in germinal center B cells. We now show that in immature/T1 B cells, but not mature B cells, B-cell antigen receptor and Toll-like receptor (TLR) signaling synergize to elicit high levels of AID expression. This synergy is restricted to intracellular TLR ligands, requires the phospholipase D-dependent compartmentalization of intracellular TLR and activation of the TLR through acidification, and acts to ensure self-tolerance through a process that requires Myd88. In a 3H9 mouse model, we show that suppression of intracellular acidification by administration of chloroquine increased the generation of immature/T1 B cells in proportion to small pre-B cells, suggesting that chloroquine treatment support the survival of autoreactive 3H9 immature/T1 B cells in bone marrow. These findings identify a novel mechanism for central B-cell tolerance and resolve several weaknesses of current models for the first tolerance checkpoint. We suggest that this AID mediated pathway may be the primary mechanism for central B-cell tolerance by deletion.
A temporal switch in the germinal center determines differential output of memory B and plasma cells (IRC3P.468)
Authors: Weisel Florian , Shlomchik Mark
Published: January 2023

Though memory B cells (MBCs) and long-lived plasma cells (LLPCs) are both thought to derive from the germinal center (GC) reaction, there is little insight into or agreement about the signals that control differentiation to one cell type or another. By performing BrdU pulse-labeling studies, GC disruption experiments and V gene sequencing, we found that the generation of these cell types is actually temporally controlled and separated during the immune response. We report that MBCs mainly derive from early GCs (much before GC peak size), while more affinity matured LLPCs are predominantly formed during late GCs - long after its peak in size. Based on these findings, we propose a new model that the GC response undergoes a temporal switch, functioning quite differently at early and late stages. Therefore the generation of MBCs and LLPCs is the consequence of a general shift in GC output over time rather than the result of specific instructive signals that are selectively delivered to GC B cells at any given time during the response. We also present direct evidence that a large fraction of long-lived IgM+ MBC, and even some IgG+ MBC, are formed at very early time points, prior to the existence of detectable GCs. The knowledge of when specific long-lived immune-effector cells are generated during an immune response has strong implications for vaccine design and understanding long-term pathogen immunity.
A novel aging-associate B-cell subset controls antitumor CD8+ T-cell response (IRC3P.469)
Authors: Lee Chang Catalina , Bodogai Monica , Moritoh Kanako , Croft Michael , Gress Ronald , Ferrucci Luigi , Hakim Fran , Biragyn Arya
Published: January 2023

CD8+CD28- T cells are considered an immune risk of mortality and morbidity in elderly. The mechanism of their enrichment and their function in elderly is poorly understood. In the present study we report a novel and evolutionary-conserved B-cell subset expressing 4-1BBL, which accumulates with aging of mammals, such as humans, rhesus macaques and mice. These cells (termed 4BL cells) induce antitumor effector CD8+ T cells by presenting endogenous antigens and utilizing 4-1BBL/4-1BB axis. 4BL cells are also responsible for the expansion of auto-reactive CD8+ T cells in patients treated with high dose chemotherapy and autologous progenitor cell transplantation. Thus, accumulation of antitumor 4BL cells explains the paradox of retarded tumor growth in the elderly, and most likely autoimmune disease outcome in the aged population. However, 4BL cell accumulation and their effects on CD8+ T-cell compartment can be eliminated by inducing B-cell lymphopoiesis in old mice (after B-cell depletion), suggesting that the aging-associated skewed cellular immune responses are reversible. We propose that 4BL cells and their 4-1BBL signaling pathway are useful targets for improved effectiveness of natural antitumor defenses and therapeutic immune manipulations in the elderly. How 4BL are generated, and their impact in the B-cell effector functions homeostasis in elderly will be further discussed.
Disparate regulatory roles for B cells during primary and secondary CD4 T cell responses (IRC3P.470)
Authors: Harrington Laurie , Mollo Sarah , Zajac Allan
Published: January 2023

The generation of CD4 T cell memory is shaped by numerous factors, such as the strength and duration of TCR signaling, as well as the priming environment, all of which can be modified by B cells. Studies using B cell-deficient mice indicate B cells play a critical role in generating effector and memory CD4 T cells; however when and how B cells are acting to promote these responses has not yet been ascertained. Moreover, memory CD4 T cells are documented to have less stringent requirements for activation than their naïve counterparts, yet it remains unknown if B cells influence secondary CD4 T cell responses. In this study, we use anti-CD20 antibody depletion of B cells at different times following Listeria monocytogenes infection to show that B cells are necessary for the induction of optimal CD4 T cell memory, but not for the transition and maintenance of this population. Importantly, the prerequisite of B cells early after infection is partially dependent on their expression of MHC class II. B cells are not only required during the priming phase, but are also necessary for the initiation of robust secondary responses by memory CD4 T cells. Interestingly, the requirement during the recall response is independent of B cell antigen presentation. Overall, these studies demonstrate the temporally and functionally distinct roles for B cells in regulating CD4 T cell responses.