bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of melanocyte-specific autoantibodies in vitiligo-prone Smyth line chickens. (BA2P.128)
Authors: Falcon Daniel , Dienglewicz Robert , Mortazavi Amir , Erf Gisela
Published: January 2023

Autoimmune vitiligo is characterized by the loss of pigment-producing melanocytes (MC) in the skin. The autoimmune vitiligo-prone Smyth line (SL) of chicken together with the parental Brown line (BL) and vitiligo-resistant Light Brown Leghorn (LBL) chickens constitutes an excellent animal model for this disorder. Although cell-mediated immunity has long been known to contribute to the etiopathology of SL vitiligo (SLV) the role of humoral immunity is less clear. We recently completed a time-course study examining levels of IgG specific to MC lysate in SL and control plasma. Results showed elevated (P<0.05) MC-lysate specific IgG levels in SL chickens 1-2 weeks prior to and throughout SLV development. To evaluate the binding characteristics of MC-specific IgG, primary MC isolated from growing feathers were incubated with IgG purified from the serum of SLV (birds with SLV) and control chickens. IgG binding was evaluated by indirect immunofluorescent staining and flow cytometry. Regardless of the source of MC (SL, BL, LBL), significant binding was only observed with SLV-IgG (mean fluorescence intensity increased approximately 100%). This effect was not observed when MC were incubated with IgG purified from egg yolk of SLV hens. Further characterization of autoantibodies from SLV chickens may provide insight into their role in the loss of MC in SLV. Studies evaluating such mechanisms as antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity are underway.
Deleted in breast cancer 1 (DBC1) is a molecular checkpoint of B cell autoimmunity (BA2P.130)
Authors: Fang Deyu
Published: January 2023

Self-reactive B cells in the periphery are maintained in a tolerized state through low levels of sCD40L and BAFF (B-cell Activating Factor) signaling. Loss of tolerance due to hyperproduction of CD40L or BAFF, or uncontrolled downstream signaling following activation is associated with multiple autoimmune diseases. In this study we identify Deleted in Breast Cancer 1 (DBC1) as a novel suppressor of B cell activation and autoantibody production. Mice with targeted deletion of Dbc1 gene have hyper-responsive B cells, which produce immunoglobulin, in particular IgG and IgA isotypes, against self-antigens when immunized with antigen without adjuvant. At the molecular level, DBC1 suppresses B cell function by regulating alternative NFΚB members RelB and p52 during the early phase of CD40 and BAFFR stimulation. In addition, IKKα regulates DBC1 interaction with RelB:p52 during B cell activation by phosphorylating a serine cluster at the C-terminus of DBC1. Finally, loss of DBC1 results in spontaneous autoantibody in aged mice. This study reveals that DBC1 functions as a critical regulator in the B cell immune tolerance checkpoint by suppressing the alternative NFκB pathway.
B cell autophagy mediates TLR7-dependent SLE and controls extramedullary hematopoiesis (BA4P.217)
Authors: Weindel Chi , Richey Lauren , Bolland Silvia , Huber Brigitte
Published: January 2023

Systemic Lupus Erythematosus (SLE) is a heterogeneous disease, defined by a loss of B cell self-tolerance resulting in the production of autoantibodies. One of the many mechanisms described resulting in a break in B cell self-tolerance is overexpression of the endosomal toll-like receptor, TLR7. We hypothesized that autophagy plays a crucial role in TLR7 activation in B cells by delivering ligand to endosomes. To test this model, we produced TLR7 transgenic (tg) mice with a B cell-specific (CD19-cre) ablation of autophagy (ATG5 ko), and compared SLE development to that of TLR7 tg mice with intact autophagy machinery. We observed that loss of ATG5 in B cells significantly increased the survival of TLR7 tg mice, reduced serum IgG and anti-nuclear autoantibodies, thereby preventing glomerulonephritis. These data suggest that B cell autophagy is required for TLR7-mediated SLE disease progression. However, while symptoms of SLE were ameliorated, extramedullary hematopoiesis (EMH) was dramatically increased with a splenic red pulp expansion in these mice. Thus, autophagy in B cells plays an important role in controlling lineage determination in hematopoiesis, likely by regulating chemokine and cytokine production, the mechanism by which is currently under investigation. A better understanding of the control steps involved in TLR7 activation and cytokine production could lead to improved therapeutics for treatment of SLE.
Endogenous IL-17A promotes pristane-induced systemic autoimmunity and experimental lupus nephritis (BA4P.218)
Authors: Kitching A Richard , Odobasic Dragana , Khouri Mary , Steinmetz Oliver , Yang Yuan , Holdsworth Stephen
Published: January 2023

IL-17A is increased in serum and in kidney biopsies from patients with lupus nephritis, but direct evidence of pathogenicity is less well established. These studies sought to define the role of IL-17A in experimental lupus induced by the administration of pristane to WT and IL-17A-/- mice. Local and systemic immune responses were assessed after 6 days and 8 weeks, and autoimmunity, glomerular inflammation and renal injury were determined at 7 months. Peritoneal IL-17A production increased significantly 6 days after pristane injection, with innate cells (neutrophils and macrophages) being the predominant source of IL-17A. After 8 weeks, while systemic IL-17A was still readily detected in WT mice, IFN-γ and TNF were diminished in IL-17A-/- mice. Seven months after pristane, humoral autoimmunity was diminished in IL-17A-/- mice, with decreased serum lgG and anti-dsDNA antibody titers. Renal inflammation and injury was less in the absence of IL-17A. Compared to WT mice, glomerular IgG, complement deposition, glomerular CD4+ T cells and intrarenal pro-inflammatory mediator expression were decreased in IL-17A-/- mice. WT mice developed progressive albuminuria, but albuminuria (7 months: WT 271±56, IL-17A 88±16μg/day; P<0.05) and histological abnormalities were attenuated in IL-17A-/- mice. Therefore, IL-17A is required for the full development of systemic autoimmunity and experimental lupus nephritis. Early in the development of autoimmunity, innate immune cells produce IL-17A.
Migration of i.c. injected BMDCs from brain to deep cervical lymph nodes is CCR7 dependent but does not promote EAE disease progression (CCR1P.244)
Authors: Clarkson Ben , Rayasam Aditya , Walker Alec , Harris Melissa , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Cell-mediated antigen (Ag) drainage from the central nervous system (CNS) is incompletely understood. In other organs, Ag-presenting cells—such as dendritic cells (DCs)—migrate from organ tissue through lymphatics to draining lymph nodes (dLNs); however, classical lymphatic vessels are not found in the CNS and few migratory tissue DCs populate neuropil. In the context of neuroinflammation associated with experimental autoimmune encephalomyelitis (EAE), intracerebral (i.c.) injection of myelin peptide-loaded bone marrow (BM)DCs exacerbates disease, suggesting that increasing the number of CNS-DCs promotes EAE progression. However, whether CNS-infiltrating DCs transport Ag to dLNs for T cell priming or restimulate myelin-specific effector T cells in situ within developing CNS inflammatory lesions is unknown. Since migration of i.c. injected BMDCs to dLNs is pertussis toxin sensitive—and thus likely regulated by G proteins/ chemokine receptors—we sought to identify key chemokine receptors that govern DC migration from CNS to dLN. Here we report that 1) CCR7+/+ but not CCR7-/- BMDCs appear in dLNs after i.c. injection, 2) CCR7-/- DCs accumulate in CNS but not dLNs during EAE compared to CCR7+/+ DCs, 3) CCR7 is not necessary for i.c. injected BMDCs to exacerbate EAE, and 4) ablation of CCR7+/+ DCs during onset of EAE leads to increased CNS T cell cytokine production. These data suggest CCR7-dependent DC migration from CNS to dLN might regulate T cell responses in early EAE.
Histone acetyl-lysine reader Brwd1 regulates Igκ accessibility and recombination (HEM2P.260)
Authors: Clark Marcus , Hamel Keith , Maienshcein-Cline Mark , Mandal Malay
Published: January 2023

The hallmark of B lymphopoiesis is sequential rearrangement of immunoglobulin heavy (Igμ) and the light chain (Igκ and then Igl). We recently demonstrated that downstream of IL-7R signaling, STAT5 represses Igκ transcription and recombination by recruiting Ezh2 and marking Igκ with H3K27me3 (Nat Immunol 12:1139). In addition to Igκ, there was a restricted program of genes similarly repressed by STAT5. Among these, Brwd1 which encodes a BROMO domain containing histone acetyl-lysine reader molecule, demonstrated a similar expression pattern to Igκ. Brwd1-/- mice had defective lymphopoiesis starting at the pre-B cell stage. Strikingly, there was a 10-fold reduction in Igκ recombination in Brwd1-/- small-pre B cells and this was associated with severely reduced H3K16 acetylation. Genome wide analyses of WT pre-B cells indicated that Brwd1 bound at the highest density to Igκ and other loci marked with both H3K9 acetylation and H3S10pK14 acetylation. Phosphorylation of H3S10 was dependent on pre-BCR mediated Erk activation. Interestingly, Brwd1 binding sites were highly enriched in extended GAGA motifs that, in Drosophila, are associated with Trl recruitment and enhanced chromosomal accessibility. These findings identify the epigenetic reader Brwd1 as a critical mediator of Igκ accessibility whose expression and recruitment to the Igκ locus are coordinately controlled by the IL-7R and pre-BCR.
Skewed homeostasis of natural killer cells in aging decreases their capacity to eliminate target cells (HEM3P.290)
Authors: Shehata Hesham , Lampe Kristin , Hoebe Kasper , Chougnet Claire
Published: January 2023

Natural killer cells (NKCs) are critical in eliminating tumors and viral infections, both of which occur at a high incidence in the elderly. Previous studies showed that aged NKCs are less cytotoxic in vitro and are less mature than young NKCs. However, the mechanisms by which aging influences NKC homeostasis and function remain poorly understood. We found that aged mice had a significant decrease in frequency of mature and educated NKCs in all lymphoid organs and aged NKCs were less efficient at eliminating allogeneic and B16 melanoma targets in vivo. This phenomenon could be explained by the impaired degranulation, particularly by mature NKCs of aged mice. Administration of exogenous IL-15 or depleting Tregs or CD8+ T cells, which may compete for IL-15, did not augment aged NKC maturation. However, T-bet and Eomes which regulate NKC maturation, were significantly decreased in bone marrow (BM) NKCs of aged mice, indicating that there may be a block in progressive differentiation of immature to mature NKCs in aging. Consistent with reports supporting T-bet mediated Granzyme B (GZB) expression, the proportion of GZB+ NKCs was significantly reduced in aged mice. The homeostatic proliferation of BM aged NKCs was also greatly diminished compared to young NKCs. Overall, our results suggest that aged BM NKCs may not respond well to maturation signals and that this impaired maturation is linked to their defective cytotoxicity.
NLRP3 deficiency reduces renal atrophy in renovascular hypertension (HUM1P.306)
Authors: Hartono Stella , Knudsen Bruce , Grande Joseph
Published: January 2023

Renovascular hypertension (RVH) affects up to 7% of individuals over 65 years of age. We have previously demonstrated that induction of oxidative stress is an early event in the development of RVH that leads to development of inflammation and renal atrophy. NLRP3 is one of the most well-known sensors of oxidative stress. However, the role of NLRP3 in the development of RVH and subsequent end-organ damage is not well understood. We therefore sought to test the hypothesis that inhibition of NLRP3 activation protects the heart and the kidneys of mice subjected to RVH. In accordance with our previous observations, wild type (WT) mice subjected to RVH developed 26±6% of renal atrophy within 1 week following cuff placement that progressed to almost 100% by 4 weeks. In contrast, although NLRP3-/- mice (KO) subjected to RVH developed similar degree of atrophy to their age-matched WT in the first week (29 ± 17 vs. 39 ± 20), the atrophy did not progress when evaluated at 4 weeks (29 ± 13 vs. 78 ± 10, p=0.013). Histologic evaluation of renal cortex of KO mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. In addition, KO mice developed no cardiac hypertrophy, no cardiac fibrosis, or aortic lesion despite similar elevation in systolic blood pressure to the WT mice. We propose that NLRP3 activation plays a critical role in the development of renal atrophy in mice subjected to RVH and represent a potential therapeutic target.
Activation of mast cells by influenza A virus stimulates virus-induced immunopathology (HYP3P.353)
Authors: Graham Amy , Hilmer Kimberly , Zickovich Julianne , Obar Joshua
Published: January 2023

Influenza A virus (IAV), a seasonal respiratory pathogen, results in severe lung pathology. Recent global analysis of lungs from mice infected with highly pathological IAV strains demonstrated enrichment of mast cells, but the role of mast cells during severe pulmonary viral infections has been under studied. We have recently shown that A/WSN/33 causes significant immunopathology in C57BL/6 mice and is mast cell-dependent. A/WSN/33(WSN) was able to directly activate mast cells to produce histamine, leukotrienes, inflammatory cytokines, and anti-viral chemokines. IAV-induced cytokine storm is thought to induce viral immunopathology, therefore we explored the importance of this response from mast cells. Mast cell cytokine production occurred in a RIG-I/MAVS-dependent fashion, but reconstitution of mast cell-deficient mice with RIG-I-/- mast cells generated lung pathology similar to wild type mast cells. Thus, it appears that mast cell degranulation rather than production of cytokines causes WSN-induced lung pathology. Mast cell degranulation occurs early in the viral entry cycle through a RIG-I/MAVS- and MyD88/Trif-independent mechanism. Using recombinant WSN strains, we found an association between the WSN hemaggultinin and neuraminidase proteins in the activation of mast cell degranulation and WSN-induced disease. We have identified a unique inflammatory cascade which could be therapeutically targeted to limit morbidity following infection with influenza virus.
Modulation of IgE mediated reactions by FcgammaRII (HYP3P.406)
Authors: Röthlisberger Silvan , Fellmann Marc , Buschor Patrick , Zellweger Fabian , Stadler Beda , Vogel Monique
Published: January 2023

Cross-linking two high affinity IgE Receptors (FcεRI) by incubation with an anti-IgE antibody binding to the receptor bound IgE’s leads to a dose dependent bell-shaped activation of basophils. Besides of the high affinity IgE receptor basophils also express two isotypes of the low affinity IgG Receptor CD32, the activatory CD32a and the inhibitory CD32b. Previous work has shown that cross-linking of FcεRI and the inhibitory CD32b decreases cell activation, hinting at the Fc part of IgG antibodies taking part in basophil modulation. To characterize the interaction between FcεRI and CD32 we used two anaphylactogenic monoclonal anti-IgE antibodies. First we produced F(ab’)2 fragments which showed an increased maximal activation of basophils compared to the full length antibodies. Blocking the binding of IgG-Fc to CD32 with an anti-CD32 molecule results as well in a higher activation, indicating an effect of IgG-Fc. Then we humanized Le27 to increase the affinity to CD32. Humanized Le27 activated basophils to the same extend as the original Le27. To investigate whether the cross-linking of FcεRI with CD32 solely has an effect on basophil activation we produced a monovalent Le27 that can cross-link FcεRI and CD32, but not two FcεRI’s. Surprisingly, this monovalent antibody did activate basophils in whole blood, but not isolated basophils. Experiments will be performed with the monovalent Le27 to investigate if it can inhibit allergen-induced basophil activation.
Molecular mechanisms responsible for low-grade cellular stress induced by super low dose lipopolysaccharide (INM2P.421)
Authors: Baker Bianca , Maitra Urmila , Li Liwu
Published: January 2023

Underlying many chronic inflammatory diseases is the persistence of low-grade inflammation. Low doses of lipopolysaccharide are capable of skewing the immune system towards a pro-inflammatory state without inducing anti-inflammatory compensation. While the cumulative effects of this low-grade endotoxemia can be detrimental, the mechanism is poorly understood. Dysregulation of mitochondrial dynamics has been linked to a variety of diseases and disorders including Parkinson’s and Alzheimer’s. We hypothesized that low-dose LPS induces mitochondrial dysregulation by altering mitochondrial dynamics through an IL-1R-associated kinase-1 and Toll-interacting protein dependent mechanism. To test this hypothesis, we utilized western blotting, immunoprecipation, and confocal microscopy techniques in wild-type and knockout bone marrow derived macrophages. In this study, we demonstrate that subclinical low-dose LPS induces mitochondrial fission while suppressing mitochondrial fusion through IRAK -1 and Tollip. Low-dose LPS causes a decrease in Drp1 phosphorylation while increasing total Drp1 protein levels thus, promoting mitochondrial fission in the wild-type cells. In addition, low-dose LPS inhibits Mfn1 levels further inducing mitochondrial fission. Our data reveals a novel role for low-dose LPS in mitochondrial dynamics, which may contribute to skewed and persistent low-grade inflammation and disease progression.
Interleukin 22 receptor abundance and signaling is regulated by the ubiquitin proteasome and glycogen synthase kinase 3β. (INM2P.422)
Authors: Weathington Nathaniel , Snavely Courtney , Zhao Jing , Chen Bill , Zhao Yutong , Mallampalli Rama
Published: January 2023

Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection at mucosal and other epithelial surfaces. Molecular regulation of IL-22 receptor (IL-22R) protein has not been characterized. In mouse lung epithelia, IL-22R is expressed as a relatively short-lived protein and is degraded by the ubiquitin proteasome. Its degradation is accelerated when cells are stimulated by IL-22. Lys 449 within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site for proteasomal degradation as deletion or mutation of K449 creates a degradation resistant IL-22R mutant protein that is not ubiquitinated in cells. IL-22 treatment of cells triggers glycogen synthase kinase (GSK)-3β inactivation, and GSK-3β phosphorylates the IL-22R within a consensus phosphorylation signature at Ser 410. GSK-3β overexpression results in accumulation of IL-22R protein while GSK-3β depletion in cells reduces levels of the receptor, and this effect is abolished on mutation of the 410-414 phosphorylation domain. Signal transduction of IL-22 appears increased when GSK-3β is overexpressed. This study shows GSK-3β as a modulator of IL-22R protein stability, which may be important for host defense and could impact therapeutic targeting of the IL-22 signaling axis.
Caspase-8, RIPK1 and FADD regulate cell death and caspase-1 activation during yersinia infection (INM3P.438)
Authors: Philip Naomi , Dillon Christopher , Snyder Annelise , Fitzgerald Patrick , Wynosky-Dolfi Meghan , Zwack Erin , Hu Baofeng , FitzGerald Louise , Mauldin Elizabeth , Copenhaver Alan , Shin Sunny , Oberst Andrew , Green Douglas , Brodsky Igor
Published: January 2023

Programmed cell death is an evolutionarily conserved response to infection that can promote host defense or microbial virulence. Pathogens manipulate various immune signaling pathways through the activity of specific virulence factors that access the host cell cytosol. Cell death is a major consequence of infection with pathogenic Y. pseudotuberculosis and requires the effector YopJ, a potent inhibitor of NF-κB and MAPK signaling. However, the pathways that regulate cell death in response to Yersinia infection and the precise mechanism by which cell death mediates protective immunity are not well understood. We find a novel requirement for caspase-8, receptor interacting protein 1 (RIPK1) and Fas-associated protein with death domain (FADD) in Yersinia-induced cell death and pro-inflammatory caspase-1 activation. Moreover, mice deficient in caspase-8 were highly susceptible to Yersinia infection and were unable to sustain innate cytokine production. We hypothesize that activation of these pathways during Yersinia infection may induce specific pro-inflammatory signals that shape innate and adaptive responses and promote microbial clearance.
A novel mechanism for IL-6 to regulate CD4 cell effector function through mitochondrial respiratory chain. (IRC2P.457)
Authors: Yang Rui , Lirussi Dario , Rincon Mercedes
Published: January 2023

Mitochondria are the essential engine of the cell to produce energy as ATP through oxidative phosphorylation (OXPHOS). Although less known, mitochondria are also a key regulator of Ca2+ homeostasis both as a major Ca2+ storage as well as a modulator of ER Ca2+. The role of mitochondria metabolism in T cells is a rapidly growing field. It has important impact on activation, differentiation and effector function of CD4 and CD8 cells. STAT3 is a transcription factor that regulates the expression of a number of genes, but it has also been found in mitochondria where it regulates ETC. Since interleukin-6 is one of the major activators of STAT3, we have investigated whether interleukin-6 could regulate mitochondria function. Our studies show that interleukin-6 is essential to maintain mitochondrial hyperpolarization during CD4 T cell activation in a Stat3-dependent manner. Hyperpolarization of mitochondria by interleukin-6 does not affect the production of ATP in CD4 T cells. However, maintaining mitochondrial hyperpolarization by interleukin-6 during activation of CD4 T cells results in increased mitochondrial Ca2+ levels. Consequently, it helps to sustain higher levels of cytosolic calcium. Increased levels of cytosolic Ca2+ facilitate the production of specific cytokines that are regulated by interleukin-6 Thus, we have identified a novel mechanism by which interleukin-6 can play a key role in the function of effector CD4 T cells through its effect on mitochondria.
Lipid raft-based membrane order is important for antigen-specific clonal expansion of CD4+ T cells (IRC2P.451)
Authors: Bamezai Anil , Schieffer Daniel , Naware Sanya
Published: January 2023

Lipid rafts contribute to the plasma membrane order and to its spatial asymmetry. Lipid rafts are cholesterol and saturated lipid-rich, nanometer sized membrane domains that are hypothesized to play an important role in spatiotemporal regulation of cell signaling. The raft nanodomains on the surface of CD4+ T lymphocytes coalesce during their interaction with antigen presenting cells (APCs). Sensing of foreign antigen by the antigen receptor on CD4+ T cells occurs during these cell-cell interactions. In response to foreign antigen the CD4+ T cells proliferate, allowing the expansion of few antigen-specific primary CD4+ T cell clones. Proliferating CD4+ T cells undergo differentiation into appropriate effectors tailored to mount an effective immunity to the pathogen. To investigate the role of lipid raft-based membrane order in the clonal expansion phase of primary CD4+ T cells, we have disrupted membrane order by incorporating an oxysterol, 7-ketocholesterol (7-KC) into the plasma membrane of primary CD4+ T cells expressing a T cell receptor specific to chicken ovalbumin323-339 peptide sequence and tested their antigen-specific response. We report that 7-KC, at appropriate concentrations, significantly diminishes the c-Ovalbumin323-339 peptide-specific clonal expansion of primary CD4+ T cells. Our findings suggest that lipid raft-based membrane order and membrane asymmetry is important for clonal expansion of CD4+ T cells in response to a model antigen.
α-galactosylceramide may lead to an enhanced affinity immune response (IRC3P.459)
Authors: Hawksworth David
Published: January 2023

α-galactosylceramide (αGalCer) has been shown to stimulate B cell proliferation in mice, leading to enhanced antibody titers. We sought to discover whether, in addition to antibody titer, we could develop a higher affinity immune response in mice administered αGalCer in the context of our standard, adjuvant based immunization methodologies. Mice were immunized three times, every 4-6 weeks, with various antigens emulsified in Freund’s adjuvant. Test animals were administered αGalCer at the time of primary immunization, in addition to the antigen. Relative affinity was determined by testing serum antibody for reactivity to limiting concentrations of solution phase antigen. Results show that, for some antigen systems, administration of αGalCer does result in a trend toward a higher relative affinity vs. control animals. Pooled data from 3 antigen systems shows an average relative affinity of 2.105 nM for αGalCer mice vs. 3.134 nM for control mice. It was also observed, for one antigen system, that only those animals administered αGalCer developed a significant immune response. Our results indicate that administration of αGalCer at the time of primary immunization may lead to a slightly higher average affinity response in mice and can also be the difference between the development of a significant immune response vs. a very weak response.
B cells potently amplify T cell IL-4 responses following non-BCR mediated cysteine protease uptake (IRC3P.463)
Authors: Dwyer Daniel , Woodruff Matthew , Carroll Michael , Austen K. , Gurish Michael
Published: January 2023

Many common allergens such as house dust mite antigen and grass pollen are complex mixes of proteins and other compounds, both of which may have innate signaling capacity. Understanding the role of each of these signals is essential to determining what separates allergens from other innocuous proteins. Papain is a cysteine protease structurally similar to proteases found in many common allergens, including grass pollen and house dust mite antigen. Papain induces a potent IL4 response directed by T cells in the popliteal lymph nodes (PLN) of mice following footpad immunization directed against the protease, but the mechanisms behind this response and all of the relevant antigen presenting cells driving this polarization have not yet been fully elucidated. We have identified a novel capacity for B cells to rapidly bind and internalize papain to endosomes in an innate manner not mediated by the specificity of the B cell receptor. B cells subsequently regulate the adaptive immune response by enhancing CD4+ T cells Inducible T cell Costimulator (ICOS) expression on CD4+ T cells and amplifying both Th2 cell and T follicular helper cell induction. Antibody blockade of ICOS ligand, expressed by PLN B cells but not DC at the peak of the immune response, inhibits this IL-4 induction in WT but not B cell-deficient μMT mice. Thus, B cells play a critical role in amplifying adjuvant-dependent Th2 polarization following non-canonical acquisition and internalization of cysteine protease.
Cellular mechanism for immunogenicity of PEGylated “stealth” liposomes (IRC3P.466)
Authors: Watanabe Akiko , Cain Derek , Hanson Melissa , Kim Mikyung , Reinherz Ellis , Irvine Darrell , Kelsoe Garnett
Published: January 2023

Liposomes have been used as a vehicle to deliver pharmaceutical agents and as a carrier of vaccine antigens for malaria, cancer and HIV. The potency of a liposome-based vaccination is affected by the lipid formulation and inclusion of adjuvants, in addition to the immunogenicity of target antigens. While liposomal vaccines elicit robust humoral responses against target antigens, recent studies have shown that lipid components, such as monophosphoryl lipid A (MPLA), or liposome adducts, such as polyethylene glycol (PEG), are also capable of being targets of induced antibody. To understand the cellular interactions and molecular pathways responsible for the anti-liposome responses, we immunized mice with PEGylated “stealth” liposomes containing lipid-arrayed membrane-proximal external region (MPER) of HIV-1, and followed the kinetics of the humoral responses against liposome components as well as MPER. Immunized mice mounted robust IgM and IgG against PEG and, to a lesser extent, MPLA. PEG IgG was dependent on CD154, yet the requirement for T cells was less stringent. Moreover, PEG IgG was generated independent of MHCII, CD1d, and TCRδ, suggesting a novel form of T-cell help. Anti-MPER IgG, in contrast, was dependent on CD154, MHCII, and LAT. These results suggest that anti-liposome IgG responses are induced by atypical T-B cognate interaction, which is independent of CD4, NKT and γδ T cells and are distinct from MPER IgG responses, which require classical T-B collaboration.
TRIF signaling is essential for TLR4-driven IgE class switching (IRM5P.707)
Authors: Janssen Erin , Ozcan Esra , Liadaki Kyriaki , Jabara Haifa , Manis John , Ullas Sumana , Akira Shizuo , Fitzgerald Katherine , Golenbock Douglas , Geha Raif
Published: January 2023

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and TRAM, which recruits TRIF. Following stimulation with LPS+IL-4, Tram-/- and Trif-/- B cells completely failed to express Cε germ line transcripts (GLT) and secrete IgE. In contrast, Myd88-/- B cells had normal expression of Cγ1 GLT, but reduced IgE secretion in response to LPS+IL-4. Following LPS+IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram-/- and Trif-/- B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram-/-,Trif-/-, and Myd88-/- B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40+IL-4. Following stimulation with LPS+IL-4, Trif-/- B cells failed to sustain NFκB p65 nuclear translocation beyond 3 hours and had reduced binding of p65 to the Iε promoter. Addition of the NFκB inhibitor, JSH-23, to wild-type B cells 15 hours after LPS+IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion, but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NFκB driven by TRIF is essential for LPS+IL-4 driven activation of the Cε locus and class switching to IgE.
RGC-32 is essential for TGF-β mediated Th17 cell differentiation (LYM3P.728)
Authors: Rus Violeta , Tegla Cosmin , Nguyen Vinh , Cudrici Cornelia , Badea Tudor , Rus Horea
Published: January 2023

Our group has cloned and characterized the Response Gene to Complement (RGC)-32 which is induced by C5b-9 and exerts an important role in cell cycle activation via cyclin B1-CDC2 activation and Akt phosphorylation. RGC-32 is also a downstream target of TGF-β and plays a role in fibrogenesis. As Th17 and induced T regulatory cells (iTreg) differentiate under the control of TGF-β, we determined whether RGC-32 plays a role in this process using RGC-32 KO mice generated in our lab. RGC-32 was not required for peripheral T cell homeostasis as we observed normal cellularity, T cell subset composition and phenotypic markers. However, RGC-32 KO CD4+ T cells failed to differentiate normally to Th17 lineage as they exhibited a significant reduction in IL-17 and RORγt mRNA and protein expression in vitro and a decreased proportion of IL-17+ cells in lamina propria lymphocytes in vivo. The defect in IL-17 expression appeared to be mediated by decreased expression of IL-6R and TGF-βRI at baseline and under Th17 conditions and was not restored by IL-2 inhibition. A trend for increased iTreg differentiation was noted in RGC-32 KO CD4+ T cells under Treg skewing conditions but the difference was not statistically significant. Differentiation of CD4+ T cells into Th1 cells was decreased in RGC-32 KO mice while Th2 cells developed normally. These results suggest that RGC-32 is a novel mediator of Th17 differentiation in vitro and in vivo through modulation of IL-6R and TGF-βRI expression.
Type I IFN regulates non-canonical inflammasome activation and impairs protective immunity during fatal ehrlichia-induced toxic shock (MPF1P.762)
Authors: Yang Qin , Stevenson Heather , Scott Melanie , Ismail Nahed
Published: January 2023

Inflammasome complexes such as NLRP3 play a major role in antimicrobial defense to infection. Canonical and non-canonical pathways activate caspase 1 and caspase 11 to secrete IL-1β and induce inflammatory cell death. Ehrlichia, obligate intracellular bacteria that cause fatal toxic shock in humans and mice, activate inflammasomes and induce expression of type 1 interferon (IFN1). The contribution of the NLRP3 and IFN1 to Ehrlichia-induced toxic shock is not known. We show that NLRP3-/- and Caspase 1-/- mice succumb to lethal Ehrlichia infection similar to wild type (WT) mice. Unlike caspase 1-/- and WT, NLRP3-/- mice have lower bacterial burdens with higher IFNγ, suggesting that NLRP3 impairs bacterial clearance in a caspase 1-independent manner. Unexpectedly, infected IFNαR-/- mice were protected against a normally lethal dose of Ehrlichia with effective bacterial clearance, altered pathology with less apoptosis, increased IFNγ, decreased IL-10 and IL-10 producing Treg cells, and increased number of protective CD4+ Th1 and NKT cells. Lack of IFN1 signaling abrogated caspase 11 activation and IL-1β secretion, suggesting that IFN1 activates the non-canonical inflammasome. Our data also show that WT, but not IFNαR-/-, cells exhibit defective autophagy. Together, these data demonstrate that IFN 1 signaling suppresses autophagy and activates the non-canonical inflammasome pathway contributing to impaired protective immunity and development of fatal Ehrlichia-induced toxic shock.
Deleterious role for type I interferon during acute blood stage malaria infection (MPF1P.767)
Authors: Spaulding Emily , Lauvau Gregoire
Published: January 2023

Malaria is a severe and life-threatening disease that affects over 219 million people worldwide every year. In patients, a robust induction of the type I IFN pathway is observed, yet studies in mouse models have failed to determine whether type I IFN contributes to host protection or pathogenesis. We thus hypothesized that type I IFN is essential during blood stage malaria infection. Using Plasmodium yoelii (Py) YM which causes an acute lethal blood infection in C57BL/6 WT mice, we found that while WT mice rapidly succumbed, mice lacking the type I IFN receptor (IFNαβR-/-) mostly survived (>80%). Innate immune cells, particularly monocytes and NK cells, underwent early and robust activation and overwhelming production of inflammatory mediators in WT mice, whereas this was prevented in IFNαβR-/- mice. We are currently investigating precisely the cellular source of type I IFN and the mechanisms that regulate the activation of type I IFN-producing cells during this infection. Our results favor a model in which type I IFN promotes over-exuberant activation of innate monocytes and NK cells, leading to overwhelming inflammation and death of Py YM-infected mice. Overall, our work highlights a novel and unsuspected role for type I IFN in this significant human parasitic disease. We believe these results contribute to our understanding of the immunological cues leading to severe disease in malaria patients.
Iron acts as a second signal during coinfection with M. tuberculosis and N. brasiliensis leading to exacerbated M. tuberculosis disease outcome (MPF1P.769)
Authors: Dietzold Jillian , Gopalakrishnan Archana , Konowich Jill , Salgame Padmini
Published: January 2023

Previously, our laboratory has demonstrated that N. brasiliensis (Nb), an intestinal helminth, is able to exacerbate Mtb burden in coinfected Balb/c mice through generation of alternatively activated macrophages (AAMs). To examine the role of host genetic background in infection we performed coinfection experiments in C57Bl/6 mice. In contrast to Balb/c, coinfected C57Bl/6 mice were able to control Mtb burden comparable to mice infected with Mtb alone, despite mounting a Th2 response. This led us to hypothesize that a factor in addition to the generation of AAMs was responsible for the increased Mtb burden seen in the coinfection model. Concurrent experiments designed to understand Mtb survival within in vitro derived AAMs showed that IL-4 and IL-13 treatment was insufficient to cause differences in Mtb growth, compared to untreated macrophages. However the addition of iron significantly increased Mtb growth in AAMs. Iron, besides being important for Mtb growth; is also present in the lungs following tissue damage caused by Nb larval migration. To determine the role of iron during Nb infection we evaluated the expression of iron responsive genes in the murine lungs at 3, 5, and 7 days post infection. Balb/c and C57Bl/6 mice exhibited distinct profiles in response to Nb infection consistent with iron recycling and iron sequestration, respectively. Future studies aim to further understand the role of iron as important cofactor in regulating bacterial burden during coinfection.
IgG anti nocardia brasiliensis antibodies block IgM mediated protective effect in BALB/c mice. (MPF2P.814)
Authors: Salinas-Carmona Mario , Perez Rivera Luz , Vazquez Anna , Welsh Oliverio
Published: January 2023

Intracellular bacteria such as, Nocardia brasiliensis, produce a chronic human disease named actinomycetoma. Diagnosis of mycetoma is made on clinical findings as swelling and deformation of infected tissue and confirmed by culturing the offending microbe or by an ELISA test. Pathogenesis of mycetoma is little known and role of antibodies is controversial but it has been published that that cell mediated immunity is responsible of host protection in other intracellular bacterial infections. We used Nocardia brasiliensis to infect BALB/c mice and studied the role of both monoclonal and polyclonal antigen-specific IgM and IgG antibodies. We investigated antibodies role in host protection using passive immunization. Mice recipients of anti N. brasiliensis antibodies were then infected to assess actinomycetoma development. We found that IgM but not IgG anti N. brasiliensis antibodies from sera of infected animals and IgM, but not IgG anti N. brasiliensis monoclonal antibodies were able to confer protection and prevented actinomycetoma infection in experimental animals. IgG antibodies blocked the IgM protective effect in vivo. Implications of these finding in vaccine development are presented.
Phenotypic and functional analysis of the immune response to chronic T.cruzi infection in C3H/HePas mice (MPF2P.805)
Authors: Fonseca Raíssa , Borges da Silva Henrique , Salgado Rafael , do Nascimento Rogério , D'Império Lima Maria , Álvarez José
Published: January 2023

C3H/HePAS mice infected with T. cruzi Sylvio X10/4 develop a chronic cardiomyopathy similar to observed in humans, associated with presence of inflammatory infiltrates in the heart, suggesting that immune responses mounted during chronic infection play an important role in disease progression. However, these immune responses are not well understood. Thus, we analyzed the activation profile of blood and spleen immune cells at 390 days post infection, compared to age matched controls. We observed, at 390dpi, an increase in spleen and blood CD8+ T cell, and a decrease in CD11b+CD11c+ blood cell frequencies. We also observed increased PDL1 expression on both CD4+ and CD8+ spleen T cells, and of PDL1 on heart of chronic mice, as assessed by qRT-PCR, indicating that PD1/PDL1 inhibitory pathway may be activated. Chronic mice had also more CD4+IFNγ+ spleen cells, which had better proliferation ability when stimulated with T. cruzi antigen. There was increased CD69 expression in splenic CD4+ and blood CD8+ T cells. Importantly, there was a positive correlation between the expression of PDL1, PD1 and CD69 in CD4+ and CD8+ spleen T cells. In blood, correlations of PD1 and PDL1 expression were found on B, CD4+ and CD8+ T cells. Our results suggest that continued activation in chronic mice may induce higher levels of PD1/PDL1, which can be important to inhibit heart infiltrating cells, favoring parasite persistence. New experiments are needed to evaluate the importance of this pathway.
Intestinal epithelial cells contribute to Citrobacter rodentium induced infectious colitis through an NF-κB-dependent mechanism (MUC2P.826)
Authors: Mackos Amy , Galley Jeff , Parry Nicola , Boyaka Prosper , Bailey Michael
Published: January 2023

Exposing mice to a psychological stressor during challenge with the enteric pathogen Citrobacter rodentium (Cr) exacerbates Cr-induced infectious colitis. Enhanced recruitment of inflammatory monocytes to the colon, via a CCL2-dependent mechanism, is essential for the exacerbation of infectious colitis by stressor exposure, however the cell type responsible for the increase of colonic CCL2 is unknown. Previous evidence demonstrates that Cr-induced CCL2 is through an NF-κB mediated mechanism, thus this study was designed to test the hypothesis that NF-κB-derived gene expression from intestinal epithelial cells is necessary for the exacerbation of colitis via the recruitment of inflammatory monocytes, as well as the corollary hypothesis that inflammatory monocytes contribute to infectious colitis also via NF-κB activation. To test these hypotheses, two different strains of mice were challenged with Cr in which classical NF-κB activation is defective via the deletion of IKKβ in either intestinal epithelial cells (IKKβΔIEC) or myeloid-derived cells (IKKβΔMY). There was a reduction in Cr-induced colon mass, colonic histopathology, and colonic inflammatory mediator gene expression in IKKβΔIEC mice as compared to wild type controls. In addition, Cr-induced colon mass, histopathology, and colonic gene expression is further reduced in IKKβΔMY mice. Together, these data illustrate the deleterious effects of pathogen induced NF-κB activation has during enteric pathogen challenge.
TGFβ converts Th1 cell into Th17 cells through stimulation of Runx1 expression under inflammatory conditions in intestines (MUC2P.819)
Authors: Liu Houpu , Feng Ting , Li Qingjie , Zhang Wenbo , Yao Suxia , Elson Charles , Cong Yingzi
Published: January 2023

Although accumulating evidence demonstrates that differentiated CD4 cells preserve plasticity to alter phenotypes under various conditions, it is still unclear how stable Th1 cells are and whether Th1 cells can convert into Th17 cells. The high stability of Th1 is supported by some epigenetic studies and numerous reports of Treg and Th17 cells conversion into Th1 cells but not vice versa. However, recent reports of Th1 cell conversion to Treg, Th2 and Tfh cells argue the absolute stability of Th1 lineage. By using IFNγThy1.1 CBir1 TCR transgenic reporter mice, whose TCR is specific for an immunodominant microbiota antigen, we investigate the stability of Th1 cells under intestinal inflammatory conditions. Transfer of purified CBir1 specific IFNγ+Th1 cells induces colitis in RAG-/- mice and IFNγ+Th1 cells convert into IL-17+ Th17 but not Foxp3+ Treg cells in the inflamed intestines. TGFβ, IL-6 and IL-2, but not hypoxia factors, differentially regulate Th1 to Th17 conversion. TGFβ induction of transcriptional factors, Runx1 and RORγt, is crucial for the conversion, in that silencing Runx1 by siRNA inhibits Th1 conversion into Th17 cells. Furthermore, using ChIP assay, we show that TGFβ enhances histone acetylation but inhibits trimethylation of Runx1 and RORγt binding sites on il-17 or rorc gene in Th1 cells. In conclusion, our data demonstrate that Th1 cells convert into Th17 cells under inflammatory conditions in intestines, which is mediated by TGFβ-induction of Runx1.
SLP76 N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal TCR signaling (TECH1P.848)
Authors: Ji Qinqin , Ding Yiyuan , Salomon Arthur
Published: January 2023

SLP76 is a cytosolic adaptor protein that plays an important role in TCR mediated T-cell signaling pathway. SLP76 consists of three functional domains: an amino-terminal acidic domain with three tyrosine phosphorylation sites, a central proline-rich domain, and a carboxy-terminal SH2 domain. The direct influence of each domain of SLP76 in T-cell signaling pathway is not well understood. In our work, phosphoproteomic methods in mass spectrometry were used to do a phosphoproteomic analysis of cells containing a mutant form of SLP76 where three tyrosine residues are replaced with phenylalanines. Thousands of unique phosphorylation sites upon a time course of T-cell receptor stimulation in these cells are identified. Mutation of the three N-terminal tyrosine residues of SLP76 phenocopied SLP76 deficient cells for the majority of tyrosine phosphorylation sites observed including feedback on proximal TCR signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr192 of Lck when comparing Y3F mutant cells to SLP76 deficient cells. In addition, the N-terminal tyrosine sites of SLP76 also perturbed phosphorylation of Tyr440 of Fyn, Tyr702 of PLCγ1, Tyr204, Tyr397 and Tyr69 of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine residues of SLP76 in T cell signaling and shed light on novel signaling networks that are uniquely regulated by SLP76 N terminal tyrosine residues.
Engineering oncogenic kinases to activate apoptotic machinery: a novel rewiring of Bcr-Abl and PKCβII (TECH1P.862)
Authors: Hoekstra David , Carlson Louise , Lee Kelvin
Published: January 2023

Oncogenic kinases are crucial for the development of several cancers and have become popular targets for small molecule based inhibitor therapy. However, despite the development of 2nd and 3rd generation inhibitors, resistance to this therapy remains a significant clinical obstacle. To overcome this resistance, we hypothesized that we could hijack oncogenic kinases to activate a rewired ‘pro-drug’, whose activation would lead to the death of the transformed cell. To test this, we selected Bcr-Abl as the oncogenic kinase, which is constitutively active and essential for chronic myeloid leukemia (CML) cell survival. For our pro-drug we selected protein kinase C, isoform βII (PKCβII) whose activation has been shown to increase apoptosis in CML cells. To test whether we could engineer PKCβII as a pro-drug to be activated by Bcr-Abl, we designed four engineered PKCβII constructs and found that were specifically and significantly activated by Bcr-Abl, with no effect on intrinsic Bcr-Abl signaling observed. Activation of the PKCβII pro-drug resulted in a significant increase in apoptosis and a significant decrease in proliferation in Bcr-Abl+ leukemic cells with no effect on Bcr-Abl- cells. These findings demonstrate that Bcr-Abl mediated activation of our PKCβII pro-drug is feasible, induces death in leukemic cells, and provides a novel mechanism by which oncogenic kinases can be re-wired to activate pro-death agents to potentially improve patient outcomes.