bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell activation is attenuated in transgenic mice rich in omega-3 fatty acids (P6227)
Authors: Jang Hyun-Young , Song Mi-Young , Lee Sang-Myeong , Park Byung-Hyun
Published: January 2023

Cytokines such as interleukin (IL)-4, IL-5 and IL-13 mainly produced by CD4+ type 2 helper T lymphocytes (Th2 cells) have an important role in pathophysiology of allergic airway inflammation. Therefore, modulation of Th2 cells may offer a novel therapeutic approach to control allergic asthma. It has been suggested that n-3 PUFA can be used as preventive or therapeutic strategy to control allergic asthma. However, mechanisms by which n-3 PUFA modulates this disease are not fully understood. Here, the effects of elevated n-3 PUFA on OVA-induced airway inflammation were investigated using Fat-1 transgenic mice which can convert n-6 PUFA to n-3 PUFA endogenously. First, this study tested whether Fat-1 expression modulates CD4+ T cell activation, proliferation, and differentiation in vitro and found that Fat-1 expression attenuated all of these CD4+ T cell responses. When Fat-1 mice were sensitized and challenged with OVA, these mice showed significantly reduced inflammatory cell recruitment into airway, lower levels of Th2 cytokines, mucin production and eotaxin in brochoalveolar lavage (BAL) and lung tissues. OVA-specific IgE were also significantly lower in Fat-1 mice. Furthermore, suppressed CD4+ T cell activation and differentiation were also observed in Fat-1 mice. Taken together, these results demonstrate that elevated n-3 PUFA play a protective role in airway inflammation by modulating CD4+ T cell activation and differentiation in Fat-1 mice.
Is the α2M/LRP-1 axis integral to the allergenicity of proteases? (P6220)
Authors: Downey Jordan , McAlees Jaclyn , Flick Leah , Herz Joachim , Wills-Karp Marsha , Karp Christopher
Published: January 2023

Allergic diseases are increasingly prevalent in Westernized environments. Recent data indicate that the molecular substrates of allergenicity are a function of direct interactions with pathways of innate immune activation that evolved to sense microbial infection. After lipid-binding proteins exemplified by Der p 2, proteases represent the next most common class of allergens. The relevant mechanisms by which proteases lead to innate immune activation remain under-defined. α2-macroglobulin (α2M) is a plasma protein that inhibits extracellular proteases, targeting them for internalization by LDL receptor-related protein 1 (LRP1). In antigen presenting cells (APC), LRP1 directs α2M-associated proteins for antigen presentation. We hypothesized that protease adjuvanticity and allergenicity result from interactions with α2M and LRP1. Thus, we intratracheally challenged mice with papain, an occupational allergen and cysteine protease. In C57Bl/6 mice, this induced eosinophil and lymphocyte recruitment to the lung and increases in total and papain-specific IgE. While α2M-deficient mice exhibited no defects in the generation of allergic responses to papain, deletion of LRP-1 on APCs resulted in increased allergic inflammatory responses after challenge. These data suggest a role for α2M/LRP-1 in the clearance of protease allergens and suppression of excessive immune activation.
Pulmonary surfactant protein A increases IL-4-induced rat alveolar macrophages’ alternative activation (P1314)
Authors: M. Minutti Carlos , García-Fojeda Belén , Casals Cristina
Published: January 2023

It is well established that IL-4 induces alternative activation of macrophages involving intracellular activation of STAT6. Furthermore, it has been suggested that this cytokine has a proliferative effect on macrophages. On the other hand, there is evidence that pulmonary surfactant protein A (SP-A) plays an important role in modulating inflammatory response of alveolar macrophages (aMϕ). However, little is known about the effect of SP-A on the alternative activation of aMϕ. The aim of this study was to determine the effect of SP-A on aMϕ stimulated with IL-4. To accomplish this, purified rat aMϕ were stimulated with IL-4, different doses of SP-A, and combinations thereof. We measured arginase activity, cell viability, and STAT6 phosphorylation. We found that SP-A increased both IL-4-induced arginase activity and STAT6 phosphorylation. Finally, we observed more viable aMϕ in the presence of IL-4 and SP-A. We conclude that SP-A has a synergistic effect with IL-4 to lead aMϕ to an alternative activation. Furthermore, SP-A together with IL-4 may have either the protective effect of preventing aMϕ death or may cause cellular proliferation. Ongoing studies will allow us to discover the mechanism by which SP-A modulates the effect of IL-4 in aMϕ and determine if SP-A together with IL-4 causes either aMϕ survival or proliferation.
Regulatory T cell dysfunction in lipocalin 2 knockout mice exacerbates serum-induced arthritis (P1326)
Authors: Shashidharamurthy Rangaiah , Bozeman Erica , Aitken Jesse , Selvaraj Periasamy , Vijay-Kumar Matam
Published: January 2023

Lipocalin 2 (Lcn2), a 25KDa innate immune protein, known to be dramatically up regulated in various inflammatory disorders including antibody-mediated arthritis, yet its biological role remains unclear. More recently our studies have suggested that the regulation might be necessary for the initiation and further resolution of inflammatory process. However, the exact mechanism involved in this process is still unknown. In this study, we report that Lcn2 is up regulated up to 5 fold during the course of serum-induced arthritis (SIA) in wild type (WT) mice. Likewise, Lcn2KO mice suffered a severe arthritic disease with elevated systemic proinflammatory cytokines as compared to their WT littermates. Immunological analysis revealed a considerable increase in granulocytic population (Gr1+, CD11b+, CD11c+), and a significant decrease (4.2 fold) in regulatory T cell (CD4+, CD25+ and FoxP3+) population in Lcn2KO arthritic mice as compared to their WT littermates. Accordingly, Lcn2KO mice exhibited a defect in T cell proliferation as compared to their WT counterparts, suggesting a novel role for Lcn2 in splenocyte proliferation and T-reg expansion. Collectively, our data suggests a crucial role of Lcn2 in resolution of arthritic inflammation via T-reg expansion, making it a promising target in designing better therapeutic strategies for the treatment of rheumatoid arthritis.
CS1 (CRACC, CD319, SLAMF7) inhibits production of proinflammatory cytokines by activated monocytes (P1335)
Authors: Mathew Stephen , Kim Jongrok , Mathew Porunelloor
Published: January 2023

CS1 (CRACC, CD319, SLAMF7) is a member of the Signaling Lymphocyte Activation Molecule family expressed on immune cells mediating host defense. CS1 is a self-ligand and has both activating and inhibitory functions in Natural Killer (NK) cells. However, induction and function of CS1 in human monocytes are currently unknown. The purpose of this study was to evaluate the control of CS1 surface expression in activated monocytes and to assess the effect of CS1 triggering on proinflammatory cytokine production to better understand the role of CS1 in the control of monocyte function. Our study revealed that adherent or LPS-activated monocytes express CS1, and CS1 induction is via NF-κB and PI3K signaling pathways. Importantly, CS1 engagement resulted in reduced production of proinflammatory cytokines TNF-α and IL12p70. Our study implicates that CS1 plays an inhibitory role in human monocytes to control proinflammatory immune responses.
Hepcidin is cytotoxic to myeloma cells (P1337)
Authors: Conrad David , Power Coombs Melanie , Hilchie Ashley , Zemlak Tyler , Doucette Carolyn , Hoskin David , Liwski Robert
Published: January 2023

Recent studies suggest that some cationic antimicrobial peptides (CAPs) contribute to innate anticancer immunity. Hepcidin is a CAP and acute phase reactant well known for its antibacterial properties and its role in iron homeostasis. No anticancer function has yet been assigned to hepcidin, although elevated hepcidin levels are found in patients with hematologic malignancies, including plasma cell myeloma. We therefore investigated the anticancer properties of hepcidin against myeloma cells in vitro. A hepcidin isoform lacking the iron-regulatory domain was used. Hepcidin toxicity was assessed by MTT survival assays and DNA fragmentation tests. Propidium iodide (PI) staining was used to measure plasma membrane damage, and scanning electron microscopy (SEM) was performed to visualize cellular membrane changes. Hepcidin impaired the survival of mouse and human myeloma cells and induced DNA fragmentation, suggesting that cytotoxicity may be due to apoptosis. Hepcidin caused substantial PI uptake in the myeloma cells, and SEM confirmed that hepcidin treatment resulted in cellular membrane pore formation. Interestingly, hepcidin was more toxic to melphalan-resistant myeloma cells than to melphalan-sensitive controls. Our data implicate a role for hepcidin in innate immunity against plasma cell myeloma. Further studies on the anticancer properties of hepcidin are warranted in order to fully understand its physiological role in the context of neoplastic disease.
The functional role of PAI-2 in IL-1β production (P1345)
Authors: Hsu Li-Chung , Chuang Shih-Yi
Published: January 2023

Sepsis caused by uncontrolled inflammation is the leading cause of death in intensive care units today, but no effective therapeutic medication is available currently. During microbial infections, pathogen-associated molecular patterns (PAMPs) are recognized by the host innate immune system, which then mounts a defensive response. Myeloid cells play central roles in PAMP recognition. Recent studies indicate that a novel system termed the inflammasome carries out processing of cytoplasmic stored pro-IL-1β, and pro-IL-18. Both IL-1β and IL-18 are highly potent proinflammatory cytokines. Dyregulation of the inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Plasminogen activator inhibitor 2 (PAI-2), a member of a large group of proteins that inhibit serine proteases, is regulated by IKKβ-dependent activation of the transcription factor NF-κB. We previous demonstrated that mice with Ikkβ deletion in myeloid cells are hypersusceptible to endotoxin (LPS)-induced septic shock, and increased LPS susceptibility is associated with elevated plasma IL-1β. Reconstitution of Ikkβ-deficient macrophages with PAI-2 blocks apoptosis and IL-1β release. We also found that depletion of PAI-2 in macrophages resulted in IL-1β secretion after LPS stimulation. Our study unraveled a novel function of IKKβ/NF-κB in the negative regulation of IL-1β production via induction of PAI-2.
Properdin is increased after allergen challenge of asthmatic patients and contributes to OVA-induced airway inflammation and AHR at the effector phase by enhancing C3a generation in mice (P1355)
Authors: Wang Yuan , Ducka-Kokalari Blerina , Redai Imre , Miwa Takashi , Sato Sayaka , Gullipalli Damodar , Haczku Angela , Song Wen-Chao
Published: January 2023

The complement alternative pathway (AP) and anaphylatoxins have been implicated in allergic asthma, but the significance and mechanisms remain to be fully characterized. We hypothesized that factor properdin (fP), the only known positive regulator of AP contributes to allergen-induced airway inflammation. In human asthmatic patients, BALF collected after allergen challenge contained higher fP levels than before challenge. Mice were sensitized and challenged with OVA and examined for fP levels and airway inflammation. High levels of fP were detected in the BALF of OVA-sensitized and challenged but not naïve mice. Compared with WT mice, fP-deficient (fP-/-) mice had significantly reduced cell counts in BALF. fP-/- mice also had significantly attenuated AHR to methacholine stimulation. Antibody blocking of fP at both sensitization and challenge or at challenge phase alone, but not at sensitization phase alone, reduced airway inflammation. Conversely, intranasal reconstitution of fP to fP-/- mice at the challenge phase restored airway inflammation to WT levels. Notably, we found C3a levels in the BALF of OVA-challenged fP-/- mice to be significantly lower than in challenged WT mice, and intranasal co-administration of anti-C3a mAb with fP to fP-/- mice prevented restoration of airway inflammation. Collectively, our data suggest a detrimental role of fP in allergen-induced airway inflammation via C3a production and therapeutic targeting of fP may be beneficial in human asthma.
Mincle signaling contributes to the inflammatory response after traumatic brain injury (P1297)
Authors: de Rivero Vaccari Juan Pablo , Brand III Frank , Berti Aldo , de Rivero Vaccari Juan Carlos
Published: January 2023

The inflammatory response after traumatic brain injury (TBI) contributes to neuronal death and progressive axonal loss to areas undamaged by the initial trauma. Moreover, anti-inflammatory therapies offer great hope to improve outcomes after TBI. Thus, it is important to find therapeutic targets to better control the inflammatory response after injury. In this study we used cells in culture, an animal model of TBI as well as brain and CSF samples obtained from patients with TBI to investigate, after injury, the role of the CLR family member, macrophage-inducible C-type lectin (mincle) in the production of the inflammatory cytokine TNF. Here we report that mincle is activated after TBI in both, humans and rodents. Our data show that moderate fluid percussion brain injury (using male Sprague-Dawley rats (250-350 g) subjected to moderate fluid-percussion brain injury (1.8-2.2 atm)) is associated with increase expression of SAP130, which activates mincle signaling resulting in phosphorylation of the enzyme syk. Moreover, we identified increased expression of SAP130 and mincle in cortical neurons after TBI. In addition, we show that SAP130 stimulates mincle signaling in primary neuronal cultures, resulting in increase expression of the inflammatory cytokine TNF. Acute TNF expression after TBI is associated with deleterious outcomes. In conclusion, these findings suggest mincle as a novel therapeutic target that can be used to lower the levels of TNF after CNS injury.
MicroRNA-let-7a promotes E2F-dependent proliferation in mouse macrophages and mesangial cells (P1301)
Authors: Chafin Cristen , Regna Nicole , Caudell David , Reilly Christopher
Published: January 2023

Purpose: Cell proliferation is a common histological feature of lupus nephritis (LN) in the autoimmune disease systemic lupus erythematosus (SLE). MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. In this study, we used in vitro approaches to investigate cell proliferation through E2F-mediated activation of miRNA-let-7a. Methods: A mouse macrophage and a mouse mesangial cell line were transfected with let-7a mimics or a non-targeting control and treated with control or stimulating media. Cell cycle distribution was measured by flow cytometry. Proliferation was measured by MTT assay. Cells were transfected with E2F2 siRNA or a non-targeting control. Let-7a and E2F2 expression were measured by real-time RT-PCR. NFκB activation and IL-6 protein levels were measured by ELISA. Results: Let-7a decreased the number of stimulated cells in G0-G1 phase, resulting in an increase in S and G2-M phase. Cell proliferation was increased in stimulated cells transfected with let-7a. Let-7a significantly increased NFκB activation and E2F2 mRNA expression in stimulated cells. Silencing E2F2 significantly decreased let-7a expression and IL-6 production in stimulated cells. Conclusion: Let-7a induces macrophage and mesangial cell growth and proliferation, in part by activating the transcription factors NFκB and E2F2. This positive feedback loop may explain one mechanism of hyperplasia in lupus.
Cooperation of CD86 signaling with TLR-2 in the activation of resting B cells: a novel BCR independent pathway of stimulating B cells (P1126)
Authors: Chodisetti Sathi Babu , Jain Shweta , Agrewala Javed
Published: January 2023

The molecules of adaptive and innate immunity cooperate with each other in inducing effective immune response. It is well established fact that B cells can be activated through BCR mediated signaling. Here, we report an alternative strategy of activating resting B cells (RB cells) through concurrent engagement of CD86 and Toll like receptor-2. Concomitant signaling through CD86 and TLR-2 significantly augmented the longevity and activation profile of RB cells. Further, these cells showed enhanced pinocytosis and receptor mediated endocytosis. Furthermore, these cells secreted high levels of IgM and IgG1 and showed better ratio of surface IgG/IgM. Moreover, this process of activation amplifies the population of marginal zone precursors, which connect innate and adaptive arms of immune response. There was also upregulation of set of genes involved in the B cell activation, proliferation, maturation and endocytosis. The data are well in agreement with the gene expression profiles and insinuates to further decipher the molecular mechanism behind this concurrence. An insight into the detailed mechanism may pave way to design vaccines based on this signaling strategy.
A unique DNA methylation signature defines human CD4 memory T cells (P1148)
Authors: Komori Heather , Hart Glenn , LaMere Sarah , Mark Adam , Salomon Daniel
Published: January 2023

Memory CD4 T cells are “primed” for rapid responses to antigen; however, the molecular basis of this “priming” is incompletely understood. Methylation of CpG dinucleotides in gene promoters is an epigenetic modification known to regulate gene transcription. Using a new protocol we developed for targeted bisulfite sequencing (Genome Res, 2011), we assessed promoter methylation in human naïve and memory CD4 T cells of 2100 genes (56,000 CpG) linked to activation by deep RNAseq. 466 individual CpGs representing 132 genes are differentially methylated between resting naïve and memory cells. 21 genes exhibited both differential methylation and gene expression before activation, linking promoter DNA methylation states to gene regulation; 6 genes encode proteins closely studied in T cells while 15 genes represent novel targets for further study. 39 genes exhibited reduced methylation in resting memory cells coupled with increased gene expression with activation compared to naïve cells including 8 chemokines and cytokines. These data reveal specific genes more rapidly expressed in memory compared to naïve cells and regulated by DNA methylation. Our findings define a DNA methylation signature unique to memory CD4 T cells and correlated with activation-induced gene expression. We are just completing a new analysis of naïve vs. memory at 3 time points (day 0, 5, 14) using a new protocol for genome-wide CpG capture and deep bisulfite DNA sequencing to validate and extend this work.
Control of the molecular behavior of SHIP by FcγRIIB and Nck (P1122)
Authors: Pauls Samantha , Ray Arnab , Marshall Aaron
Published: January 2023

Leukocyte activity is controlled by balancing activating and inhibitory signals in order to generate effective immune responses while avoiding tissue damage and autoimmunity. This project examines an important regulatory circuit in B cells that involves the lipid phosphatase SHIP. Localization, phosphorylation, and binding to interaction partners are important factors in the regulation of this protein, however knowledge of their inter-relationships is incomplete. Here, we examine the influence of two interaction partners on the molecular behaviour and function of SHIP. First, we examine in detail how the inhibitory receptor FcγRIIB controls SHIP localization dynamics. We have observed that co-engagement of FcγRIIB along with the B cell receptor in A20 cells does not influence the magnitude or kinetics of SHIP-EGFP recruitment to the membrane as assessed by confocal microscopy, however it does alter mobility at the cell periphery as measured by fluorescence recovery after photobleaching. Next, we probe the influence of a novel binding partner, the adaptor protein Nck. We have demonstrated an interaction between SHIP and Nck by both Biacore affinity analysis and pull-down assays. Functional relevance will be addressed through mutagenesis experiments. With these aims we hope to enhance our understanding of an interaction that is already recognized as relevant and investigate the function of a novel interaction that occurs at a previously uncharacterized regulatory site.
Tregs suppress CD8 T cell effector function in inflamed tissue in a mouse model of autoimmune diabetes (P1018)
Authors: Mahne Ashley , Chou Annie , Tang Qizhi
Published: January 2023

The aim of this study was to determine the means by which Tregs control disease in the NOD mouse model of autoimmune diabetes. Treatment of pre-diabetic mice with ongoing islet destruction with islet antigen-specific Tregs expanded from BDC2.5 TCR transgenic mice led to a rapid restoration of insulin production and a concomitant selective downregulation of a cytotoxic T lymphocyte signature, which included CD8α, IFNγ, granzymes A and B, and Cxcl9. This downregulation could be partially attributed to a decrease in intra-islet CD8 T cell numbers due to impaired recruitment of CD8 T cells to the islets. In addition, Treg treatment also reduced effector function of persisting CD8 T cells. Two-photon imaging showed that Tregs did not significantly disrupt interactions between islet CD8 T cells and dendritic cells, but abrogated IFNγ production by CD8 T cells. This suppression appeared to take place at multiple stages of CD8 T cell differentiation. Tregs inhibited differentiation of newly recruited CD8 T cells to IFNγ producers completely, but prevented IFNγ protein expression without affecting IFNγ message in pre-activated CD8 T cells. Altogether, these results indicate that CD8 T cells are the primary early targets of Treg control of tissue inflammation and rapid disease resolution by Tregs can be attributed to efficient control of CD8 T cell infiltration, reactivation in the tissue, and their expression of effector functions.
The innate-like T cells are required to modulate acute inflammatory response (P1050)
Authors: Tang Hong , Feng JIn , Zhang Chao
Published: January 2023

Macrophages infiltration and activation in myocardium is a pivotal immunopathological lead to hypertensive cardiac micro-injury, but underlying mechanisms remain elusive. We have found that CD8 KO or CD8+ T cells depletion by antibody significantly reduce cardiac pro-fibrotic inflammatory responses induced by angiotensin II (Ang II) infusion, whereas CD8 KO mice reconstituted with CD8+ T cells became sensitive. More importantly, CD8+ T cells are required for macrophage infiltration in myocardium and subsequent activation to express pro-inflammatory cytokines and chemokines, including MCP-1. Furthermore, transwell experiments showed that macrophage activation requires direct contact with activated CD8+ T cells, but with TCR dispensable. TCR-independent activation of macrophage is further confirmed in vivo, where OT-I transgenic mice show a similar cardiac pro-inflammatory response to Ang II as wt mice. Finally, IFNγ seems required for influx and activation of CD8+ T cells in myocardium in response to Ang II, that subsequently activate macrophages in the onset of cardiac inflammation. Thus, TCR-independent innate nature of CD8+ T cells is both necessary and sufficient for macrophage-induced hypertensive cardiac fibrosis. In conclusion, TCR-independent activation of macrophages by CD8+ T cells casts yet a novel innate function of T cells that is required to activate inflammatory response of macrophages to danger signals.
Choline acetyltransferase+ T cells represent a discrete lymphocyte subset that regulates innate immunity. (P1008)
Authors: Olofsson Peder , Oswald Michaela , Hanes William , Lu Ben , Chavan Sangeeta , Gregersen Peter , Tracey Kevin
Published: January 2023

Neural circuits regulate cytokine production and inflammation. A vagus nerve circuit inhibits TNF production by acetylcholine signaling through the α7 nicotinic acetylcholine receptor on macrophages in spleen, but spleen nerves lack the machinery for acetylcholine biosynthesis. Instead, the nerve signal is relayed by CD4+CD44hiCD62Llow choline acetyltransferase+ (ChAT+) T cells, which produce the required acetylcholine. Understanding of the ChAT+ T cell phenotype is incomplete. mRNA levels in ChAT+ and ChAT- splenic T cells were measured by gene array. Hierarchical clustering of significantly different transcripts rendered four clusters. Expression data from the ChAT+/ChAT- dataset was also analyzed together with publicly available data on immune cell mRNA expression from the ImmGen consortium. In principal component analysis of this dataset, NK-cells, dendtritic cells/macropages, B cells and T cells formed separate clusters. ChAT+ T cells clustered among T cells, but separately from other T cell subpopulations, and were distinguished by the first principal component. Gene ontology analysis of this component showed enrichment of the G-protein coupled receptor signaling pathway, proliferation, and metabolism terms. In vitro, presence of ChAT+ T cells inhibited macrophage TNF production. Taken together, these data indicate that ChAT+ T cells are phenotypically distinct from other cell types in the ImmGen dataset and suggests that ChAT+ T cells are a novel T cell subset.
Host resistance to a gastrointestinal parasite regulated by Mina, an immunoregulatory member of the JmjC protein family (P1014)
Authors: R.Pillai Meenu , Van Wyhe Renae , Vogel Peter , Glaysher Bridget , Coleman Mathew , Ratcliffe Peter , Coles Mark , Bix Mark
Published: January 2023

T helper (Th) 1 and Th2 responses while critical for clearance of pathogens, when dysregulated results in autoimmune disease or allergy and asthma respectively. The genetic predisposition of certain inbred mouse strains towards developing Th2 as opposed to Th1 response (Th2-bias) has long been associated with differential susceptibility across a wide range of immunological diseases. However, its molecular genetic basis and thus the causal relationship to disease states are poorly understood. Prior investigations into the nature of this relationship resulted in the physical isolation of Dice1.2 on chromosome 16 and identification of Mina as a Dice1.2 resident gene exhibiting Il4 repressor activity. Thus, we hypothesized that Mina’s physiological role is to negatively regulate Th2 responses. To test this, we generated Mina KO mice and studied their response to the intestinal nematode T. muris. We found that Mina KO mice are hyper-resistant to T. muris infection. Surprisingly, the elevated T. muris resistance of Mina KO mice was not associated with an increased Th2 response but a defect in Th1 response. This correlated well with reduced inflammation in the cecum and reduced expression of antigen specific serum IgG2a. Further analysis revealed diminished expression of epithelial cell-specific Relmβ in Mina KO mice. These results highlight the importance of Mina in immune regulation and provide insight into the complex immune network perturbed by its expression and function.
Enhancement of vaccine-induced respiratory syncytial virus-specific memory CD8 T cell responses by targeting 41BB (P6158)
Authors: Lee Sujin , Moore Martin
Published: January 2023

Respiratory syncytial virus (RSV) causes significant morbidity and mortality in children and the elderly. There is no RSV vaccine in use. One of the major obstacles to RSV vaccine development is that RSV re-infects humans throughout life due to lack of durable protective immunity. We hypothesized that maintenance of robust and long-lasting memory CD8 T cells would be the key factor to prevent RSV infection. Recently, we generated a TriVax vaccination approach (peptide, polyI:C, and agonistic α-CD40 mAb). TriVax vaccination in mice completely protected against RSV infection at the effector phase, but protection against RSV was partial during the memory phase. 41BB (CD137) plays a key role in the survival of memory CD8 T cells. To determine whether co-administration of α-41BB in conjunction with TriVax could improve the maintenance of RSV-specific CD8 T cells at memory phase, we administered α-41BB (clone 3H3) or control IgG on the day of TriVax vaccination, and again 2 days later. Data indicated that co-administration of α-41BB and TriVax maintained 30% RSV-specific CD8 T cells until day 35 after the boost. Administration of TriVax+α-41BB followed by challenge using RSV rA2-line19F strain resulted in increased CD8 T cell cytokine expression and protection against RSV infection at memory phase as compared to TriVax+isotype control. Collectively, these data suggest that 41BB may be a promising candidate for the induction of long-lasting RSV-specific memory CD8 T cell immunity.
Dissecting the potential of resident CD11c-positive and CD11c-negative cells to support vitamin A-driven virus-specific immune responses in the upper respiratory tract (P6139)
Authors: Rudraraju Rajeev , Jones Bart , Surman Sherri , Sealy Robert , Woodland David , Hurwitz Julia
Published: January 2023

Morbidity and mortality due to viral infections are major health concerns, particularly when individuals are vitamin A (VitA) deficient (VAD). Previous literature has suggested that CD11c+ dendritic cells in the gastrointestinal tract are largely responsible for VitA metabolism and the associated homing and function of gut lymphocytes. To determine if/how VitA impacts virus-specific immunity in the upper respiratory tract (RT), we administered virus intranasally (IN) to VAD mice. We found that virus-specific B- and T-cell responses were altered, with a significant down-regulation of IgA in the nasal wash of VAD mice compared to controls. We next asked if RT cells could autonomously support VitA-driven IgA production. We harvested CD11c+ and Cd11c- cells from diffuse nasal associated lymphoid tissues and found that both expressed RALDH mRNA, as required for VitA metabolism. Furthermore, preliminary data showed that both populations enhanced IgA in activated splenocyte cultures. To investigate which CD11c- cells were pertinent to function, we tested a RT epithelial cell line. These cells: (i) expressed RALDH mRNA and (ii) enhanced IgA in the presence of VitA A derivatives. In total, data highlighted the autonomous potential of RT cells to support a VitA-driven immune response. Results may ultimately instruct a clinical healthcare initiative to supplement VAD individuals with VitA by the oral or IN route, to enhance RT immunity toward viral infections and viral vaccines.
The DNA sensing inflammasome, AIM2, is critical for protection from influenza infection (P6140)
Authors: Schattgen Stefan , Kurt-Jones Evelyn , Fitzgerald Katherine
Published: January 2023

Sensing of viral-derived nucleic acids following entry into cytosol of the host cell triggers inflammasome activation leading to maturation of interleukin 1β (IL-1β) and IL-18. In addition to activating the inflammasome directly within infected cells, viral infection can cause extensive tissue damage resulting in the accumulation of endogenous danger-associated molecular patterns (DAMPs), including self-DNA, which may provide signals in triggering inflammasome activation in neighboring uninfected cells. Though several studies have shown inflammasome activation within infected cells, it is unknown whether the sensing of DAMPs by uninfected bystander cells and the subsequent inflammasome activation provides an important proinflammatory signal in establishing an effective antiviral response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate AIM2 is required for protective immunity to influenza A virus (IAV), an orthomyxovirus with an ssRNA genome and a strictly RNA replication cycle. In addition to their increase in mortality and morbidity, Aim2-deficient mice show a defect in levels caspase-1-dependent cytokines IL-1β and IL-18, both important in the activation of cellular antiviral responses, following IAV challenge. Collectively, these data findings suggest that the sensing of self-DNA by AIM2 plays a critical role in triggering innate immune responses following infection with an RNA virus.
Stay off my lawn: T cell territorialism in virus-infected tissues of the respiratory tract (P6150)
Authors: Hurwitz Julia , Sealy Robert , Surman Sherri , Thomas Paul , Dayton Barry , Rudraraju Rajeev
Published: January 2023

Little is known of the population dynamics that influence lymphocyte positioning in virus- infected tissues. To address this issue, we primed animals with Sendai virus (SeV) and harvested cervical lymph nodes (CLN) or spleens 10 days later. Cells were unenriched or enriched for CD3, CD4 or CD8 phenotypes and transferred intraorbitally to GFP-SeV infected mice. Cell positions were then examined by confocal microscopy. Results showed that donor cells were configured in infected tissues with limited overlap. In most infected fields, the mean distance between a donor cell and its nearest neighbor was greater than would be predicted by random uniform distribution. To explain these findings, we hypothesized that a newly resident lymphocyte may instruct expression of unique chemokines/cytokines (c/c) in its immediate vicinity to mark its territory and inhibit co-localization by other cells. In vitro tests supported this hypothesis in that when virus-primed CLN were co-cultured with stroma and SeV, there was a synergistic increase in c/c production. Transwell experiments then showed that cell migration was intricately affected by the c/c milieu at local and distant sites. We therefore propose that a newly-resident lymphocyte in an infected tissue creates a c/c halo to identify and claim its territory. Lymphocyte cross-talk may ensure a spatial distribution of effectors to avoid redundancies and enhance the efficiency with which effector cells clear their virus infected targets.
Exhaustion of influenza-specific CD8+ T cells during primary infection leads to severe disease (P6152)
Authors: Rutigliano John , Morris Melissa , Oguin Thomas , McClaren Jennifer , Doherty Peter , Thomas Paul
Published: January 2023

It remains unclear why some strains of influenza induce severe disease while others cause more mild illness. We approached this question by investigating the CD8+ T cell response in a mouse model system comparing high and low pathological influenza infections. We discovered that early viral titer was not the determining factor in the outcome of disease. Instead, increased numbers of antigen-specific CD8+ T cells and elevated effector function on a per cell basis were found in low pathological infection and correlated with reduced illness and later time point viral titer. High pathological infection was associated with increased PD-1 expression on influenza-specific CD8+ T cells, but blockade of PD1-L in vivo did not rescue severe disease, and CD8+ T cell effector function was not restored. However, after anti-PD-1L treatment CD8+ T cell numbers were dramatically increased in high pathological infection, but not in low pathological infection. Additional studies in IL-6-deficient mice and WT mice treated with anti-G-CSF antibody showed a partial reversal of the severe disease phenotype, but these treatments failed to fully rescue severe disease. These data suggest that high pathological acute influenza infection is associated with CD8+ T cell exhaustion and an attendant increase in inflammation that was observed in the airway microenvironment during the early stages of infection. As a result, therapeutic approaches specifically aimed at modulating airway inflammation may promote efficient CD8+ T cell activity, with improved morbidity and mortality of severe influenza infections.
Toll like receptor 7 antagonist IRS661 inhibits insulitis and autoimmune diabetes in non-obese diabetic mice (P5227)
Authors: Zhang Yiqun , Chang Emily , Dutz Jan
Published: January 2023

Toll like receptor 7 (TLR7) stimulation activates DCs and T cells to promote autoimmune diabetes in non-obese diabetic (NOD) mice. The aim of the study was to investigate effects of TLR7 antagonist IRS661 on type 1 diabetes onset, insulitis and immune responses in NOD mice. Female NOD mice were treated with IRS661 at ages of 4, 6 and 8 weeks or with either ODN1982 or PBS as control groups. Blood glucose was measured weekly, and pancreas, pancreatic lymph nodes (PLNs) and spleens were obtained for histology analysis or FACS analysis. This treatment significantly inhibited the onset of diabetes, as 6/15 NOD mice treated with IRS661 developed diabetes by 30 weeks of age, compared to 8/8 ODN1982-treated or 13/15 PBS groups (p<0.001). Further, insulitis scores were lower and pancreatic islets were preserved in NOD mice treated with IRS661. To determine effects of IRS661 upon immune responses, NOD PLN cells or splenocytes were analyzed after the treatment. IRS661 diminished CD86 expression on DCs and IFN-α expression in plasmacytoid DCs in NOD PLNs. Further, IRS661 treatment decreased IGRP V7+ CD8 T cells and CD69 expression on CD8 T cells, and inhibited adoptively transferred BDC 2.5 CD4 T cell proliferation in NOD PLNs. No difference in CD25+Foxp3+ CD4 T regulatory cells between IRS661-treated or ODN1982-treated NOD splenocytes was detected. We conclude that IRS661 administration inhibits activation of DCs and CD8 T cells so as to decrease insulitis and diabetes onsets.
Fms-like tyrosine kinase 3 ligand-Fc chimera suppresses autoimmune demyelinating disease (P5228)
Authors: Burke Thomas , Tu Hua , Lewen Susanna , Huang Kexin , Carlson Jeffrey , Sobel Raymond , Zabel Brian
Published: January 2023

Dendritic cells (DC) are versatile immune cells that can promote protective anti-pathogen immunity or dampen destructive autoimmune responses. Fms-like tyrosine kinase 3-ligand (FLT3L) signals through the FLT3 receptor to drive DC differentiation and proliferation. We generated a novel mouse-FLT3L—human-FC-domain (FLT3L-FC) fusion construct that, when combined with hydrodynamic gene delivery (HDT), induces robust DC expansion in vivo. In particular, FLT3L-FC HDT induced expansion of surface marker-defined tolerogenic DC subsets. Given the central role of DC in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS), we investigated the action of FLT3L-FC in EAE. Prophylactic treatment of mice with FLT3L-FC HDT (a single i.v. injection of 10 ug of plasmid DNA) significantly suppressed the onset and clinical severity of EAE, and significantly reduced the number of inflammatory foci in CNS parenchyma. FLT3L-FC HDT in EAE mice induced expansion of peripheral (spleen) and CNS-infiltrating tolerogenic DC, as well as peripheral CD4+ regulatory T cells (Tregs), which we speculate underlies the cellular mechanism of EAE suppression by FLT3L. Our results therefore support the emerging paradigm that the fundamental action of FLT3L in vivo is immune suppression.
Characterization of TCR retrogenic mice that have a YFAK-specific regulatory T cell population. (P5230)
Authors: Ng Sze-Ling , Strominger Jack
Published: January 2023

T cell receptor (TCR) transgenic mice have been instrumental to the understanding of T cell biology. A valuable alternative to transgenic animals is the generation of TCR retrogenic mice using retrovirus-mediated gene transfer of the TCR chains into donor hematopoietic stem cells (HSCs) prior to reconstitution of irradiated recipients. However, to date, these genetically modified mice centered on the cloning and study of TCRs from effector T cells. Here, we isolated several TCRs from a YFAK-specific regulatory T cell (Treg) line in order to study Treg development, function, and maintenance. YFAK is a random amino acid copolymer identified in our lab to be more effective in amelioration of experimental autoimmune encephalomyelitis than the copolymer YEAK (Copaxone), which is currently used in the therapy of human multiple sclerosis. The TCR retrogenic mice generated had successful engraftment of the YFAK-specific Treg population in the thymus. These Tregs were also present in peripheral blood, spleen and lymph nodes. In addition, these cells secreted high levels of anti-inflammatory cytokines. Full characterization of these retrogenic Treg cells will be reported. Thus, the retrovirus-mediated expression of Treg-derived TCR chains in HSCs and transplantation into mice resulted in the in vivo differentiation of these cells into Tregs. This technique has potential in the therapy of autoimmune diseases such as multiple sclerosis by increasing the numbers of Tregs produced.
LEAPS peptide vaccination alters T cell phenotype and suppresses joint inflammation in a murine model of rheumatoid arthitis (P5223)
Authors: Zimmerman Daniel , Mikecz Katalin , Kurko Julia , Glant Tibor , Steiner Harold , Carambula Roy
Published: January 2023

Introduction. A major therapeutic goal in autoimmune inflammatory diseases such as rheumatoid arthritis (RA) is to suppress responses against (auto)antigens present in the target organs. Antigen-specific therapies such as the Ligand Epitope Antigen Presentation System (LEAPS) technology offer such a possibility. Materials and Methods. We evaluated LEAPS therapy in the cartilage proteoglycan (PG) induced arthritis (PGIA) model of RA. After the onset of PGIA, mice were divided in 3 groups, each with a similar mean arthritis index (AI). One group was vaccinated with adjuvant only, and the other groups with one of two LEAPS conjugates of a PG epitope 70 (J-PG70 or derG-PG70) in adjuvant. The same vaccination was repeated two weeks later. AI was measured by visual scoring every other day for 5 weeks. Upon euthanization, the proportions of Th1, Th2, Th17, and regulatory T cells (Tregs) in the spleen were determined by flow cytometry, and PG-specific lymphocyte proliferation by [3H]thymidine uptake. Results. Disease severity (AI) was dramatically suppressed in mice vaccinated with the LEAPS conjugate derG-PG70 as compared with controls or animals receiving J-PG70 vaccine. Protection was associated with reduced PG-specific lymphocyte proliferation, decreased proportions of Th1/Th17 cells, and increases in Th2 cells and Tregs. Conclusion. The derG-PG70 vaccine is therapeutically effective in PGIA (a Th1 dominated disease) by inducing immune deviation in favor of Th2 and Treg cells.
Tregitopes: The active ingredient in IVIG-mediated tolerance induction? (P5224)
Authors: Cousens Leslie , Moise Leonard , Martin William , De Groot Anne
Published: January 2023

In the course of screening IgG sequences for T cell epitopes, we identified Treg epitope peptides, now called Tregitopes, contained in conserved framework regions of Fab and Fc. Tregitopes may provide one explanation for the expansion and stimulation of Treg following intravenous immunoglobulin (IVIg) therapy. Their distinguishing characteristics include in silico signatures of high-affinity binding to multiple human HLA class II DR, and conservation across IgG isotypes as well as mammalian species. Tregitopes induce expansion of CD4+/CD25hi/FoxP3+ T cells and suppress immune responses to co-incubated antigens in vitro. Comparing human Tregitopes (hTregitopes) to murine sequences, we identified class II-restricted murine Tregitope homologs (mTregitopes). We provide evidence that when APCs present Tregitopes to natural (n) Treg, APC expression of MHC II, CD80, and CD86 are decreased. Moreover, Tregitopes increase proportions of IL-10-producing nTreg. We propose the mechanism of Tregitope-mediated tolerance induction to be as follows: 1) APC present Tregitopes to nTreg, 2) nTreg are activated, proliferate and produce IL-10, 3) nTreg provide tolerogenic feedback signals to APC, modulating the APC phenotype, and 4) nTreg and tolerogenic APC together suppress antigen-specific T cell responses. The discovery of Tregitopes in IgG and other autologous proteins may lead to the development of new insights as to the role of Tregs in autoimmune diseases.
Aryl hydrocarbon receptor negatively regulates type I interferon production in murine lupus (P5225)
Authors: Lee Soyoung , Ripley Barry , Millrine David , Chinen Ichino , Kishimoto Tadamitsu
Published: January 2023

Systemic lupus erythematosus is a multi-organ disorder characterized by the production of diverse autoantibodies. Recent studies suggest an important role for type I IFN in the pathogenesis of this disease. Production of type I IFN is induced by the transcription factor IRF7, while type I IFN signaling requires the STAT1 transcription factor. However, it is unclear how type I IFN is negatively regulated. Here, we show that production of type I IFN in cultured pDCs from aryl hydrocarbon receptor knockout mice is significantly higher compared to wild-type mice stimulated with TLR 7/9 agonists. Furthermore, treatment of pDCs from wild-type mice with the aryl hydrocarbon receptor agonist L-kynurenine significantly inhibits TLR 7/9-mediated type I IFN production. We demonstrate that through TLR 7/9 signaling, expression of aryl hydrocarbon receptor is induced, which in-turn forms an inhibitory interaction with IRF7 and STAT1, thus attenuating both type I IFN production and signaling. In addition, we found that production of type I IFN and expression of interferon stimulation genes in aryl hydrocarbon receptor knockout mice is higher than in wild-type mice when we induce lupus-like disease by pristane treatment. Our results suggest that aryl hydrocarbon receptor is a critical negative regulator of TLR-mediated type I IFN production and inhibits type I IFN signaling in murine lupus. We are evaluating protective effects of aryl hydrocarbon receptor agonists in murine lupus.
Novel LMW inhibitors of ITPKb block autoimmune disease by promoting calcium-induced T lymphocyte death (P5165)
Authors: Miller Andrew , Dahlberg Carol , Wen Ben , Sandberg Mark , Beisner Daniel , Parker Albert , Schmedt Christian , Stinson Monique , McPate Mark , Tranter Pamela , Groot-Kormelink Paul , Gosling Martin , Dawson-King Janet , Pan Shifeng , Tian Shin-Shay , Seidel H. Martin , Cooke Michael
Published: January 2023

Lymphocyte antigen receptor-mediated production of Ins(1,4,5)P3 induces the release of Ca2+ from intracellular stores, resulting in the opening of store-operated Ca2+ (SOC) channels. Mice deficient in inositol(1,4,5)P3-3 kinase B (ITPKb), which converts inositol(1,4,5)P3 (IP3) to inositol(1,3,4,5)P4 (IP4), exhibit a complete block in T cell positive selection. Previous studies demonstrated that IP4 is an inhibitor of SOC channels. To understand the role of ITPKb in mature peripheral lymphocytes, inducible ITPKb-/- mice were generated. Deletion of ITPKb in mature lymphocytes reveals that ITPKb is required for mature T cell function and T-dependent antibody responses. Following antigen receptor activation, the loss of ITPKb leads to enhanced Ca2+ levels and the induction of death effector gene expression resulting in apoptosis. We further demonstrate that IP4 is an inhibitor of open-state Orai1 channels. LMW ITPKb inhibitors were identified using a high-throughput compound screen. Application of ITPKb inhibitors to lymphocytes enhanced Ca2+ responses following antigen receptor stimulation, similar to ITPKb-/- cells. Treatment of mice with ITPKb inhibitors recapitulated the block in T cell development observed in ITPKb-/- mice and inhibited antigen-induced arthritis formation in rats. These data identify ITPKb and IP4 as crucial mediators of lymphocyte development and activation, and suggest that inhibition of ITPKb may provide a novel mechanism to treat autoimmune disease.