bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide inhibits the NLRP3 inflammasome (P1270)
Authors: Tsuchiya Kohsuke , Hernandez-Cuellar Eduardo , Hara Hideki , Fang Rendong , Sakai Shunsuke , Kawamura Ikuo , Mitsuyama Masao
Published: January 2023

Activation of the inflammasome leads to caspase-1 activation and maturation of pro-inflammatory cytokines, IL-1β and IL-18. The inflammasome plays a pivotal role in host defense against microbial pathogens. However, its uncontrolled activation is associated with inflammatory disorders, suggesting that regulation of inflammasome activation is important to prevent detrimental effects. In this study, we found that endogenous and exogenous NO inhibits the NLRP3 inflammasome. iNOS was involved in the regulation of NLRP3 inflammasome activation by either IFN-β pretreatment or long-term LPS stimulation. Furthermore, the NO donor SNAP markedly inhibited NLRP3 inflammasome activation, while the AIM2 and NLRC4 inflammasomes were only partially inhibited by SNAP. An increase in mitochondrial reactive oxygen species induced by ATP was only modestly affected by SNAP treatment. S-nitrosylation of NLRP3 was detected in macrophages treated with SNAP, and this modification may account for the NO-mediated mechanism controlling inflammasome activation. Taken together, these results revealed a novel role of NO in regulating the NLRP3 inflammasome.
Memantine modulation of LPS-induced NLRP3 inflammasome and pro-inflammatory cytokine expression (P1278)
Authors: Chang Sulie , Mao Xin , Sarkar Sraboni
Published: January 2023

The bacterial endotoxin, lipopolysaccharide (LPS), can induce the production of pro-inflammatory cytokines. Pro-inflammatory signaling is partly mediated through multiprotein complexes known as inflammasomes, including the NLRP3. Recent studies report that systemic treatment with low doses of the drug, memantine, decreases cytokine levels. In this study, LPS induction of several pro-inflammatory cytokines and the NLRP3 inflammasome were examined, with and without memantine treatment. Male F344 rats were treated with LPS (500 μg/kg) or saline intraperitoneally (i.p). Thirty minutes after treatment, the rats were given (i.p.) either memantine (0.2, 5, or 10 mg/kg) or saline. Blood and spleen were collected 1.5 h after treatment. Serum cytokine levels were measured using electrochemiluminescent assays; IL-1β and NLRP3 expression in the spleen was determined using real-time PCR. LPS-induced IL-1β, IL-5, and KC/GRO serum levels were significantly increased, and reduced by only the 5 and 10 mg/kg doses of memantine. LPS-induced IL-1β expression in the spleen was also significantly increased, but reduced only following a 10 mg/kg memantine dose. NLRP3 expression in the spleen was significantly increased after LPS, and decreased significantly by all doses of memantine. Our findings suggest that inhibition of the NLRP3 inflammasome may be one of the mechanisms underlying memantine’s anti-inflammatory actions.
The PYHIN family member IFI205 regulates immune signaling via transcriptional regulation of the inflammasome adapter ASC (P1280)
Authors: Ghosh Sreya , Carpenter Susan , Fitzgerald Katherine
Published: January 2023

Members of the PYHIN protein family such as AIM2 and IFI16, function as DNA sensors in innate immunity. Here we investigated the role of Ifi205, a murine PYHIN family member in innate immunity by generating murine macrophages lacking Ifi205 and monitoring immune signaling in these cells. Macrophages lacking Ifi205 had abrogated caspase-1 dependent processing and production of the pro-inflammatory cytokine IL-1β in response to cytosolic double stranded DNA and Nigericin, that activate the AIM2 and NLRP3 inflammasomes, respectively. Surprisingly, expression of the inflammasome adapter ASC was completely abolished at both the protein and mRNA levels in all Ifi205 knockdown cells generated. Reconstitution of the knockdown cells with ASC rescued IL-1β production attributing the knockdown phenotype to the loss of ASC expression. We observed a similar defect in ASC expression in cell lines lacking Ifi205 by targeting the 3’-UTR of the ifi205 gene. Reconstitution of these cells with Ifi205 rescued ASC mRNA expression. We also found that ectopic expression of Ifi205 augmented the activation of an ASC promoter-luciferase reporter gene in a dose-dependent manner in HEK 293T cells. The data suggest that ASC expression might be controlled at the transcriptional level. Thus, Ifi205 regulates inflammasome activation by controlling transcriptional regulation of ASC.
Gardnerella vaginalis causes inflammasome recruitment and inflammatory cytokine release in human monocytes (P1282)
Authors: Vick Eric , Huff Krista , Ouellette Corbett , Park Erin , Farone Mary , Farone Anthony
Published: January 2023

Intrauterine inflammation during pregnancy can cause prenatal brain injury, and is associated with preterm birth. Gardnerella vaginalis (GV) is a gram variable rod associated with bacterial vaginosis, pelvic inflammatory disease, bacteremia, and preterm birth. Bacterial vaginosis samples demonstrate up-regulation of Toll-like Receptor 2 and 4 mRNA and secretion of IL-1β in cultured trophoblasts. It has not been clearly demonstrated that isolated GV specimens are capable of the characteristic inflammatory downstream effectors in monocytes. We set out to determine if GV causes inflammatory effector recruitment and expression of proinflammatory cytokines in a human monocyte cell line, THP-1. An ASC-YFP overexpression system was used for immunofluorescent detection of inflammasome components, the Annexin-V apoptosis assay was used for viability, and cytokine levels were quantified by ELISA. Immunofluorescent detection showed co-localization of both NLRP3 and NLRC4 with ASC-YFP in GV-treated and LPS/ATP-treated cells. GV-treated cells exhibited a statistically significant IL-1β, IL-18, and TNFα response over untreated cells, however, IL-18 was significantly increased compared to LPS/ATP treatment. GV-treated cells remained viable through 24 h which demonstrates that GV causes significant cytokine elevation in THP-1 cells, without significant cell death. These results suggest that GV infection may lead to significant sequelae by production of proinflammatory cytokines.
Foxo1 is essential for effector-to-memory transition in CD8+ T cells (P1420)
Authors: Hess Michelini Rodrigo , Doedens Andrew , Goldrath Ananda , Hedrick Stephen
Published: January 2023

The forkhead DNA binding domain, O subgroup of transcription factors (FOXO) integrate a range of extracellular signals including growth factor and nutrient availability, and assimilate these to subsequent downstream FOXO TF activity to modulate cell survival, cell cycle progression, and a multitude of cell-type specific responses yet to be unraveled. CD8+ T lymphocytes are essential for the clearance of intracellular pathogens, and they undergo a rapid expansion and arming of effector function within days of pathogen exposure. Upon pathogen clearance, most cells die off while a subset remain with short-term or long-term memory function; this contraction is thought to depend on availability of growth factors/inflammatory cytokines and TCR stimulation, all of which regulate FOXO TFs. Using LCMV Armstrong as an infection model, we found that upon the transition from effector to memory FOXO1-/- CD8+ T cells contract slowly. In addition, memory precursors KLRG1lo CD8+ T cells retain higher GranzymeB and display an enhanced effector phenotype. Consistent with this phenotype, FOXO1-/- CD8+ T cells fail to upregulate memory-associated Tcf7, Eomesodermin, Bcl2, and a multitude of lesser known memory signature genes. As a result, FOXO1-/- CD8+ T cells were unable to properly respond to a secondary infection. Our results demonstrate an intrinsic role for FOXO1 in regulating the post-antigenic crash of CD8+ T cells, and is essential for the transition from effector to true memory.
Progressive memory differentiation of CD8 T cells is regulated by de novo DNA methylation (P1426)
Authors: Youngblood Benjamin , Hale Jeffery , Xu Xiaojin , Ahn Eunseon , Araki Koichi , West Erin , Konieczny Bogumila , Ahmed Rafi
Published: January 2023

Memory CD8 T cells generated from acute infection or vaccination have the potential to provide the host with life-long immunity against re-infection, yet one of the least understood and most remarkable attributes of memory T cells is their ability to progressively regain select naïve cell functions following the effector stage of the response. Presently debated is whether or not CD8 T cell memory arises through progressive modification of effector programs. To ascertain if effector programs are progressively modified during the effector to memory stage of the response, we performed longitudinal analyses of acquired epigenetic programs at the L-selectin (CD62L) promoter in virus-specific CD8 T cells during acute LCMV infection of mice. We observed that the CD62L promoter acquires a repressive de novo DNA methylation program in both terminal (TE) and memory precursor (MP) effector CD8 T cells. Demethylation of the de novo program in MP effector cells results in re-expression of CD62L at the memory stage of differentiation and occurs in the absence of cell division. Conditional deletion of the DNA methyltransferase 3a blocks methylation of the CD62L promoter in effector cells resulting in faster CD62L re-expression during effector to memory cell conversion. These data provide proof-of-principle that progressive modification of epigenetic programs in CD8 T cells undergoing memory differentiation is utilized to re-acquire naïve-like properties to enhance the recall response.
Determining molecular mechanisms of Tc17 plasticity in vitro and in vivo (P1414)
Authors: Ceccato Christina , Nirschl Christopher , Durham Nicholas , Alme Angela , Jackson Christopher , Drake Charles
Published: January 2023

We and others have shown that CD8+ T cells cultured in the presence of IL-6 and TGF-β acquire the capacity to secrete IL-17 (Tc17). Several in vivo tumor models have shown that adoptively transferred Tc17s have an equivalent if not superior anti-tumor response compared to adoptively transferred cells activated in the presence of IL-12 (Tc1). Since the efficacy of Tc17s correlates to conversion to an IFN-γ secreting phenotype, we sought to understand the mechanism of IL-17+ to IFN-γ+ conversion (plasticity). In this report, we show that antigen-specific, sorted IL-17+IFN-γ- (pure Tc17) cells that are re-cultured in vitro can convert to an IFN-γ secreting phenotype. Surprisingly, this conversion occurs not in the presence of Tc1 skewing cytokines but rather in the presence of IL-2 or IL-7. This conversion in vitro appears to be controlled by the balance of the transcription factors Tbet and RORγT. When ova-specific Tc17s are adoptively transferred in vivo they maintain a stable phenotype, but convert during re-stimulation with Vaccinia-Ova. The balance of the transcription factors Eomesodermin and RORγT likely controls this in vivo conversion, where IL-17 is not expressed in Eomesodermin positive CD8 T cells. Our data provide insight into the nature of these cells that could be important in clinical scenarios such as chronic infectious diseases or adoptive T cell immunotherapy.
CD8 T cells express randomly selected KIRs with distinct specificities compared to NK cells (P1418)
Authors: Björkström Niklas , Beziat Vivien , Cichocki Frank , Liu Lisa , Levine Jeffrey , Larsson Stella , Koup Richard , Anderson Stephen , Ljunggren Hans-Gustaf , Malmberg Karl-Johan
Published: January 2023

Epistatic interactions between killer cell immunoglobulin-like receptors (KIRs) and their cognate HLA class I ligands have important implications for reproductive success, anti-viral immunity, susceptibility to autoimmune conditions and cancer, as well as for graft-versus-leukemia reactions in settings of allogeneic stem cell transplantation. Although CD8 T cells are known to acquire KIRs when maturing from naïve to terminally differentiated cells, little information is available about the constitution of KIR repertoires on human CD8 T cells. Here, we have performed a high-resolution analysis of KIR-expression on CD8 T cells. The results show that most CD8 T cells possess a restricted KIR expression-pattern, often dominated by a single activating or inhibitory KIR. Furthermore, the expression of KIR, and its modulation of CD8 T cell function, was independent of expression of self-HLA class I-ligands. Finally, despite similarities in the stochastic regulation of KIRs by the bi-directional proximal promoter, the specificity of inhibitory KIRs on CD8 T cells was often distinct from that of NK cells in the same individual. The results provide new insight into the formation of KIR repertoires on human T cells.
Curcumin protects microglia and primary cortical neurons against HIV-mediated inflammation and apoptosis (P6181)
Authors: Dong Jun , Guo Luyan , Xing Yanyan , Jiang Mingliang , Lin Liqing , Wang Junbing , Xiong Guoying
Published: January 2023

Curcumin is a substance in turmeric root with anti-inflammatory, antioxidant, and anti-tumor properties, and has been widely used both as an herbal drug and a food additive to treat or prevent neurodisorders. To explore whether curcumin is able to treat HIV-associated neurocognitive disorders (HAND), we treated murine microglial cell line (N9) and primary rat cortical neurons with curcumin in the presence and absence of HIV gp120 (V3 loop) protein. We found that HIV gp120 profoundly induced N9 cells to produce reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1). HIV-1 gp120 also induced apoptosis of primary rat cortical neurons. Curcumin exerted powerful inhibitory effect against gp120-induced neuronal damage, reducing production of ROS, TNF-α and MCP-1 by N9 cells, and inhibiting apoptosis of primary rat cortical neurons. Curcumin may exert its biological activities through inhibition of gp120-mediated rectification and transient outward potassium current, as curcumin effectively reduced gp120-mediated elevation of potassium channel current of neurons. Conclusions: HIV gp120 increases ROS, TNF-α and MCP-1 production in microglia, and induces cortical neuron apoptosis by affecting the voltage-gated potassium channel current. Curcumin protects microglia and cortical neurons against HIV-mediated inflammation and apoptosis, probably through inhibition of gp120-induced elevation of the voltage-gated potassium current.
Immune exposure to a specific sequence of antibody escape variants could program long-term potential for neutralization breadth in subtype A HIV-1 infection (P6161)
Authors: Murphy Megan , Yue Ling , Pan Ruimin , Boliar Saikat , Sethi Anurag , Tian Jianhui , Karita Etienne , Allen Susan , Cormier Emmanuel , Robinson James , Gnanakaran S. , Hunter Eric , Kong Xiang-Peng , Derdeyn Cynthia
Published: January 2023

Approximately one quarter of HIV-1 infected individuals will generate broadly neutralizing antibodies, but the exact mechanisms for triggering and maturation of these responses are presently undefined. We consequently investigated the roots of neutralization breadth. In a subtype A HIV-1 infected Rwandan seroconverter, we pinpointed the primary neutralizing antibody target and the successive routes of viral escape using envelope glycoproteins from the transmitted/founder virus and longitudinal escape variants along with autologous plasma and monoclonal antibodies. Initially, a single mutation at one of three clustered residues proximal to gp120’s third hypervariable loop conferred viral escape. This putative epitope subsequently elicited at least two somatically related monoclonal antibodies, which bound and neutralized the established escape mutations. Resistance to this secondary wave of immune pressure then arose in later viral envelopes through introduction of two glycans that obscured this consistently targeted space. At 16-months post-infection, what had been a narrow, regional response evolved to force recognition and neutralization of distinct envelope portions, which resulted in moderate cross-clade humoral breadth. Our data suggest that unveiling a certain chain of envelope mutations could drive B cells toward the production of broadly neutralizing antibodies. Appreciating this knowledge during immunogen construction could positively impact HIV-1 vaccine design.
Differential function and maintenance of distinct precursor T cell subsets in unimmunized mice with aging (P1302)
Authors: Renkema Kristin , Smithey Megan , Nikolich-Zugich Janko
Published: January 2023

While age-related T cell defects in the context of infection are being elucidated, changes in the naïve T cell pool with aging remain poorly understood. We recently showed that unimmunized old mice lose >70% of Ag-specific CD8 T cell precursors, and many of the remaining precursors acquire a “virtual memory” (CD44hiCD62Lhi) phenotype. Here, we demonstrate that unimmunized TCR transgenic mice also undergo massive virtual memory conversion with age. Similar to non-transgenic mice, TCR transgenic virtual memory cells expressed increased levels of IL-15 receptor and were capable of rapid effector function upon both TCR and cytokine triggering. TCR transgenic virtual memory cells exhibited age-related delayed proliferation, suggesting an onset of proliferative senescence. This reduced proliferation could not be explained by the modest increase in expression of exhaustion molecules PD-1and LAG-3. Importantly, in Rag-sufficient OT-I mice, the highest rate of conversion to virtual memory occurred in cells that replaced the transgenic TCRα chain with endogenously recombined TCRα chains; indeed, these cells randomly expressed alternate Vα rearrangements. In contrast, OT-I Rag-/- mice, which cannot rearrange endogenous Vα, contained a small population (<5%) of virtual memory cells, which did not increase with aging. These results identify a new subset of Ag-specific CD8 precursors in unimmunized mice, with distinct age-related maintenance requirements and functional capabilities.
Rapid proliferation of memory phenotype CD4+ T cells is primarily driven by self-antigens (P1330)
Authors: Becklund Bryan , Surh Charles
Published: January 2023

The memory phenotype (MP) CD4+ T cell pool is a heterogeneous population of cells produced upon exposure to foreign antigens and through homeostatic proliferation during transient periods of lymphopenia. Previous studies aimed at identifying the mechanisms regulating the homeostasis of the MP CD4+ T cell pool have shown that a subset of MP CD4+ T cells undergoes a rapid rate of proliferation that is dependent on T cell receptor interactions with peptides bound to MHC-II. However, it is unclear whether the rapidly proliferating MP CD4+ T cells are responding to foreign or self-derived peptide antigens. To address this question, we have analyzed the proliferation of adoptively transferred MP CD4+ T cells in a host which predominantly displays a single self-peptide bound to MHC-II and in germ-free hosts which have a dramatically reduced foreign antigen load. Our results indicate that self-peptide/MHC-II complexes are primarily responsible for driving the rapid proliferation of MP CD4+ T cells in a lymphopenic host. The vigorous response to self-antigens and acquisition of effector functions by the rapidly dividing MP CD4+ T cell subset could potentially play a role in mediating the breakdown in peripheral self-tolerance underlying lymphopenia-associated autoimmune diseases.
The forkhead transcription factor, Foxo1, fine-tunes sensitivity to homeostatic signals during calorie restriction in CD8+ cytotoxic T cells (P1292)
Authors: Gubbels Bupp Melanie , Litvin Seth , Robinson Victoria
Published: January 2023

In addition to longevity enhancement, calorie restriction has been reported to slow the appearance of aging-related T cell abnormalities. However, the molecular mechanism underlying this phenomenon is ill-defined. We report that short-term calorie restriction in mice conferred protection to naïve CD8+ T cells from death-by-neglect, as compared to cells isolated from mice fed ad libitum. An ortholog of the forkhead transcription factor, Foxo1, is required for the longevity benefits of calorie restriction in C. elegans. Therefore, mice with a conditional, T cell-specific deficiency of Foxo1 were also subjected to calorie restriction. Calorie restriction in the absence of Foxo1 did not result in reduced naïve CD8+ T cell death by neglect. Naïve CD8+ T cells isolated from calorie restricted mice express higher levels of the cell surface receptors, CD8 and CD127, both of which are responsible for receiving homeostatic survival signals in vivo. Thus, Foxo1 coordinates an intrinsic transcriptional response to calorie restriction that alters the sensitivity of naïve CD8+ T cells to homeostatic signals. Additional studies revealed that full calorie restriction (as opposed to restricting one component of the diet) is required for this phenomenon. To our knowledge, Foxo1 is the first transcription factor identified to contribute to altered naïve CD8+ T cell homeostasis during calorie restriction in mice.
The role of Ubiquitin Specific Protease 1 in promoting Id protein stability during T cell responses (P1300)
Authors: Omilusik Kyla , Yang Cliff , Goldrath Ananda
Published: January 2023

CD8+ T cells are necessary for immune responses against intracellular pathogens and tumors. The activation of T cells and subsequent differentiation into memory populations involves dramatic changes in gene expression. E protein transcription factors and their inhibitors, Id proteins, play key roles in lymphocyte development, differentiation, and maintenance. Specifically, Id2 is upregulated in effector CD8+ T cells and maintained in memory cells presumably to induce effector function. Interestingly, Id2 overexpression is also associated with lymphomas. The mechanisms controlling normal Id2 expression and how misregulation leads to transformation is largely unknown. Recently, USP1, a deubiquitinase, was shown to stabilize Id2 in osteosarcomas resulting in decreased E protein activity. Analogously, we propose a role for USP1 in posttranslational control of Id2 in T cells. Here, we show that USP1 is upregulated in CD8+ T cells upon infection and this expression change is likely mediated through the T cell receptor. Furthermore, we establish a direct interaction of Id2 and USP1 following T cell activation. By defining a regulatory role for USP1, we will have a more complete understanding of mechanisms required for controlled survival and expansion of healthy T cells and how failure of this process can lead to tumorigenesis. Ultimately, identifying USP1 as an oncogene in lymphomas will provide a novel target for therapeutic interventions.
Carbohydrates in dust mite allergen modulate Th2 immune responses (P6019)
Authors: Gour Naina , Wills-Karp Marsha
Published: January 2023

Asthma is a chronic inflammatory disease of the airways, driven by a Th2 immune response to allergens like house dust mite (HDM). While Th2 immune responses (IL-13 in particular) alone can induce allergic asthma, Th17 cytokines (IL-17) can exacerbate the disease. Although, the components of HDM that drive asthma remain unclear, HDM is a complex mixture including immunogenic structural moieties like carbohydrates. Such carbohydrate moieties can be recognized by pattern recognition receptors on dendritic cells (DCs) like C-type lectin receptors (CLRs). Recently, CLRs like Dectin-1 & Dectin-2 have been implicated in driving Th2 & Th17 responses against various infections. However, the role of carbohydrate recognition in modulation of HDM-induced allergic asthma remains unclear. Therefore we hypothesized that Dectin-1 & Dectin-2 induce Th2 & Th17 responses following HDM exposure. Surprisingly, Dectin-1 KO mice had exacerbated Th2 immune responses leading to enhance airway resistance. In contrast, mice in which Dectin-2 was blocked (using monoclonal antibody) had diminished HDM-induced Th2 immune responses and airway reactivity. Interestingly, neither Dectin-1 nor Dectin-2 modulated Th17 responses. Overall, our data suggests that while Dectin-1 protects against allergic asthma by negatively regulating Th2 immune responses, Dectin-2 promotes asthma by enhancing Th2 immune responses.
Cannabinoid CB2 receptors as novel target for inhibiting house dust mite induced allergic airway inflammation (P6023)
Authors: Ferrini Maria , Hong Soram , Roberts Kevan , Jaffar Zeina
Published: January 2023

Asthma is a chronic lung inflammatory disorder characterized by airflow obstruction, airway hyperreactivity (AHR) and bronchial inflammation in response to environmental stimuli including allergens. Allergic asthma is associated with the presence in the airways of CD4+ Th2 cells and eosinophils, together with goblet cell hyperplasia and epithelial desquamation. Recent studies have indicated that cannabis-derived compounds and cannabinoid receptor agonists have immunosuppressive and anti-inflammatory properties. In this study, we examined the effect of CB2 selective agonists on allergic airway inflammation using Derp house dust mite (HDM)-induced model of asthma. Our results revealed that the intranasal administration of nonpsychoactive CB2 agonists was highly effective at attenuating allergic airway inflammatory responses. Specifically, CB2 agonists caused a marked inhibition in the accumulation of CD4+ T cells and eosinophils into the airways and the level of Th2 cytokine production in the lungs of mice immunized and challenged with HDM. Moreover, treatment with CB2 agonists ameliorated HDM-induced AHR and mucus production. CB2 receptors were highly expressed by antigen-presenting cells in the airways during allergic inflammation. Collectively, these results demonstrate that CB2 activation leads to suppression of allergen-induced airway inflammation and AHR and may provide a novel approach for the treatment for lung inflammatory diseases such as asthma.
SPA0355, a thiourea analog, suppressed T cell activation and reduced airway inflammation in mice (P6027)
Authors: Jang Hyun-Young , Lee Sang-Myeong , Park Byung-Hyun
Published: January 2023

SPA0355, a thiourea analog, has reported to inhibit the nuclear factor-κB (NF-κB) pathway by targeting IκB kinase. Because NF-κB activation plays a pivotal role in the pathogenesis of allergic airway inflammation and CD4+ type 2 helper T lymphocytes (Th2 cells) cytokines production, the effect of SPA0355 on an ovalbumin-induced airway inflammation model in mice was examined. Airway hyperresponsiveness, cytokines, mucin, and eosinophilic infiltration were analyzed in bronchoalveolar lavage fluid and lung tissue. An intraperitoneal administration of SPA0355 suppressed allergen-induced NF-κB activation in the lung of sensitized mice. SPA0355 also inhibited inflammatory cell recruitment into the airways as well as the production of Th2 cytokines in bronchoalveolar fluid. In addition, SPA0355 suppressed mucin production and prevented the development of airway hyper-responsiveness. Further in vitro studies showed that SPA0355 suppressed CD4+ T cell proliferation, activation, and differentiation into Th1 and Th2 cells. These results suggest that immunomodulatory effects of SPA0355 in vivo and in vitro may provide a basis for the development of a new therapy for the allergic airway inflammation.
Intestinal epithelial cells IKKβ regulate allergic responses in the skin (P6053)
Authors: Bonnegarde-Bernard Astrid , Jee Junbae , Fial Michael , Aeffner Famke , Boyaka Prosper
Published: January 2023

The sites where immune responses are initiated are believed to play a key role at shaping subsequent responses at distant mucosal sites. We have previously shown that IKKβ in intestinal epithelial cells (IECs) shapes the immune responses to ingested antigens and influences airway responses to subsequent antigen exposure via regulation of IgA and Th17. People with food allergy often suffer from allergic airway and/or skin diseases like asthma or atopic dermatitis but it is unknown whether T cells sensitized in the gut promote inflammation at different mucosal surfaces through the same mechanism. Using a model of oral antigen sensitization and mice with cell-specific deletion of IKKβ, we addressed the contribution of IECs to regulatory crosstalk between the gut and skin. Control C57BL/6 mice and mice deficient in IKKβ in IEC (IKKβΔIEC) were orally sensitized by administration of ovalbumin and cholera toxin as adjuvant. All groups developed similar levels of antigen-specific IgE and IgG1 responses, but IgG2b responses were enhanced in IKKβΔIEC. Subsequent epicutaneous challenges of control mice with ovalbumin induced skin inflammation characterized by increased epidermal thickness and dermal infiltration of eosinophils. Interestingly, both the epidermal thickness and dermal infiltration of eosinophils were reduced in IKKβΔIEC. Ongoing studies will determine mechanisms underlying the protective effect of IKKβ in intestinal epithelial cells against allergic responses in the skin.
Insulin Receptor Substrate-2 negatively regulates alternative macrophage activation and allergic lung inflammation. (P6009)
Authors: Keegan Achsah , Dorsey Nicolas , Qi Xiulan , Smith Elizabeth , Dasgupta Preeta
Published: January 2023

Insulin Receptor Substrate (IRS)2 is an adaptor that mediates anti-apoptotic signals in response to IL-4 or IL-13 in addition to regulating insulin signaling. Studies have shown that IRS2 plays a role in IL-4-induced proliferation of CD4+ T cells and secretion of TH2 cytokines in vitro. However, the functional significance of this pathway in induction of allergic lung inflammation has not been assessed. We previously demonstrated that IL-4 induced greater phosphorylation of IRS2 and expression of genes associated with the alternative activation of macrophages (AAM) when compared to IL-13, suggesting that IRS2 may be required for AAM differentiation. To examine the role of IRS2 in AAM gene induction and pulmonary inflammation, we utilized IRS-2-/- mice. Surprisingly, IRS2-/- mice developed enhanced pulmonary inflammation with significantly increased recruitment of eosinophils and macrophages in comparison to IRS2+/+ mice. Additionally, greater numbers of FIZZ1, YM1 and Arginase1 transcripts were detected in response to IL-4 or IL-13 in macrophages lacking IRS2 in vitro. This also correlated to increased YM1 protein expression in macrophages in vivo in the allergic airways in IRS2-/- mice. Interestingly, IRS2+/- mice demonstrated a gene dosage effect; they developed intermediate levels of inflammation, cellular infiltration and AAM gene/protein expression. Thus, this study identifies a previously unrecognized role for the IRS2 pathway in suppressing asthma responses.
PTX3 modulates airway hyperresponsiveness and immune response to OVA in a murine model of asthma (P6011)
Authors: Balhara Jyoti , Shan Lianyu , Soussi Gounni Abdelilah
Published: January 2023

Pentraxin-3 (PTX3) is a member of the long pentraxins family. It activates the innate and adaptive immune systems, playing an important role in providing immunity against various pulmonary infections. Considering its role in fostering lung immunity, we examined the role of PTX3 in asthma. We have recently demonstrated an enhanced expression of PTX3 in bronchial biopsies of allergic asthmatics that correlated with the disease severity. In this report, we assessed the effect of PTX3 deficiency in a murine model of OVA-induced asthma. Sensitized PTX3 Knockout (KO) mice exhibited an enhanced airway and tissue resistance in response to methacholine (MCh) in contrast to their WT counterparts. We observed an increase in the infiltration of inflammatory cells in BALF obtained from PTX3 KO mice upon OVA sensitization/challenge as compared to their WT littermates. However peribronchial and perivascular inflammation and goblet cells hyperplasia showed no significant difference in both mouse strains. Further we found an enhanced induction of IL-4, IFN-gamma, IL-17 and IL-10 production in the lungs of PTX3 KO mice as compared to WT mice upon OVA challenge. In lymph node, IL-17A recall response was found to be greater in PTX3 KO mice than WT mice but no difference was observed in term of IL-4, IFN-gamma and IL-10 production. Taken together, we conclude that lack of PTX3 predisposes mice to airway hyperresponsiveness and an enhanced inflammation.
CCL3 and CCL4 secretion by T regulatory cells attracts CD4+ and CD8+ T cells (P1077)
Authors: Wang Adele , Patterson Scott , Marwaha Ashish , Tan Rusung , Levings Megan
Published: January 2023

FOXP3+ T regulatory cells (Tregs) function to restrain immune responses. Accumulating evidence has shown that Tregs mediate their suppression by migrating to tissues based on their distinct expression of chemokine receptors. Yet how Tregs ensure they are close to their targets is unknown. Using ELISA and intracellular staining we discovered that both human and mouse Tregs produce CCL3 and CCL4, ligands for CCR5. Since chemokines recruit immune cells and the broad suppressive activities of Tregs require intimate cell proximity, we hypothesized that the secretion of the pro-inflammatory chemokines may bring immune cells closer to Tregs to be suppressed. To determine whether Tregs can recruit CD4+ and CD8+ cells via a CCR5-dependent mechanism, we performed migration assays and showed CCL3- and CCL4- containing-supernatants from Tregs attracted both subsets of T cells as well Tregs. Additionally, CCR5-/- T cells had a significantly reduced capacity to migrate towards Tregs in vitro and in vivo, supporting that chemokine signalling through CCR5 is critical. Furthermore, decreased proportion of FOXP3+CCL3+ Tregs was observed in patients with type 1 diabetes compared to healthy controls, implying that the altered chemokine secretion could contribute to the inadequate Treg function. These results suggest that active recruitment of their targets of suppression may be a novel mechanism of action by Tregs and that this pathway could be a potential therapeutic target in autoimmunity.
Elucidation of the mechanism of cell type specific IRF-4-mediated transcription in CD4+ T cells (P1081)
Authors: Spolski Rosanne , Li Peng , Liao Wei , Wang Lu , Leonard Warren
Published: January 2023

IL-21 plays a key role in Th17 differentiation. Interferon (IFN) regulatory factor 4 (IRF4) is a critical regulator of differentiation in B and T cells, and Th17 differentiation is defective in Irf4 KO CD4+ T cells. In B cells, IRF4 is known to cooperatively bind DNA in partnership with the Ets family protein PU.1. However, PU.1 is not expressed in most T cells even though IRF4 is active within these cells, and the functional transcription factor complex that regulates Th17-specific genes was not known. Genome-wide Chip-Seq analysis was used to demonstrate that in T cells, IRF4 binds to a novel composite DNA motif that includes AP1 motifs and which we named AP1-IRF4 composite elements (AICEs). Moreover, IL-21-regulated genes in Th17 cells have co-localized binding of IRF4, BATF, and JUN family proteins, and the genes encoding these transcription factors are themselves regulated by IL-21 in CD4+ T cells. RNA-seq analysis revealed subsets of genes whose regulation by IL-21, either positive or negative, depends on IRF4, and reporter studies demonstrated that IL-21-induced transcription of these genes was dependent on both IRF4 and AP1 transcription factor binding. Our results demonstrate that by utilizing distinct binding partners and DNA sequences, IRF4 mediates cell-type and stimulus-specific functions in multiple lymphocyte lineages.
Redirecting antigen specificity to nonspecific CD8+25+ regular T cells through antigen-specific peptide/MHC complex arming. (P1085)
Authors: Wang Lu , Zhang Xueshu , Mulligan Sean , Xiang Jim
Published: January 2023

Compared with CD4+25+ regulatory T (Tr) cells, the mechanisms for immune suppression of natural CD8+25+ Tr cells are less studied. In this study, we demonstrate that CD8+25+ Tr cells inhibit in vitro T cell proliferation via cell-contact fashion, but independent upon expression of suppressive IL-10, TGF-β and CTLA-4. CD8+25+ Tr-treated T cells express CD73 and FR-4 and become resistant to cell proliferation by dendritic cell (DC) stimulation, whereas CD8+25+ Tr-treated DCs down-regulate expression of DC-maturation markers and inflammatory cytokines, and lack stimulatory effect on T cell proliferation. CD8+25+ Tr cells suppress ovalbumin (OVA)-pulsed DC (DCOVA)-stimulated CD8+ cytotoxic T lymphocyte (CTL) responses and antitumor immunity as well as myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). Importantly, CD8+25+ Tr cells, when pulsed with OVA323-339 and MOG35-55 peptide, exert enhanced inhibition of antitumor immunity and EAE, respectively. Interestingly, OVA323-339-pulsed (armed) CD8+25+ Tr cells also reduce CD4+ T cell interactions with DCOVA via increasing CD4+ T-cell velocities assessed by two-photon microscopy. Therefore, redirecting antigen specificity to nonspecific CD8+25+ Tr cells for enhanced immune suppression can be achieved via antigen-specific peptide/MHC complex arming. This principle may have great impact in natural Tr cell-mediated immunotherapy for autoimmune diseases.
NK cell licensing modulates NK cell conjugation to target cells via altered activation receptor function (P1063)
Authors: Thomas Louis , Long Eric
Published: January 2023

MHC class I recognition by inhibitory receptors on natural killer (NK) cells promotes a state of increased NK cell responsiveness through a process known as licensing/education. In the absence of either MHC class I expression or inhibitory receptors, NK cells exhibit a decreased responsive state. Thus NK cell licensing is a critical aspect of NK cell biology. However, understanding how licensing modulates natural cytotoxicity is limited. Using pure populations of unlicensed human NK cells, we were able to evaluate the effect of licensing on several NK cell functions. Our findings reveal that NK cell licensing does not affect lytic granule polarization but significantly enhances NK cell conjugation to target cells. Furthermore, our data show that activation receptors in unlicensed NK cells fail to provide strong ‘inside-out’ signaling for the beta2 integrin LFA-1. NK cell adhesion to target cells is a crucial initial step in NK cell-mediated cytotoxicity. The reduced conjugation of unlicensed NK cells may contribute to their hyporesponsive state.
Exploration and blockade of the decoy strategy: crystal structure of the LIGHT:DcR3 complex and rational design of DcR3 antagonists (P1067)
Authors: Liu Weifeng , Zhan Chenyang , Nathenson Stanley , Almo Steven
Published: January 2023

LIGHT is a member of the tumor necrosis factor (TNF) family that initiates intracellular signaling via engagement of the two TNF receptors HVEM and Lymphotoxin beta Receptor (LTbetaR). In humans, LIGHT is neutralized by a soluble TNF receptor denoted as decoy receptor 3 (DcR3), which tightly binds LIGHT and inhibits its interactions with HVEM and LTbetaR, resulting in blockade of the corresponding signaling pathways. In addition to LIGHT, DcR3 also binds and neutralizes two other TNF ligands, FasL and TL1A, thus further modulating the immune response. Because of its remarkable ability to neutralize three different ligands, DcR3 is thought to play an important role in regulating the immune response, especially in cancer immunology as suggested by elevated expression levels and the associated poor prognosis in cancer patients. However, the mechanisms that support the broad specificity of DcR3 (i.e., recognition of three different TNF ligands) remain to be fully defined. We have determined the structure of the LIGHT:DcR3 complex, revealing the structural basis for the DcR3-mediated neutralization of LIGHT. Comparison with our previously reported structure of the TL1A:DcR3 complex affords new insights into DcR3 function and selectivity. Based on these structures, we designed a novel LIGHT mutant that specifically recognizes DcR3 and exhibits no interaction with HVEM; this reagent dissects the multiple biochemical functions of LIGHT and offers unique clinical opportunities.
CD11c+ dendritic cells are required for IL-33-mediated expansion of ST2+Foxp3+ regulatory T cells in vivo (P1075)
Authors: Matta Benjamin , Lott Jeremy , Mathews Lisa , Rosborough Brian , Turnquist Heth
Published: January 2023

IL-33 is an IL-1 cytokine that signals via ST2, which is expressed on T cells and myeloid cells. Although IL-33 promotes Th2 responses, its administration potently expands CD4+Foxp3+ regulatory T cells (Treg). We examined if IL-33 expands murine Treg directly or indirectly through its impact on CD11c+ DC. The ability of IL-33 to facilitate CD3/CD28-stimulated proliferation of wild-type (WT) or ST2-/- Treg was compared to IL-2. The impact of IL-33 on the capacity of DC to expand Treg was defined in vitro on bone marrow (BM)-generated DC from WT or ST2-/- mice and in vivo using CD11c-DTR BM chimeras administered diphtheria toxin (DT) to deplete CD11c+ cells during IL-33 treatment. The capacity of IL-33-expanded Treg from Foxp3-RFP reporter mice to suppress effector T cells was assessed. We found that IL-33 directly expands Treg in vitro, including an ST2+ subset absent from IL-2-treated cultures. IL-33-exposed DC generate ST2+ Treg from naïve T cells, and is dependent on ST2 expressed by DC. IL-33 failed to expand Treg in vivo, especially ST2+ Treg, in the absence of CD11c+ cells. In conclusion, IL-33 promotes the expansion of suppressive Foxp3+ Treg, including an ST2+ subset in vitro and in vivo. This results from IL-33 activity directly on Treg, but more significantly, indirectly through its impact on CD11c+ cells. These findings have important implications for further characterization of ST2+ Treg and the development of novel therapeutics aimed at promoting immune tolerance.
Local changes in lipid environment of T cell receptor microclusters regulate membrane binding by the CD3epsilon cytoplasmic domain (P1103)
Authors: Gagnon Etienne , Wucherpfennig Kai
Published: January 2023

The CD3epsilon and zeta cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane, and a previous NMR structure showed that both tyrosines of the CD3epsilon Immuno-tyrosine based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3epsilon residues were previously shown to be essential for CD3epsilon and zeta membrane binding. Phosphatidylserine is the most abundant negatively charged lipid on the inner leaflet of the plasma membrane and makes a major contribution to membrane binding by the CD3epsilon cytoplasmic domain. Here we show that T cell receptor triggering by peptide-MHC complexes induces dissociation of the CD3epsilon cytoplasmic domain from the plasma membrane. Release of the CD3epsilon cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and phosphatidylserine density in T cell receptor microclusters. The localized reduction in phosphatidylserine density is explained by reduced diffusion of this lipid into synapses. These changes in the lipid composition of T cell receptor microclusters even occur when receptor signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of the microclusters thus render the CD3epsilon cytoplasmic domain accessible during early stages of T cell activation.
An altered relationship of influenza vaccine-specific IgG responses with T cell immunity occurs with aging in humans (P4293)
Authors: Lee Naeun , Kang Ki Soo , Shin Min Sun , Mohanty Subhasis , Belshe Robert B. , Montgomery Ruth R. , Shaw Albert C. , Kang Insoo
Published: January 2023

Alterations in T cell immunity occur with aging. Influenza causes significant morbidity and mortality in the elderly. We investigated the relationship of serum IgG responses with hemagglutinin inhibition (HI) antibody titers and the frequency of distinct T cell subsets in young and elderly people who received the inactivated influenza vaccine. Influenza vaccine-specific IgG responses correlated with the increase of HI antibody titers and the frequency of CD4+ T cells producing IFN-γ and IL-17 in young, but not elderly, people. Also, only in young people, such IgG responses correlated with the frequency of memory T cells, especially central memory cells, CD45RA- effector memory CD8+ T cells and IL-7 receptor alpha high effector memory CD8+ T cells with potent survival and proliferative capacity. These findings suggest that aging alters the association of influenza-vaccine specific IgG responses with HI antibody titers, cytokine-producing capacity and proportions of memory T cells in humans.
Influenza antigen microarrays reveal reactivity signatures associated with effective response to seasonal trivalent influenza vaccination (P4295)
Authors: Price Jordan , Jarrell Justin , Furman David , Kattah Nicole , Newell Evan , Dekker Cornelia , Davis Mark , Utz Paul
Published: January 2023

Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. In a cohort of 76 young and elderly subjects administered with the 2007/2008 trivalent influenza vaccine, we observed reactivity to influenza whole-protein and peptide array features that correlated significantly with age and post-vaccine titer as assessed through a standard microneutralization assay. One reactive peptide correlated positively with age and negatively with seasonal H1N1 neutralization titer, implicating reactivity to this epitope in age-related defects in response to H1N1 influenza. We also generated models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy. Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.
Cross-reactive influenza H1N1 T cell epitopes identified by immunoinformatic methods stimulate CD4+ T cell responses (P4303)
Authors: De Groot Anne , Boyle Christine , Ardito Matthew , Terry Frances , Latimer Howard , Tassone Ryan , Cote Mindy , Moise Leonard , Martin William
Published: January 2023

Immune responses to cross-conserved T cell epitopes in novel H1N1 influenza might explain reports of diminished influenza-like illnesses and confirmed infection among older adults, in the absence of cross-reactive humoral immunity, during the 2009 pandemic. We set out to identify and characterize cross-conserved H1N1 T cell epitopes to develop a universal H1N1 influenza vaccine. An immunoinformatic analysis was conducted using all available pandemic and pre-pandemic HA-H1 and NA-N1 sequences dating back to 1980. From 5,738 HA-H1 and 5,396 NA-N1 sequences, 13 HA and 4 NA immunogenic consensus sequences (ICS) were selected that each cover >84% of pre-pandemic and pandemic H1N1 influenza strains, bear EpiMatrix scores ≥95th percentile and cover ≥4 HLA Class II archetypal alleles. HLA binding assays for 6 Class II archetypal alleles showed that immunoinformatic predictions were 78% accurate. Individual ICS peptides were immunoreactive in cultured human IFNy ELISpot assays after antigen-specific in vitro expansion. Intracellular cytokine staining showed the magnitude of IL-2, IFNy and/or TNFa expressing CD4+ T cells was boosted by 2011 seasonal trivalent influenza immunization for vaccine-matched and ICS HA peptide pools. Immunoinformatic methods identify cross-reactive influenza H1N1-specific CD4+ T cell epitopes. This approach can be applied to other influenza subtypes to develop a universal influenza vaccine that will protect against antigenically novel influenza viruses.