bluuebunny/crossref_metadata_2025 huggingface dataset:
Allergic lung disease is enhanced by antigen-specific B cells (P6033)
Authors: Lindell Dennis , White Maria
Published: January 2023

Previous studies from our laboratory have demonstrated that B cells play an important role in the initiation and maintenance of chronic allergic lung disease (asthma), and ascribed much of this role to antigen presentation (Lindell et al. PLoSONE 2008). Although present at higher frequency than other organs, antibody secreting B cells in the lungs represent a small proportion (<0.1%) of the overall B cells in the lungs. As alternative strategies to study the role of B cells in this disease, we have used novel flow cytometric techniques to detect the de novo expansion of antigen-specific B cells during allergic lung disease, as well as utilizing adoptive transfer of naive antigen-specific B cells. Mice were immunized with hen egg lysozyme and a cohort of these mice received adoptive transfers of HEL-specific B cells from MD4 mice. Our data indicate that the addition of antigen-specific B cells augmented mucus production in the lungs. Mechanistically, this was associated with increased T cell responses in the lungs (a 4-fold increase in CD69+ CD4+ and CD8+ T cells), significantly higher levels of IL-13 and IL-17a, as well as overall inflammation, although very few HEL+ B cells were found in the lungs, and we observed no increase in HEL-specific antibodies. These data suggest that B cells predominantly contribute to allergic disease by amplification of the T cell response, rather than via the production of antibodies.
Divergent roles of two neuroimmune semaphorins, Sema4A and Sema4D, in regulation of experimental asthma severity (P6041)
Authors: Chapoval Svetlana , Nkyimbeng-Takwi Eusebius-Henry , Shanks Kathleen , Smith Elizabeth , Iyer Apoorva , Lipsky Michael , DeTolla Louis , Keegan Achsah
Published: January 2023

Allergic asthma is characterized by exaggerated Th2 inflammation resulting from interaction between DC and T cells. Molecules acting in this interaction, regulating cell activation and differentiation are desirable therapeutic targets. Based on the previously published data and our results, we hypothesized that neuroimmune semaphorins play critical costimulatory roles in DC - T cell interaction, thereby influencing allergic lung inflammation. To test our hypothesis, we used Sema4A-/- and Sema4D-/- mice in the mouse model of OVA-induced allergic lung response. Our results have shown that both molecules play critical but opposite roles in disease severity. It was significantly potentiated by Sema4A deficiency, what included increased eosinophilic infiltration, AHR, local/systemic IL-13 levels, sera OVA-specific IgG1/ IgG2b/IgE levels, and decreased Treg numbers. Moreover, we defined that Sema4A expression on CD4+ T cells but not on DC was important for Th2 response regulation. In contrast, we found a decrease in lung inflammation and BAL Th2 cytokine levels in allergen-treated Sema4D-/- mice relative to WT mice. In addition, T cell proliferation in OVA323-339 - restimulated Sema4D-/- cell cultures was downregulated suggesting a costimulatory role of Sema4D for T cell activation. However, AHR was not affected by Sema4D deficiency suggesting its negligible role in airway physiology. These data define both molecules as important regulators of Th2-driven lung pathophysiology.
Differential regulation of IDO activity in human dendritic cells through ligation of different c-type lectins: implications for allergic sensitization (P6055)
Authors: Ghaem Maghami Amir , Salazar Lizama Fabian , Skahib Farouk
Published: January 2023

Indoleamine 2,3-dioxygenase (IDO), a key enzyme in tryptophan metabolism, plays a critical role in regulating immune responses. IDO has been shown to have a protective effect in different models of experimental allergy and asthma however its role in human allergy has remained elusive. Recent work by our group showed that Der p 1 (a major house dust mite allergen), through engaging mannose receptor (MR) on dendritic cells (DCs), could modulate IDO activity. This in turn was shown to bias immune responses towards a Th2 phenotype. Here we studied the relationship between two major ‘allergen receptors’ on DCs namely MR and DC-SIGN in regulating IDO activity in order to further define the role of these receptors in controlling allergen induced Th2 immune responses. Our data indicates that IDO expression and activity in human DCs can be differentially regulated by engagement of MR and DC-SIGN. Intriguingly, we have shown that simultaneous activation of DCs with LPS (a known inducer of IDO activity) and either MR or DC-SIGN ligands has opposing effects on IDO activity in DCs with the MR ligation causing a decrease in IDO activity whilst DC-SIGN engagement causing an increase. Such opposing effect is likely due to differential activation of a number of downstream signalling molecules. Better understanding of these pathways could shed light on a range of host-pathogen interactions involving these key receptors including those occurring in the context of allergic sensitisation.
Phenotypical characterization of Treg cells in ocular allergy patients. (P6059)
Authors: Galicia-Carreón Jorge , Palomares-Ordóñez Jose , Hong Enrique , Pérez-Tapia Sonia , Jiménez-Martínez María
Published: January 2023

Purpose: To evaluate frequency of T regulatory cells (Tregs) and homing receptor expression in patients with allergic conjunctivitis (AC). Methods: Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors (HD) and AC-patients and labeled with mAbs against CD4, CD25, FOXP3, CCR4, CCR5, CCR7, CCR9, CD103 and CD108 before and after antigen stimulation with Dermatophagoides pteronyssinus (Der p). All cells were analyzed by flow cytometry and culture supernatants were recovered to analyze IL-10 and TGF-β. Results. AC-patients showed 55-times more CD4+ CD25+ cells than HD. Most of CD4+ CD25+ cells were FOXP3-, also these cells were CCR4+ and CCR9+ cells. After culture, no changes were detected in frequency of CD4+ CD25+ FOXP3+, nor FOXP3- T cells. Interestingly, we observed increased expression of CD103 and CD108 on T cells and IL-10 only in AC-patients. Conclusions. Circulating Tregs were decreased in allergic conjunctivitis patients, and these cells were expressing homing receptors associated to mucosa migration; after specific stimuli, these cells were able to express CD103 and CD108 on its surface; taken together these results suggest that in allergic conjunctivitis patients these cells could be migrating to conjunctival mucosa associated-lymphoid tissue remaining a phenotype related to Tr1 cells.
The leucine zipper transcription factor BATF induces IL-10 and IL-3 in allergic asthma (P6061)
Authors: Sopel Nina , Übel Caroline , Hildner Kai , Finotto Susetta
Published: January 2023

The transcription factor BATF is essentially involved in the differentiation of Th17 and follicular T helper cells. In B cells BATF influences the class-switch recombination of immunoglobulins. In an experimental model of allergic asthma we previously observed that BATF-/- mice do not develop asthma. We found that airway inflammation, hyperresponsiveness and eosinophilia were decreased, concomitant with lower serum IgE levels and reduced Th2 and Th17 cytokine secretion in the lung. Here we show an inhibition of IL-10 secretion in in vitro differentiated Th17 cells lacking BATF, while the expression of IRF4 mRNA was not altered. We confirmed a defect of IL-10 secretion in supernatants of total lung cells isolated from BATF-/- mice sensitized and challenged with OVA. Furthermore, IL-3, an important growth factor for the survival of eosinophils, basophils and mast cells, was also reduced in the supernatants of lung CD4+ T cells isolated from BATF-/- mice. Consistently, in BATF-/- mice treated with OVA, the number of lung mast cells expressing the IL-3 receptor α-chain was decreased. This defect was also confirmed in bone marrow derived mast cells. Taken together, BATF seems to play an important role not only in Th17 and Tfh differentiation but also in mast cell development. Therefore, local inhibition of BATF expression in the lung might be a feasible strategy for treating airway inflammation seen in allergic asthma.
Glucocorticoid induces pentraxin 3 expression through mitogen-activated protein kinase pathway in human airway smooth muscle cells (P6013)
Authors: Zhang Jingbo , Koussih Latifa , Shan Lianyu , Soussi Gounni Abdelilah
Published: January 2023

Long pentraxin 3 (PTX3) is a multifunctional soluble pattern recognition receptor that links innate immunity, inflammation, and matrix deposition. Glucocorticoids (GC) are an important anti-inflammatory treatment in asthma. Previous studies have demonstrated that GC had divergent effects on PTX3 production in diverse cell types. In this study, we first evaluated whether GC alone induced and, under inflammatory conditions, enhanced and extended PTX3 production in Human airway smooth muscle cells (HASMC) and investigated the plausible molecule mechanism regulating the induction of PTX3. HASMC transfected with PTX3 promoter constructs were treated with dexamethasone (Dex) or and TNF. We demonstrated Dex induced and synergized with TNF to augment PTX3 protein levels. However, Dex does not influence the mRNA levels and promoter activity of PTX3 alone, but it decreased the effect of TNF-induced PTX3 promoter activity. Further results had shown that Dex increased TNF-induced PTX3 mRNA stability. Primary HASMC pretreated with inhibitors of p42/p44 ERK, but not p38 or JNK-MAPK, significant decrease in PTX3 release on Dex- and TNF-treatment. Taken together, our data provide the first evidence of Dex-induced PTX3 expression in HASMC maybe occurred posttranscriptional mechanisms and via p42/p44-MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in airway inflammation via PTX3-dependent pathway.
Interferon α/β receptor signaling plays a critical role in regulatory T cell development and function (P1093)
Authors: Metidji Amina , Punkosdy George , Shevach Ethan
Published: January 2023

Type I IFNs are a family of cytokines with both antiviral and immunomodulatory properties. While the antiviral effects are appreciated, the immunomodulatory properties are less clear. Given the immunomodulatory properties of Treg, we have considered a possible role for IFNα/βR signaling in Treg development/function. Treg from WT and IFNα/βR deficient (-/-) mice were equal in number and in vitro/in vivo suppressive activity. Therefore, we set up a model to test IFNα/βR-/- Treg fitness in a competitive environment using bone marrow chimeras reconstituted with a 1:1 mixture of WT and IFNα/βR-/- cells. While the WT to IFNα/βR-/- ratio of all T cells was significantly skewed toward WT in these mice, skewing was most pronounced in the Treg compartment. The disadvantage of the IFNα/βR-/- Treg in the competitive environment was likely due to a defect in development and/or survival, as the ratio of IFNα/βR-/- Treg was markedly decreased in the thymus, and peripheral IFNα/βR-/- Treg had a significantly higher frequency of active caspase positive cells. The fitness of IFNα/βR-/- Treg was further examined in a disease model by testing their capacity to rescue the scurfy disease phenotype. IFNα/βR-/- Treg in chimeric mice reconstituted with scurfy and IFNα/βR-/- bone morrow were unable to control effector T cell activation and tissue inflammation. Collectively, these data demonstrate a critical role for IFNα/βR signaling in the development and/or survival of Treg.
CCR7 coordinates paracrine interleukin-2 cross-talk between central memory and regulatory T cells and controls the homeostatic balance between regulatory T cell subsets (P1065)
Authors: Smigiel Kate , Richards Elizabeth , Thomas Kerri , Campbell Dan
Published: January 2023

The importance of IL-2 in immune suppression mediated by Foxp3+ Treg cells is well established. However, IL-2 is not required for Treg cell development, in vitro suppression, or to maintain total cell number; thus, the mechanism by which IL-2 controls Treg cell activity remains unclear. We demonstrate that CD44lo CD62Lhi lymphoid tissue-resident Treg cells require IL-2 for survival and maintenance, whereas CD44hi CD62Llo Treg cells with a peripheral tissue homing phenotype are IL-2 independent. We termed these subsets central (cTr) and effector Treg (eTr) cells, respectively, due to their unique homeostatic requirements and migratory potential. Furthermore, we show that the chemokine receptor CCR7 provides cTr cells access to IL-2 by coordinating their localization in proximity to IL-2-producing CCR7+ Foxp3- central memory cells in the T cell zones of secondary lymphoid tissues. Collectively, our data demonstrate that the factors governing the homeostasis and maintenance of different Treg cell subsets are highly dependent on their tissue and microenvironment localization and indicate that loss of immune tolerance in the absence of IL-2 or CCR7 is due to an imbalance of cTr and eTr cells.
Role of immunological synapse and kinapse in peripheral tolerance. (P1105)
Authors: Beuneu Helene , Dustin Michael
Published: January 2023

In the lymph node, T cells can receive signals through the recognition of pMHC during both long-lived (>5 minutes) synapses or short-lived (<5 minutes) kinapses with antigen presenting cells (APC). It has been shown that injection of low dose antigenic peptide can lead to T cell tolerance and T regulatory cells induction in vivo. Here, we are evaluating the impact of MHC-peptide potency, peptide dose and APC frequency on induction of tolerance and Treg in relation to the dynamics of T cell-antigen presenting cell (APC) interactions. We hypothesize that induction of tolerance will be a function of signal integration by T cells and that the formation of synapses will favor tolerance induction when the frequency of APC is low based on the ability of rare APC to capture T cells with MHC-peptide potency and dose supporting synapse formation. In vitro, the high potency MHC-peptide has an advantage over the low potency MHC-peptide for Treg induction when APC frequency is low. In vivo, the low potency MHC-peptide induces Treg and tolerance through kinapses. We are now in the process of characterizing, in vivo, the tolerance and Treg induction in condition where the number of APC is limited using a mixed bone marrow chimera approach.
Transient alteration in cytokine patterns following influenza vaccination detected by Ki-67 expression and automated multiparameter analysis of flow cytometry. (P4311)
Authors: Mosmann Tim , Li Xi , Zand Martin , Wu Hulin , Weaver Jason , Qi Yilin
Published: January 2023

We previously showed that seasonal Trivalent Inactivated Influenza Vaccine (TIV) induces a strong, transient CD4 T cell response in healthy adults, measured at days 4-6 after vaccination as influenza-specific CD4 T cells expressing Ki-67. We have now examined the phenotype of the vaccine-responsive cells, using a whole blood assay, high-dimensional flow cytometry and automated population identification by the SWIFT algorithm, which quantifies responding populations, and helps reveal unexpected population changes. A strong Ki-67+ CD4 T cell response was detected in 10/10 subjects. At day 0, Influenza-specific CD4 memory cells mostly produced Interferon gamma, Tumor Necrosis Factor alpha and Interleukin 2 in response to in vitro restimulation. After 4-6 days, the Ki-67- influenza-specific T cells expressed a similar cytokine pattern as before vaccination, whereas the Ki-67+ cells produced more Interferon gamma but much less Tumor Necrosis Factor alpha and/or IL-2. Our interpretation is that influenza-specific CD4 T cells are recruited to lymph nodes, proliferate and express Ki-67, then return to the circulation at about 4 days, still expressing Ki-67. The dominant Interferon gamma single-positive phenotype in Ki-67+ population may be a transient state of the normal multiple cytokine producers, or a transient effector population that dies, or rapidly leaves the circulation to reside in tissues.
Neonatal immunization with respiratory syncytial virus glycoprotein fragment induces protective immunity in the presence of maternal antibodies in mice (P4277)
Authors: Kim Jae-Ouk , Noh Youran , Shim Byoung-Shik , Cheon In Su , Rho Semi , Kim Heejoo , Kang Chang-Yuil , Chang Jun , Song Man Ki
Published: January 2023

Respiratory syncytial virus (RSV) is the major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of an RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies affects the responsiveness and protection efficacy of Gcf in newborn mice, since infants can have RSV-specific maternal antibodies since adults are frequently reinfected with RSV. Intranasal administration of Gcf induced antibody response and IFNγ secretion and protected mice against RSV challenge without lung eosinophilia even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period.
A proteome-wide comparison of human antibody responses to the standard polyclonal vaccine for smallpox prevention and the monoclonal replacement ACAM2000 (P4321)
Authors: Ulrich Robert , Pugh Christine , Keasey Sarah , Pittman Phillip
Published: January 2023

Smallpox was eradicated in the 1980s due to the success of vaccines such as Dryvax (Wyeth Laboratories, Inc., U.S.). However, production of an improved live-virus vaccine was needed due to the limited amount of Dryvax doses available, as well as manufacturing concerns over the sterility and the clonal heterogeneity of the vaccine. Clones isolated from Dryvax produce variable numbers of antigens important for protective immunity or contributing to reactogenicity. In 2008, ACAM2000 (Acambis, Inc™), a single-plaque-purified vaccinia virus derivative of Dryvax, replaced the first-generation, polyclonal smallpox vaccine. We examined human responses to Dryvax and ACAM2000 by using a protein microarray comprised of approximately 90% of the poxvirus proteome (245 proteins) to measure protein-specific IgG in sera of 71 individuals responding to a single vaccination. We observed robust antibody responses to 21 poxvirus proteins in recipients of either product (p-value ≤0.0002), while other protein antigens could be used to distinguish vaccination with Dryvax from antibody responses to the monoclonal vaccine.
Immunosuppressive effects of early-life exposure to combustion-derived particulate matter: implications for enhanced severity due to respiratory tract infections (P3218)
Authors: Saravia Jordy , You Dahui , Lee Greg , Cormier Stephania
Published: January 2023

The association of elevated levels of ambient particulate matter with increased morbidity and mortality due to respiratory tract infections has been well documented. Interestingly, infants and young children appear to be the most vulnerable to these adversities. Using mice to model early-life exposure to combustion-derived particulate matter (CDPM), we demonstrate the development of an immunosuppressive environment in the lung as evidenced by increases in tolerogenic dendritic cells, regulatory T cells (Tregs), and IL-10 production and impairment of T effector proliferation in response to antigen. Specifically, infant mice co-exposed to CDPM and house dust mite allergen failed to develop certain hallmarks of asthma (e.g. airway hyperresponsiveness, eosinophilia, Th2 inflammation) while maintaining others (e.g. increased mucus production, elevated serum IgE). To investigate the effects of CDPM-induced immunosuppression on viral infections, exposed neonatal mice were infected with influenza (H1N1). CDPM-exposed and infected mice had significantly reduced pulmonary cytotoxic/helper T cell effector responses (Tc1, Tc2, Tc17/Th1, Th2; respectively) and significantly elevated Tregs compared to unexposed and infected mice. Consequently, this impairment in effector responses led to increased viral loads in the lungs of CDPM-exposed mice. Our data suggest a mechanism of exposure-mediated modulation of pulmonary disease in infants.
Distinct LLO-specific CD4 T cells infiltrate the intestinal mucosa following oral Listeria monocytogenes infection. (P3224)
Authors: Fu Han-Hsuan , Sheridan Brian , Pham Quynh-Mai , Schubert Wolf-Dieter , Freitag Nancy , Lefrançois Leo
Published: January 2023

Listeria monocytogenes (LM) is an enteroinvasive pathogen and the cause of listeriosis, a highly fatal foodborne infection. Using an oral LM infection model, we show that a subset of blood-borne listeriolysin O (LLO)-specific CD4 T cells transiently expressed the α4β7 integrin early after infection. Antigen-specific CD4 T cells rapidly accumulated in the mucosa and mounted a robust recall response following secondary infection. On day 9 post LM infection, LLO-specific CD4 T cells in the mucosa are almost exclusively Ly6Clow/CD27low and this population persists throughout memory. In contrast, LLO-specific CD4 T cells isolated from the spleen and MLN were heterogenous for CD27 and Ly6C expression. Mucosal LLO-specific CD27lowLy6ClowCD4 T cells produced IFN-γ and IL-2 and a small subset also produced IL-17. Interestingly, IL-17 production increased in mucosal memory CD4 T cells. Furthermore, IL-17 production was increased in mucosal CD4 T cells in CD69-deficient mice. However, mixed bone marrow chimera studies indicated that this phenomenon is independent ofCD69 expression on responding CD4 T cells. Depletion of CD4 T cells during memory led to higher bacterial burden in the gut and spleen. These results indicate that mucosal effector and memory CD4 T cells are phenotypically and functionally distinct from their splenic counterparts. Thus, our findings demonstrate an important role for mucosal memory CD4 T cells in protection against oral infection.
Osteopontin receptor expression on immune effector cells in neonatal RSV infection (P3232)
Authors: Durre Kainath , Qureshi Nagib , Gevorkian Natalie , Qureshi Mahboob
Published: January 2023

Respiratory Syncitial Virus (RSV)-induced bronchiolitis-pneumonia during early infancy predisposes asthma in later life. Augmented Th2 response and skewed polarization of Dendritic cells (DC) towards plasmacytoid DC (pDC) are associated. In addition, Osteopontin (OPN) serves as an immunomodulator, which is relatively deficient in RSV- infected neonatal lungs. Exogenous OPN inhibits skewed pDC polarization in RSV- infected neonatal mice. In the present study, we examined the expression of OPN receptors (CD44 and CD51/61) by flowcytometry on pDC, myeloid DC (mDC), T and B cells in response to RSV infection in the presence and absence of NKT cells using wild type (WT) and NKT cell KO (NKT-/-) mouse models. Neonates and adults are shown to exhibit NKT and NK dominant responses, respectively, in RSV infections. Expression of CD44 and CD51/61 decreased on mDC, pDC, T and B cells in RSV infected WT pups compared to the uninfected pups particularly early on infection (day 2 and 4 post-infection). On the other hand, expression OPN receptors on DC, T and B cells were increased in the RSV-infected NKT-/- pups compared to the infected WT and uninfected NKT-/- pups. This augmented expression was abolished by day 10 post-infection. Increased expression of OPN receptors in absence of NKT cells signifies their inhibitory role. Further studies examining the roles of NKT cells in OPN treatment would help understand the underlying mechanisms for developing new therapeutic strategies.
IL-21 is required for maintaining mucosal Th1 and Th17 responses during Helicobacter pylori infection (P3184)
Authors: Carbo Adria , Hontecillas Raquel , Washington M. Kay , Chaturvedi Rupesh , Olivares-Villagómez Danyvid , Wilson Keith T. , Bassaganya-Riera Josep , Algood Holly M.
Published: January 2023

CD4+ T cells play a major role in the modulation of immune responses at the gastric mucosa during Helicobacter pylori infection. The relative contributions of CD4+ T cell subsets to gastritis and control of H. pylori colonization and clearance are not completely understood. Expression of IL-21, which is produced by a number of T cell subsets, increases during H. pylori infection. To elucidate the mechanisms of action of IL-21 in effector and regulatory CD4+ T cells during H. pylori infection, we combined computational modeling of CD4+ T cell differentiation and in vivo challenge studies in mice. Our computational modeling predicted activated production of IFNγ and IL-17 by IL-21, and the expression of T-bet, RORγt and phosphorylation of STAT3. To investigate the role of IL-21 in vivo, we infected IL-21-/- and wild-type mice with H. pylori SS1, and assessed colonization, gastric inflammation, cellular infiltration and cytokine profiles in the gastric mucosa. During chronic infection, IL-21-/- mice had higher H. pylori colonization, less gastritis and reduced expression of inflammatory cytokines and chemokines. Moreover, H. pylori infected IL-21-/- mice had reduced levels of CD4+ T cell specific RORγt and T-bet when compared to infected wild-type mice. Our results indicate that IL-21 contributes to the control of infection and severity of gastritis, suggesting a role for induction of gastritis in response to H. pylori through activation of both Th17 and Th1 effector responses.
Inflammasome activation in Histoplasma stimulated-dendritic cells (P3071)
Authors: Tzu-Hsuan Chang , Betty A Wu-Hsieh
Published: January 2023

Histoplasma capsulatum is an opportunistic dimorphic fungal pathogen. Dendritic cells take up Histoplasma yeasts and present antigens to activate both CD4 and CD8 T cells. Therefore, the interaction of dendritic cells with the fungus is critical to host defense against Histoplasma infection. Here, we investigated the mechanism of inflammasome activation in dendritic cells after infection with Histoplasma. Interaction of bone marrow-derived dendritic cells with Histoplasma activated caspase-1 and induced the secretion of IL-1β. Using blocking antibodies we also found that Dectin-2 regulated pro-IL-β gene transcription and caspase-1 activation after Histoplasma infection in dendritic cells. Histoplasma interaction with Dectin-2 triggered both the first (transcription of pro-IL-1β) and the second (activation of caspase-1) signals in response to Histoplasma stimulation. Cells with NLRP3-deficiency failed to activate caspase-1 and to process pro-IL-1β, showing that NLRP3 is an important pattern recognition receptor in the cytosol to control inflammasome activation by Histoplasma. Thus, our results demonstrated that both Dectin-2 and NLRP3 play important roles in inflammasome activation after Histoplasma infection.
IgM recognition of glycan antigens is impaired by IRAK-4/MyD88 deficiency (P3077)
Authors: Maglione Paul , Bossuyt Xavier , Meyts Isabelle , Picard Capucine , Cunningham-Rundles Charlotte
Published: January 2023

About 75% of patients with IRAK-4 or MyD88 deficiency develop overwhelming infections caused by S. pneumonia or S. aureus in early childhood. While IgG production is not compromised, TLR signaling mediated by IRAK-4 and MyD88 has broad effects upon the immune system, including stimulation of T-independent IgM antibody. In fact, it has recently been shown that IRAK-4/MyD88 deficiency reduces a putative reservoir of IgM production, circulating CD27+, IgD+, IgM+ B cells. Using an array of 610 glycans covering diverse structures (functionalglycomics.org), we measured serum IgM binding glycans from 8 age-matched control, 8 IRAK-4-deficient, and 3 MyD88-deficient patients. Serum IgM from these patients bound a significantly reduced number of glycans relative to controls (p < 0.05). Using a microbial glycan database (csdb.glycoscience.ru) we found that control IgM bound 18 glycans expressed by S. pneumonia or S. aureus, while IgM from IRAK-4/MyD88-deficient subjects bound only 1. The reduced recognition of glycans by IgM in IRAK-4/MyD88-deficient patients was supported by ELISA, with significant reduction in IgM binding S. pneumonia capsular glycans (p = 0.004), cell wall glycan (p <0.05), and phosphorylcholine (p <0.05), as well as S. aureus teichoic acid (p < 0.0001). These results indicate that IRAK-4/MyD88 deficiency impairs IgM recognition of glycans expressed by S. pneumonia and S. aureus, providing an explanation for heightened susceptibility to these encapsulated pathogens.
Whole Blood Assay detects endotoxin and non-endotoxin pyrogens (P3098)
Authors: Vysyaraju Kranthi , Rivera-Mariani Felix , Negherbon Jesse , Hogberg Helena , Hartung Thomas
Published: January 2023

Pyrogenic contaminations may precipitate adverse effects that range from fever to death. There is limited surveillance on non-endotoxin pyrogens and a sensitive technique to detect them is essential. Cells of the immune system come in contact with pyrogens; this causes the release of mediators that induce fever. The Whole Blood Assay (WBA) exploits this human physiological response; human whole blood is incubated with test samples, and interleukin-1 beta (IL-1β) induced in the presence of pyrogenic contamination is measured by ELISA. In this study, we demonstrate the ability of WBA to detect non-endotoxin pyrogens. Human whole blood was incubated with different doses of the following non-endotoxin pyrogenic stimuli: lipoteichoic acid (gram positive bacteria cell wall), Pam2CSK4, FSL-1 (bacterial diacylated lipoproteins), zymosan (fungal [1, 3] - beta glucan) and fungal spores. Levels of induced IL-1β were compared with endotoxin stimulated blood and unstimulated blood as positive and negative controls respectively. All tested stimuli showed pro-inflammatory response compared to the negative control (p < 0.05) confirming that WBA effectively detects non-endotoxin pyrogens. In conclusion, WBA is a sensitive and valuable assay relevant to the human response to a broad range of pyrogenic stimuli. It can also be easily adapted for application in quality assessment of biotherapeutics and immunotoxicology.
Bioinformatic meta-analysis identifies a new role for macrophage E2F transcription factors during interaction with intracellular pathogens. (P3102)
Authors: Gregory David , Kramnik Igor , Kobzik Lester
Published: January 2023

We sought to identify the portion of the macrophage genome that is involved - for good or ill - in the macrophage’s interactions with intracellular pathogens. We therefore analysed publically available gene expression datasets for macrophages infected with Mycobacterium tuberculosis, Listeria monocytogenes, Francisella tularensis, Bacillus anthracis, Yersinia spp., Brucella melitensis, or Leishmania ssp.. To reflect the diversity of interactions and contexts that may influence the importance of different genes, we included data from a variety of human and mouse primary cells and lines, along with experiments comparing resistant to susceptible genetic backgrounds or the effect of IFNγ. We combined these diverse datasets to identify genes that were significantly regulated in multiple contexts. We define this group of ~3500 genes as the MacPathome: the subset of the genome particularly relevant to macrophages and their intracellular pathogens. A search for enriched regulatory motifs in the promoters of these genes found, in addition to known immune regulators, an unexpected over-representation of binding sites for the E2F transcription factor family. Initial validation by qPCR showed inhibition of E2F expression following infection of macrophages with various intracellular bacteria. An inhibitor of E2F activity (HLM006474) decreased bacterial load. These data indicate a novel role for E2F family members in the macrophage response to intracellular pathogens.
Acute cytokine responses of human monocytes to Toxoplasma gondii (P3104)
Authors: Bagriacik Emin , Hasgur Suheyla , Yavuz Emine , Atak Aysegul , Delibasi Tuncay
Published: January 2023

Toxoplasma gondii is an intracellular protozoan parasite. The parasite is capable of penetrating to human monocytes. Some studies showed immune responses to Toxoplasma gondii infection. However, acute cytokine responses of monocytes from sero negative individuls to the parasite are still elusive. Therefore, we studied cytokine responses of enriched human monocytes to tachyzoites in culture medium as an in vitro model of acute Toxoplasma gondii infection. Human monocytes were stimulated by tachyzoites in culture medium. Cytokine productions were determined by ELISA. Viability of cells was determined to find whether the monocytes were alive and functioning following the parasite infection. Infected monocyte viability was more than 98% after 18 hours indicating that they were functional cytokine secreting cells. Infection by the parasite increased proinflammatory cytokines such as IL-6 and TNFa significantly. Similarly IL-17 production increased after the infection. Anti-inflammatory cytokines such as IL-10 and TGFb productions were altered significantly as well. These results indicated that cytokines released by infected monocytes might contribute regulatory mechanisms of T cell activations during acute infections of Toxoplasma gondii.
CD40 induces anti-T. gondii activity in non-hematopoietic cells via the autophagy pathway (P3050)
Authors: Muniz-Feliciano Luis , Van Grol Jennifer , Subauste Carlos
Published: January 2023

Toxoplasma gondii infects both hematopoietic and non-hematopoietic cells and can cause cerebral and ocular toxoplasmosis. Host protection likely acts at both cellular levels to control the parasite. CD40 is key for protection against cerebral and ocular toxoplasmosis. We determined if CD40 induces anti- T. gondii activity at the level of non-hematopoietic cells. Engagement of CD40 on various endothelial cells including human brain microvascular endothelial cells, human umbilical vein endothelial cells, a mouse endothelial cell line, as well as human and mouse retinal pigmented epithelial cells resulted in killing of T. gondii. CD40 stimulation increased expression of the autophagy proteins Beclin 1 and LC3 II, enhanced autophagy flux and led to recruitment of LC3 around the parasites. The late endosomal/lysosomal marker LAMP-1 accumulated around the parasite in CD40 stimulated cells. This was accompanied by killing of T. gondii dependent on lysosomal enzymes. Accumulation of LAMP-1 and killing of T. gondii were dependent on the autophagy proteins Beclin 1 and Atg7. Together, these studies revealed that CD40 induces toxoplasmacidal activity in various non-hematopoietic cells dependent on proteins of the autophagy machinery.
Dual role of type I IFN during plasmodium infection (P3056)
Authors: Chan Jennie , Liehl Peter , DeOliveira Rosane , Sharma Shruti , Golenbock Douglas , Mota Maria , Fitzgerald Katherine
Published: January 2023

The molecular mechanisms regulating the inflammatory response during malaria are still poorly defined. Inflammatory cytokines and type I IFNs induced when innate immune sensors recognize components of the malaria parasite can contribute to clearance of the parasite and in some circumstances these same effectors lead to experimental cerebral malaria (ECM). Infection of C57BL/6 mice with Plasmodium berghei ANKA (PbA) leads to ECM where animals succumb to infection and die. Upon infection with PbA infected red blood cells (iRBCs), C57/Bl6 mice succumb to death 6-12 days post-infection. Recently, we have found that mice lacking the type I IFN receptor, IFNAR-/-, are protected from ECM-mediated death, implicating an important role of type I IFNs in exacerbating the ECM phenotype. Alternatively, when mice are infected with liver-tropic PbA sporozoites, a type I IFN response is induced while the parasites develop inside hepatocytes. This host response is responsible for upregulating interferon stimulated genes (ISGs) and limit the parasite load in the liver. The expression of ISGs are abrogated in IFNAR-/- mice. This protective phenotype is dependent on IRF3/7 and the adaptor MAVS suggesting that parasite RNA is recognized by host cells. Collectively, these findings reveal a dual role of type I IFNs that contribute to ECM-mediated death during PbA blood stage infections, but are also responsible for reducing parasite load during a liver stage infection.
Medullublastoma expresses CD1d and can be targeted for immunotherapy with NKT cells (P4453)
Authors: Liu Daofeng , Song Liping , Brawley Vita , Robison Nathan , Wei Jie , Gao Xiuhua , Ahmed Nabil , Asgharzadeh Shahab , Metelitsa Leonid
Published: January 2023

Medulloblastoma (MB) is the most common brain tumor of childhood. Current therapies are toxic and often not curative that necessitates development of targeted immunotherapy. However, little is known about immunobiology of this tumor. In this study, we show that MB cells in 9 of 20 primary tumors express CD1d, an antigen-presenting molecule for Natural Killer T cells (NKTs). Quantitative RT-PCR analysis of 61 primary tumors revealed a high level of CD1d mRNA expression in a molecular subgroup characterized by overactivation of Sonic Hedgehog (SHH) oncogene. CD1d-positive MB cells cross-presented glycolipid antigens to activate NKT-cell cytotoxicity. Intracranial injection of human NKTs resulted in regression of orthotopic MB xenografts in NOD/SCID mice. Importantly, the numbers and functional properties of peripheral blood type-I NKTs were preserved in MB patients. Therefore, CD1d is expressed on tumor cells in a subset of MB patients and represents a novel target for immunotherapy of this brain tumor.
A novel CEA-based cancer vaccine that blocks tumor implantation and tumor foci formation in vivo (P4459)
Authors: Gariepy Jean , Mallick Amirul , Huang Eric , Abdul-Wahid Aws
Published: January 2023

Metastatic forms of cancer account for most cancer-related deaths. Essentially all patients with advanced metastatic cancer are given palliative care in the absence of any useful curative strategies. Since tumour foci formation and expansion are directly linked to cell-cell adhesion events, we have developed a human carcinoembryonic antigen (CEA)-based subunit vaccine using only an altered-self form of its IgV like N domain responsible for the homotypic adhesion of CEA-expressing cancer cells. We now report that simply vaccinating CEA-expressing transgenic mice intramuscularly with this subunit and MF59 (squalene) engenders anti-adhesive as well as cytocidal (CDC and ADCC) antibodies in these transgenic animals that effectively block the subsequent lodging and formation of tumour foci at sites such as the peritoneal cavity and kill CEA-expressing murine tumour cells. This vaccine formulation may represent a simple, safe, preventive strategy against the long-term establishment of metastases in patients diagnosed with CEA-expressing colon, lungs, ovarian and breast cancer.
CD4 T cells control the induction, magnitude, and phenotype of the primary CD8 T cell response following adenovirus vector immunization (P4465)
Authors: Provine Nicholas , Penaloza-MacMaster Pablo , Larocca Rafael , Borducchi Erica , McNally Anna , Parenteau Lily , Barouch Dan
Published: January 2023

Despite the widespread use of replication-incompetent recombinant Adenovirus (Ad) vectors as candidate vaccine platforms for the elicitation of CD8 T cell responses, the mechanism by which these vectors elicit a CD8 T cell response remains poorly understood. Our data demonstrate that the induction of a primary CD8 T cell response by Ad immunization is dependent on CD4 T cell help. CD4 and MHC class II knockout mice had substantial defects in the primary CD8 T cell response following Ad vaccination. Furthermore, The CD8 T cell response generated in the absence of CD4 T cells exhibited a less effector-like phenotype, as assessed by surface staining, cytokine production, and expression of transcription factors. The unhelped CD8 T cell population also exhibited increased apoptosis and increased PD-1 expression. Intriguingly, longitudinal removal of the CD4 T cell population by monoclonal antibody depletion demonstrated that CD4 T cell help is required for at least eight days post-immunization to facilitate normal expansion and effector differentiation of the responding CD8 T cell population. Collectively, these data suggest that CD4 T cell help is needed not only at the time of initial antigen exposure but also during the first week of induction of CD8 T cell responses. Future experiments will aim to identify the mechanism(s) by which CD4 T cell help is provided in this system.
Neutrophil Gelatinase Associated Lipocalin (NGAL) regulates the onset of serum autoantibodies in pristane induced lupus (P4059)
Authors: Pawar Rahul , Goilav Beatrice , Xia Yumin , Zuang Haoyang , Reeves Westley , Putterman Chaim
Published: January 2023

NGAL (lipocalin-2), a member of the lipocalin superfamily, is expressed by neutrophils, hepatocytes, and renal resident cells following inflammatory conditions, and is involved in innate immunity during infection. Previously, we found that NGAL worsens renal disease in nephrotoxic nephritis. However, its relevance to adaptive immunity has not been fully explored. To investigate a possible role for NGAL in autoreactive antibody responses in experimental lupus, we injected pristane into B6 (n=10) and NGAL-deficient B6 (NGAL-/-) (n=10) mice. Analyzing the levels of serum autoantibodies at 3 months post injection, we found that B6 NGAL-/- mice had significant increases in IgG2a and IgG2b anti-double stranded DNA antibodies. Similarly, the titers of IgG2a and IgG2b antibodies to single stranded DNA and histone were significantly increased in pristane challenged NGAL-/- mice as compared to NGAL sufficient mice. Furthermore, elevated serum anti-ribonucleoprotein antibodies were found in pristane treated NGAL-/- mice by immunoprecipitation. Although autoantibody titers in NGAL-/- mice were not increased at baseline, our results are consistent with the apoptotic defects and accumulation of lymphoid cells reported previously in these mice. We also found significant upregulation of IRF5, IP-10, CXCL13, CXCR5, CxCR3, PD-1, PD-L1, and activation induced cytidine deaminase (AID) in spleens of NGAL-/- mice compared to WT mice. In vitro, we found that pristane and other inflammatory mediators promoted NGAL secretion by splenocytes. In conclusion, NGAL deficiency accelerates the onset of anti-nuclear antibodies in pristane induced lupus, suggesting that NGAL may regulate the spontaneous or induced humoral autoimmunity.
Early events in immunologic tolerance to apoptotic cells are dependent on marginal zone macrophage CCL22 production. (P4081)
Authors: Ravishankar Buvana , Chandler Philip , McGaha Tracy
Published: January 2023

Apoptotic cells (ACs) promote immunologic tolerance but the early innate mechanism(s) involved in the process are not known. Here we report that administration of ACs i/v induced rapid splenic expression of the regulatory T cell chemokine CCL22 by CD169+ marginal zone macrophages (MZMs). Similarly, in-vitro culture with ACs lead to expression of CCL22 in purified MZMs, but not CD11c+ dendritic cells (DCs) and conditioned media from MZM cocultures induced migration of Tregs in a CCL22 dependent manner. Administration of a soluble antagonist for the CCL22 receptor (i.e. CCR4) skewed the early innate immune response to ACs in-vivo shifting the balance from regulatory (IL-10, TGF-β) to inflammatory (TNF-α, IL-6, IL-12) cytokine production in splenic DCs and macrophages. Similarly, CCR4 blockade inhibited splenic accumulation of Tregs after apoptotic cell injection i/v and enhanced effector T cell responses to AC-associated antigens. Short-term CCR4 inhibition at the time of i/v apoptotic cell challenge (i.e. blockade only at the time of apoptotic cell administration) reversed AC-mediated tolerance to skin allografts in both primary recipients and in Treg adoptive transfers to secondary recipients instead promoting more rapid rejection. Suggesting a primary role for CCL22 in AC tolerance. Thus, we show for the first time that selective induction of CCL22 by MZMs is an essential early innate step in the generation of infectious immune tolerance to apoptotic self.
Interferon alpha deregulates endothelial nitric oxide synthase (P4085)
Authors: Buie Joy , Oates James
Published: January 2023

Background: Recent in vitro studies suggest that Type I interferons promote endothelial progenitor cell dysfunction and apoptosis while others have shown that Type I interferon gene signature correlates with increased endothelial dysfunction in systemic lupus erythematosus patients. Although causes of ED are likely multifactorial, all pathways converge on the diminished activity of endothelial nitric oxide synthase. We examined the effects of IFN-alpha on eNOS gene expression, phosphorylation, and NO production. Methods: Changes in eNOS expression in response to IFN-a (100, 1000 IU) were quantified by real-time PCR. Functional changes in response to vascular endothelial growth factor were assessed by immunoblot. We also evaluated the effect of serum from patients that induce a type I IFN signature from 5 patients and 2 controls on eNOS expression and VEGF mediated function. Gene expression with PCR and phosphorylated serine 1177 eNOS expression with immunoblot were assessed . Type I IFN neutralization studies (IFN-a Ab, IFNAR Ab) were also performed and eNOS expression was assessed. Results: HUVECs treated with IFN-a at 100IU and 1000IU exhibited a significant reduction in eNOS gene expression, VEGF induced phosphorylation at the serine 1177 site after 24 hours, and NO production. SLE serum also caused a reduction in eNOS gene and phosphorylated eNOS protein expression. Moreover, addition of neutralizing antibodies partially reversed observed affects on eNOS gene changes.
Determining the role of the Axl receptor tyrosine kinase in beta cell autoimmunity (P4089)
Authors: Manzoor Fatima , Kroger Charles , Wang Bo , Tisch Roland
Published: January 2023

Type I diabetes (T1D) is an autoimmune disorder characterized by the T cell mediated destruction of the insulin-producing beta cells. Mechanisms involving the breakdown of self-tolerance in T1D are ill defined. We have previously shown that T1D progression is inhibited in NOD mice lacking expression of Mer, a member of the TAM family of receptor tyrosine kinases. Mer is expressed by dendritic cells (DC) and is critical for mediating the tolerogenic effects of apoptotic cells (AC) on DC. Axl, another TAM receptor is co-expressed by DC and binds AC. The relative role of Mer and Axl in mediating the tolerogenic effects of AC on DC, and regulating beta cell autoimmunity are unknown. Accordingly, we assessed the effects of AC on bone marrow-derived and splenic NOD DC lacking Axl expression. Pretreatment with AC of DC showed that Axl was required for AC-mediated inhibition of DC activation and maturation. Axl-deficient DC failed to activate c-Src and STAT3, which are key signaling molecules needed for AC-induced inhibition of DC. Furthermore, incidence of T1D in NOD mice lacking Axl was significantly reduced. Collectively, these findings indicate that Axl contributes to AC-induced inhibition of DC, and has a role in regulating T1D progression.