bluuebunny/crossref_metadata_2025 huggingface dataset:
EFFECTS OF B CELL COX-2/PGE2 ON ACTIVATION-INDUCED CYTOSINE DEAMINASE (AID) (35.4)
Authors: Mongini Patricia K. A.
Published: January 2023

The rate-limiting enzyme for prostaglandin E2 (PGE2) production, i.e. COX-2, was recently found to be expressed and functional within human B cells. In B2 cell cultures activated by limiting BCR:CD21 ligand and IL-4, delayed signals from BAFF and APRIL heightened COX-2 expression and ensuing PGE2 production from replicating blasts (Mongini et al., 2006, J. Immunol. 176:6736). The present study has begun to evaluate whether BAFF/APRIL-promoted COX-2/PGE2 contributes to quantitative/qualitative changes in human B cell expression of activation-induced cytosine deaminase (AID). This possibility was suggested from observations of others that

BAFF and APRIL upregulate AID and promote isotype switching andexogenous PGE2 promotes the heightened production of IgG and IgE in murine B cell cultures stimulated by LPS and IL-4.

Present studies with CFSE-labeled human B2 cells activated by BCR:CD21L, IL-4, and BAFF/APRIL indicate that by day 6 of culture, replicating blasts express significantly elevated levels of AID protein, while activated but non-replicating cells do not. AID levels were highest in daughter cells that had undergone the most divisions. While exogenously added PGE2 increased the level of AID detected within replicating blasts, COX-2 function was not essential for AID expression since COX-2 inhibitors did not block AID upregulation. Whether PGE2 may modulate the phosphorylation (activation) status of the expressed AID is being investigated. In sites of chronic inflammation, BAFF/APRIL-upregulated PGE2 in activated B cells might contribute to isotype switching and somatic hypermutation in the Ig locus, and potentially, lymphomagenic mutations in oncogenes.
The three different promoters regulate the expression of mouse 4-1BB (35.48)
Authors: Kim Jung D , Kim Chang H , Kim Young H , Kwon Byoung S
Published: January 2023

4-1BB(CD137) is a member of the tumor necrosis factor receptor superfamily and is expressed on avtivated T cells, monocytes and natural killer (NK) cells. The expression of 4-1BB has been known to be activation dependent except regulatory T cells. Interestingly, we found that 4-1BB has three promoter regions which could regulate 4-1BB mRNA level. Thus, we investigated expression level of three different transcripts of 4-1BB in naïve T cells and EL4 cell lines. It was reported that 4-1BB expresson was increased by several anti-cancer drugs in human peripheral blood mononuclear cells and CEM cells. We also test whether the treatment with anti-cancer drugs could induce 4-1BB expression in naïve T cells and EL4 cells. The data indicated that 4-1BB expression increased by agents such as mitomycin C, 5-FU, and doxorubicin. Especially, type II transcript of 4-1BB was preferentially induced in naïve T cells stimulated by anti-CD3 because of NFkB binding site in type II promoter region. It suggests that type II promoter region is major promoter which could be activated in naïve T cells by TCR signaling. We also found a splicing variant deleted exon 7 including transmembrane domain region could inhibit the 4-1BB signaling. Our studies demonstrated that 4-1BB has three promoter could induce the expression of 4-1BB mRNA but the type II promoter is a inducible promoter in naïve T cell and costimulatory effect through 4-1BB receptor could be modulated by soluble 4-1BB produced by splicing variant without transmembrane domain induced in activation of naïve T cells.
Alleles and isoforms of human membrane-bound IgA1 (35.50)
Authors: Hung Alfur Fu-Hsin , Chen Juin-Bo , Chang Tse Wen
Published: January 2023

In humans, IgA has two subclasses, IgA1 and IgA2, which contain α1 and α2 heavy chains, respectively. For both subclasses, there are also membrane-bound forms, mIgA1, and mIgA2, containing mα1 and mα2 heavy chains. Mα1 and mα2 differ from α1 and α2 by having a “membrane-anchor” peptide segment extending from the CH3 domain of α1 and α2. The membrane-anchor segment has three parts: an extracellular, a trans-membrane, and an intracellular segments. It was found previously that mα1 exists in a short and a long isoforms, referred to as mα1(I) and mα1(II), with the latter containing extra 6 amino acid residues, GSCSVA (residues 453–458), at the N-terminal of the extracellular segment. By studying the genomic and mRNA sequences of mα1 and mα2 from 30 individuals residing in Taiwan, we have found that, in addition to mα1(456S) allele, mα1 has a previously unknown allele, referred to as mα1(456C), which is present in the donor population at about the same proportion as mα1(456S). Moreover, mα1(456C) allele exists in the long isoform only and does not yield a short isoform corresponding to mα1(I). Furthermore, we have clarified that mα2 exist as short isoforms only. Future studies will examine the variations in mIgA1 in other races and the possible association of such variations with the regulation of IgA synthesis.

This research is supported by a grant, #94-2320-B007-004, from the National Science Council, Taiwan.
Regulation of Antigen Presentation by Amyloid Precursor-Like Protein 2 (36.1)
Authors: Tuli Amit , Sharma Mahak , Menezes Miriam Rose , Naslavsky Naava , Caplan Steve , McIlhaney Mary M , Solheim Joyce C.
Published: January 2023

The amyloid precursor-like protein 2 (APLP2) is a secreted protein that is ubiquitously expressed. We have found that APLP2 over-expression decreases the quantity of H2-Kd molecules on the plasma membrane. In addition, Kd thermostability was improved by APLP2 down-regulation, suggesting that APLP2 affects Kd peptide selection and/or retention. The effects of APLP2 on Kd are likely related to their physical association, as pulse-chase experiments demonstrated interaction for up to two hours after Kd synthesis. Furthermore, significant co-localization of Kd molecules with APLP2 was observed in vesicular structures both at steady state and during endocytosis of Kd molecules from the cell surface. Mutagenesis studies revealed that APLP2/MHC association is influenced by sequences in the peptide-binding region and the α3 domain of the MHC class I heavy chain. β2m has an impact on APLP2/MHC interaction, since introduction of human β2m into mouse fibroblasts increased the level of interaction between H2-Dd and APLP2. In summary, APLP2 affects MHC class I cell surface expression and thermostability by an interaction controlled by more than one domain of the MHC class I heavy chain and by β2m. These data support a model in which APLP2 regulates MHC class I trafficking, and thereby antigen presentation. (NIH Grants AI54645, GM57428, and GM74876)
Multiple round of stimulation renders B cells capable to drive type 2 helper T (TH2) cell response (36.13)
Authors: Nam Sang-Yun , Do Jeong-Su , Seo Hyo-Jung , Hwang Jin-Ki , Kim Jun-Hee
Published: January 2023

Using hybrid mice of HEL-specific BCR Tg mice and OVA-specific TCR Tg mice, we investigated whether activated B cells may modulate T cell differentiation in addition to antigen presentation. Analysis of cytokine secretion has revealed that primarily stimulated (1°) B cells are more potent APC than naive B cells. However, second (2°) and further round of stimulation renders B cells capable to strongly drive polarization of naive CD4 T cells toward TH2 response, and concomitantly inhibitory to TH1 response. In the presence of 2° B cells as APC, activation of STAT-4 and T-bet was selectively inhibited during Ag-specific priming of T cells, whereas STAT-6 and GATA-3 were promoted, compared to 1° B cells. Altered MHC expression on 2° B cells was uncoupled with TH2 polarization, because they similarly work in nonspecific CD3 stimulation of T cells. Second round of stimulation regulates expression of costimulatory molecules on B cells, but not positively any TH2-related ones. Rather, 2° B cells were more likely to drive toward TH2 response via soluble mediators. 2° B cells produced much lower level of IFNγ and TNFα, but markedly higher levels of TH2 cytokines including IL-4 and IL-10, which largely contributed to 2° B-mediated TH2 polarization. This study suggests a possible therapeutic application of activated B cells against diseases caused by inappropriate TH1/2 polarization.
IL-12-producing CD11c+NK1.1-DC predominate in the liver during protective immunity induced with radiation-attenuated Plasmodium berghei sporozoites (36.15)
Authors: Jobe ousman.jobe , Donofrio Gina , Schwenk Robert , Williams Jackie , Krzych Urszula
Published: January 2023

Immunizations with radiation-attenuated Plasmodium berghei (γPb) sporozoites (spz) culminate in protection against wild-type Pbspz challenge. Immune protection is considered to be largely mediated by Ag-specific, IFNγ-producing liver CD8+T cells; however the role of APC in the induction of CD8+T cells remain unexplored. In vivo deletion of DC results in loss of protection against spz challenge. Therefore, in this study we investigated the possible involvement of liver DC in the induction of liver CD8+TEM cells. γPbspz immunization induced CD11c+CD8α+DC which represented ~75% of the hepatic CD11c+NK1.1-DC. Coincidental with the appearance of CD11c+CD8α+DC, was a 7-fold increase in liver CD8+TEM cells. Adoptive transfer of γPbspz-immune liver CD11c+NK1.1-DC to naïve mice reduced parasite burden (4-fold) after challenge and the recipient mice survived 14 days beyond the infectivity controls. CD11c+CD8α+DC from γPbspz-immune mice produced IL-12 and activated naive CD8+T cells to exhibit a TEM phenotype in vitro. Thus, we hypothesize that IL-12-producing hepatic CD11c+CD8α+DC induced by γPbspz activate liver CD8+T cells that are the key cellular component of protective immunity to liver-stage parasites.

Supported by NIH NIAID, FNIH and the USAMC.
Antigen encapsulated in biodegradable nanospheres are cross-presented in a proteosome-independent, TAP-dependent pathway (36.2)
Authors: Young-Hee Lee , Trumunkh Gerelchuluun , Young-Ran Lee , Hyunjin Kim , Sun-A Im , Chong-Kil Lee
Published: January 2023

Various mechanisms have been proposed to explain cross-presentation such as the classical cytosolic, TAP-dependent MHC-I processing pathway, the noncytosolic alternate MHC-I pathway, and the fusion of the endoplasmic reticulum (ER) with the endosome/phagosome. Here we show that antigens encapsulated in biodegradable nanospheres are cross-presented in a cytosolic pathway that does not require proteosome. Nanospheres containing OVA were prepared with poly(DL-lactide-co-glycolide). OVA containing in the nanospheres were efficiently captured, processed and presented on MHC-I molecules by DCs. MHC-I presentation of OVA-nanospheres was resistant to lactacystin, a proteosome inhibitor, and was sensitive to brefeldin A, an inhibitor of anterograde endoplasmic reticulum-Golgi transport. Chloroquine, which inhibits phagolysomal enzymes by increasing phagolysomal pH, inhibited MHC-I-restricted presentation of OVA. MHC-I presentation of OVA-nanospheres was greatly reduced in DCs generated from TAP−/− mice. These results demonstrate that antigens encapsulated in the biodegradable nanospheres are cross-presented by a mechanism involving phagolysosomal degradation, subsequent release of the degraded peptides into the cytosol, and transport of the peptides from the cytosol into ER via TAP.
Human skin migratory dendritic cells that do not secrete IL-12p70 stimulate both Th17 and Th1 allogeneic responses (36.20)
Authors: Mathers Alicia R , Morelli Adrian E , Janelsins Brian , Rubin Joseph P , Tkacheva Olga A , Falo Louis D , Larregina Adriana T
Published: January 2023

We analyzed the ability of human skin migratory dendritic cells (smiDC) to stimulate allogeneic (allo)-CD4+ T helper cell responses and to induce differentiation of Th1 and Th17 cells. SmiDCs obtained from cultures of human skin explants consisted of Langerhans cells and dermal DCs, which synthesized IL-8, IL-15, IL-23, IL-10 and TGF-β1, but not IL-12p70, even after exposure to DC1-driving stimuli. Regardless of the lack of IL-12p70 smiLC and DDC induced differentiation of effector memory Th cells that expressed CD45RO, the skin homing molecule CLA, and decreased CD62L and CCR7. SmiDCs stimulated proliferation of allo-CD4+ T cells that differentiated into Th1 and Th17 cells during 5 day-MLCs. T cell proliferation depended on the expression of MHC-II, costimulatory and adhesion molecules by smiDCs. The Th1 biasing function of smiDCs depended on the production of IL-23. The Th17 response was supported by the proinflammatory cytokines IL-8 and IL-15 and was significantly increased by the blockade of IL-6, whereas the blockade of IL-23 did not affect the secretion of IL-17. Levels of IL-17 and IFN-γ secretion reached their maximum at day 4 and 5, respectively. IFN-γ (Th1) and IL-17 (Th17) was produced by two distinct Th cell subsets as demonstrated by flow cytometry. Therefore, human smiDCs support the induction and coexistence of Th1 and Th17 effector T cells by mechanisms distinct from those described in the mouse model.
A novel Lactococcus lactis antigen delivery system induces activation and maturation of neonatal dendritic cells and T cell stimulation. (36.22)
Authors: Ditamo Yanina , van Roosmalen Maarten L , Leenhouts Kees , Ramirez Karina , Pasetti Marcela F
Published: January 2023

Neonatal immune responses to vaccine antigens are usually short lived and Th-2 biased. This has been attributed, in part, to the immaturity of neonatal dendritic cells (DC) which lack full capacity for antigen presentation and T cell stimulation. Effective priming of the neonatal immune system can be achieved when antigens are delivered in the presence of adequate immunostimulatory signals. We investigated the capacity of a novel L. lactis-derived delivery system, designated Gram-positive Enhancer Matrix (GEM) particles, to stimulate neonatal immune cell populations. This system had been shown to enhance immunity to protein antigens when given mucosally in adult mice. To assess their capacity to activate the neonatal immune system, DC and macrophages derived from newborn mice were cultured in the presence of GEM particles. Adult-derived DC were included as controls. We showed that GEM particles induced upregulation of activation and maturation markers (CD80, CD86, CD40, MHCII) in neonatal DC and macrophages. Similar experiments were performed with human DC; GEM particle treatment also increased expression of activation and maturation markers of human cord blood- as well as adult-derived DC and increased allogeneic human T cell stimulation. These results indicate that GEM particles are proficient stimulators of neonatal immune cell populations, and likely to enhance in vivo immune responses in neonates.
Evaluation of function vs. purity of mouse bone marrow derived dendritic cells by analyzing FACS and ELISA profiles of CD11c-selected and non-selected populations (36.26)
Authors: Chou Ping-Jen , Newton Cathy , Perkins Izabella , Friedman Herman , Klein Thomas
Published: January 2023

Bone marrow derived dendritic cells are APCs and serve as “immunological sensors” that detect microbial invasion. The hallmark of BMDC purity is >90% positive for the CD11c molecular marker and this level of purity can be achieved by the use of costly cell sorters. A confound to the methods of isolation and purification is that growing BMDC in GM-CSF enriched culture medium can increase the CD11c expression <75%. In this study, BMDC were harvested and enriched by incubation for 9 days with GM-CSF medium followed by CD11c IMag™ Magnetic Particle selection. The selected and non-selected cells were analyzed for percentage expression of CD11c. The DCs were then treated with effective doses of anthrax toxins (200ng/ml PA with LF and EF at 25ng/ml each) for 5 hours prior to Legionella pneumophila infection for 18-hour incubation. The culture supernatants, examined by ELISA, showed that the proinflammatory cytokine response following various treatments, namely IL-12, IL-6, IL-1β, and TNF-α, was essentially the same between the selected and non-selected populations. In addition, flow data showed an increase in CD11c expression as DCs become mature upon stimulation, suggesting this marker is inducible. Overall, our data show that BMDC populations of >65% and >90% CD11c positive have a similar cytokine response following stimulation and can be generated by a much more cost effective technique.

Supported by NIAID grant AI45169.
Evaluation of Adjuvant Regimens for Antibody Development in Rabbits (36.29)
Authors: Hawksworth David , Tyner Joan
Published: January 2023

Several adjuvant regimens were assessed for their ability to safely develop a high titer, high affinity antibody response in rabbits. Female, New Zealand White (NZW) rabbits were given five monthly 20 ug immunizations with a 25 KDa recombinant protein using one of four adjuvant programs:

Adjulite® Complete and Adjulite® Incomplete Freund’s adjuvants,Alhydrogel® Aluminum hydroxide gel adjuvant in combination with oligodeoxynucleotides containing unmethylated CpG dinucleotides (Alum/CpG),Difco Freund’s adjuvant alternating with MPL+TDM+CWS orDifco Freund’s adjuvant alternating with Quil A supplemented with CpG-ODN.

Sera samples taken following all booster immunizations were evaluated for antibody titer and relative affinity in a microtiter enzyme immunoassay by testing for antibody reactivity to limiting amounts of the administered antigen. Animals were also monitored during the immunization period for any reaction at the sites of injection. Results show that the Adjulite® animals elicited a higher average titer compared to animals immunized using any of the alternate strategies. Additionally, Adjulite® rabbits developed an average relative antibody affinity similar to that for the Alum/CpG animals and higher than either of the groups employing Difco Freund’s adjuvant. One Adjulite® rabbit developed a temporary nodule at the site of injection compared to none of the Alum/CpG animals. Two of the five animals using Difco Freund’s adjuvant developed a nodule, both of which progressed to lesions. This study demonstrates that Adjulite® adjuvant can serve as a safe alternative to other adjuvant regimens for the development of a high titer, high affinity antibody response in rabbits.
Oxidant production by APCs in response to glycoantigens (36.9)
Authors: Lewis Colleen J. , Cobb Brian A.
Published: January 2023

Nitric oxide (NO) and reactive oxygen species (ROS) are generated during the oxidative burst and are thought to have redundant bactericidal functions. However, zwitterionic polysaccharide antigens (glycoantigens), such as polysaccharide A from the capsule of Bacteroides fragilis, are processed within APCs in a NO-dependent fashion leading to T cell activation and abscess formation. That observation suggests differential functions for ROS and NO in APCs. In order to understand these roles, the oxidative response to glycoantigens in APCs has been measured. We found that murine and human macrophage lines produce NO over a 48-hour period in an antigen dose-dependent manner, and this production can be blocked with the NO synthase inhibitor L-NAME. The macrophages also produce ROS, which can be ablated with apocynin, an inhibitor of superoxide production. Interestingly, maximal ROS production occurs earlier (T½ = 15.8 hrs) but requires a higher antigen concentration (24.8 μg) than NO production (T½ = 24.5 hrs; 14.6 μg). In addition, B cells produce small amounts of ROS and no detectable NO, despite their ability to process glycoantigens, revealing a NO-independent processing mechanism. These data suggest that NO and ROS are controlled independently and could serve different processing roles in macrophages and B cells during T cell responses to glycoantigens.

This work was supported by a grant to B.A. Cobb (AI062707).
Identification of a novel subset of activation induced deaminase (AID)-dependent B-1 cells that mediate Initiation of Contact Sensitivity (37.10)
Authors: Kerfoot Steven M , Szczepanik Marian , Tung James , Herzenberg Leonore A , Askenase Philip W
Published: January 2023

Contact sensitivity (CS) to haptens is related to delayed type hypersensitivity (DTH) and is a well characterized prototype of T cell mediated inflammation. CS is additionally dependent on a parallel immune pathway. In response to sensitization, a population of B-1 cells produces antigen-specific IgM which mediates a rapid inflammatory response to hapten re-exposure. Interference with this pathway prevents the full development of the classic delayed response and is therefore termed the “CS-Initiation” pathway. The identity of the B-1 cell subset responsible for CS-Initiation is not known. Here, we show that CS-Initiation is dependent on activation induced deaminase (AID), an enzyme central to the process of somatic hypermutation (SHM). Using adoptive transfer experiments, we demonstrate that the defect is specific to B-1 cells as transfer of wild type B-1 cells reconstitutes CS-Initiation in deficient recipients. We went on to identify and characterize a unique subpopulation of B-1 cells in the spleens of sensitized mice that possess initiation activity. Detailed analysis of B cell receptor genes isolated from these cells revealed evidence of AID-mediated SHM. The sensitivity of CS-Initiation to very low amounts of hapten and the dependence on AID suggests that these cells are selected to generate antibodies capable of mediating CS-Initiation.

This work funded by the Canadian Institutes of Health Research, the American Academy of Allergy, Asthma and Immunology and the National Institutes of Health.
Effect of staphylococcal enterotoxin B antigen on atopic dermatitis-like lesion induced by contact allergen in hairless mice (37.13)
Authors: Lee Jong Kwon , Ju Jung Hun , Kim Jinho , Kim Jung Rae , Chun Eun Young , Kim In Young , Sohn Kyung Hee , Kim Seung Hee , Park Kui Lea
Published: January 2023

Atopic dermatitis (AD) is a common pruritic disease and its incidence is increasing in infancy and childhood. Patients with AD have reported cutaneous to both environmental and superantigen-producing strains of Staphylococcal aureus. In this study, we investigated the effect of staphyloccoal enterotoxin B (SEB) antigen on atopic dermatitis-like lesion induced by dinitrochlorobenzene (DNCB) in hairless mice. We treated the DNCB or DNCB+SEB topically on the skin of hairless mice. Repeated epicutaneous sensitization with DNCB or DNCB+SEB induced AD-like skin lesions including erythema followed edema, excoriation and scaling. The histologic features of the skin site in mice treated with DNCB or DNCB+SEB showed epidermal hyperplasia with hyperkeratosis, severe infiltration of eosinophils and increase of mast cells in dermis. The DNCB+SEB-sensitized skin site showed slightly increase in infiltration of eosinophils in dermis compared with DNCB-sensitized skin sites. The serum level of IgE in DNCB+SEB group showed slightly increase compared to the DNCB group. The serum level of IgG2a in DNCB+SEB group showed significantly increase compared to the DNCB. The serum level of IgG1 and IgG2b in DNCB+SEB group showed slightly increase compared to the DNCB. These results suggest that topical exposure to SEB slightly provokes AD-like lesion and hypersensitivity response induced by DNCB in mice.
Shift from IL-3- to IgE-induced IL-4 production as basophils become a predominant source of IL-4 (37.15)
Authors: Yang Junqi , Khodoun Marat , Orekov Tatyana , Mohrs Katja , Potter Crystal , Mohrs Markus , Finkelman Fred D.
Published: January 2023

We compared CD4+ T cell and basophil contributions to in vivo IL-4 production and evaluated stimuli that induce basophil IL-4 production during immune responses to a protein Ag, goat anti-mouse IgD antiserum (GaMD), and a nematode parasite. IL-4 production by basophils preceded CD4+ T cells, but CD4+ T cells produced most IL-4 secreted during the primary response to GAMD and the secondary response to Ag challenge 4–5 d after priming. In contrast, basophils produced most of the IL-4 secreted in mice challenged 12–14 d after priming. Basophil IL-4 secretion was IL-3-dependent but influenced by IgE during the primary response and IL-3- and CD4+ T cell-dependent/IgE-independent during the d 4–5 secondary response, but was IgE-dependent and IL-3/CD4+ T cell-independent during the d 12–14 secondary response. Basophil IL-4 secretion during a primary infection with Trichinella spiralis was localized to the liver and was partially IL-3- and IgE-independent. Thus, IL-3-dependent basophil IL-4 production precedes and supplements the primary T cell IL-4 response to a protein Ag; basophil IL-4 production can exceed T cell IL-4 production in animals challenged with Ag after basophils are primed by Ag-specific IgE; and helminthic parasites can stimulate basophil IL-4 secretion through a distinct mechanism.
CD8+ Tc1 and TIL-17 cells mediate allergic contact hypersensitivity by different mechanisms (37.18)
Authors: He Donggou , Wu Lizhi , Kim Hee Kyung , Li Hui , Elmets Craig A , Xu Hui
Published: January 2023

Allergen induced contact hypersensitivity(CHS) in the skin has been considered to be mediated by CD4+ Th1 and CD8+ Tc1 cells that produce IFN-γ. Our recent studies have demonstrated that CD8+ IL-17 producing T cells (TIL-17) play important roles in the elicitation of CHS. Allergen sensitization induces the development of CD8+ Tc1 and TIL-17 cells that are distinct T cell subpopulations. Here we demonstrate that both CD8+ Tc1 and TIL-17 cells play a role in the elicitation of CHS. However, they mediate inflammatory reactions in the skin by different mechanisms. CHS was reduced but not abolished in mice that were defect in the receptor for IFN-γ (IFN-γR−/−) or IL-17 (IL-17R−/−) compared to wild type controls. Similar results were observed in the adoptive transfer of CHS in the gene knockout mice that were transferred with allergen primed wild type CD8+ T cells. However, neutralization of IL-17 in IFN-γR−/− or IFN-γ in IL-17R−/− recipient mice abrogated the response. It implicates that both IFN-γ and IL-17 signals are involved in the elicitation of CHS and are required for optimal responses. Immunohistochemical analysis indicated that the infiltration of Gr-1+ granulocytes and CD11b+ macrophages in the allergen challenged skin of IL-17R−/− mice was significantly reduced whereas it was slightly affected in IFN-γR−/− mice. Further analysis using real time RT-PCR showed different patterns of chemokines and inflammatory cytokines in the allergen challenged skin of IFN-γR−/− or IL-17R−/− mice. These data indicate that both CD8+ Tc1 and TIL-17 cells take part in the elicitation of CHS and that specific mechanisms orchestrate Tc1 and TIL-17 mediated inflammatory reactions in the skin. The result of current studies may provide new insights into therapeutic strategies for T cell mediated inflammatory disorders.
THE ACETYLCHOLINESTERASE INHIBITOR MALATION IS NOT TOXIC TO THYMOCYTES, BUT MAY ALTER THE DEVELOPMENTAL PROGRAM AT LOW DOSES. (37.2)
Authors: Broussard Christine , Varney Jessica
Published: January 2023

The incidence of allergy and asthma among children has risen 150% in the last two decades in children under five years old, and environmental pollutants are suspected to be contributors to this rise in incidence. Environmental pollutants, such as pesticides, are known to have a negative impact on the environment as well as many living organisms. However, there are a limited number of studies that have investigated the impact pesticides have on the immune system of mammals. Futhermore, even fewer studies exist that examine the impact of environmental toxicants on the immune system of the developing embryo and young animal, though the immune systems of the young are more vulnerable to perturbation than that of adults. Therefore risk assessment studies addressing fetal immune development are important tools for better understanding whether pesticides and other environmental toxicants contribute to immune system diseases that are widely common today. We set up an in vitro model system to assess the risk of exposure to the pesticide Malathion. Malathion was chosen for the study because of its widespread use in agriculture and the known role it plays as an acetylcholinesterase inhibitor. We analyzed the response of day 16–18 embryonic thymocytes to varying concentrations of Malathion. We hypothesized that greater concentrations of the pesticide would have a greater toxicity in T cells. Flow cytometric analysis was used to determine the level of toxicity and maturation of the embryonic T cells following culture.

J.V. is a ULV undergraduate researcher.
Rgs13 inhibits IgE-mediated allergic responses (37.5)
Authors: Bansal Geetanjali , Xie Zhihui , Druey Kirk
Published: January 2023

Mast cells (MCs) evoke immediate hypersensitivity by degranulating in response to antigen (Ag) exposure and releasing preformed mediators such as histamine and leukotreines. Classically, the high affinity immune receptor for immunoglobulin E (FcεRI) mediates MC degranulation and anaphylaxis in response to antigen. In addition, various G protein coupled receptors (GPCRs) on MCs such as those for adenosine and sphingosine-1-phosphate (S1P) may also activate MCs. Since GPCR signaling is inhibited by Regulator of G protein Signaling (RGS) proteins, we studied the potential regulation of MC activation by RGS proteins. We identified Rgs1, 8, 13 and 18 in murine bone marrow-derived MCs (BMMCs). Rgs13−/− (KO) mice with LacZ knock-in were generated. Surprisingly, G protein-mediated degranulation induced by either adenosine or S1P was similar in WT and KO mice. In contrast, IgE/Ag -dependent degranulation and intracellular Ca++ flux as well as in vivo passive cutaneous anaphylaxis responses were significantly higher in KO BMMCs suggesting that Rgs13 affects IgE/Ag dependent pathway rather that GPCR-mediated pathway leading to MC activation. Reconstitution of KO BMMCs with WT Rgs13 inhibited degranulation to IgE/Ag. Analysis of effectors downstream of FcεRI revealed that Rgs13 interacts with PI3K and inhibits its activation in response to IgE/Ag. Thus, Rgs13 could represent a new therapeutic target for MC-dependent allergic responses.

Work was funded by DIR, NIAID/NIH.
The role of Rab27A in human eosinophil exocytosis (38.3)
Authors: Coughlin Jason J , Moqbel Redwan
Published: January 2023

Eosinophils play an effector role in airway damage in asthma and related disorders, since their secreted granule-stored products can induce many of the clinical features of asthma. Degranulation of eosinophils requires the activity of several GTP-binding proteins, many of which remain unidentified. We hypothesized that Rab27A is one of these GTP-binding proteins because, in other cells, it interacts with effectors that regulate vesicle motility, docking and fusion. Humans with Rab27A-deficiency develop Griscelli syndrome which is characterized by immunodeficiency and partial albinism due to secretory defects in various cell types. We have established that Rab27A is expressed in human peripheral blood eosinophils as determined by RT-PCR and Western blotting. To asses Rab27A activation we developed a novel assay allowing the pulldown of the active, GTP-bound form. Rab27A was activated in several leukocyte populations upon cell stimulation with degranulatory stimuli. In human eosinophils, Rab27A was transiently activated by secretory-IgA stimulation. Rab27A activation followed a temporal pattern similar to that of degranulation, suggesting that Rab27A activation induces secretory granule movement and exocytosis. A clearer understanding of granulocyte exocytosis may contribute to novel therapeutic strategies in asthma, allergic inflammation and related conditions.

Funding provided by the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research
Skewed polarization of pulmonary dendritic cells in RSV-infection and susceptibility to asthma (39.2)
Authors: Qureshi Mahboob Hossain , Durre Kainathk , Yang William
Published: January 2023

Respiratory syncytial virus (RSV) infection accounts for significant morbidity in young infants. The survivors become susceptible to develop asthma later in life. Studies have shown increased airway responsiveness in these individuals and a concomitant Th2 response in their lungs. However, the underlying mechanism (s) responsible for this pulmonary Th2 response in post- RSV infection period is not well understood. In this study, using a mouse model, we examined whether differentiation of lung dendritic cells (DCs) was skewed due to RSV infection and whether a Th2 cytokine response was associated. Neonatal mice were sensitized with intraperitoneal ovalabumin (ova) and infected with intranasal RSV followed by subsequent challenge with intranasal ova. Flowcytometric analysis of lung parenchymal cells determined the proportions of CD11bloCD11chiCD45R- myeloid DCs (mDCs) and CD11blo CD11chi CD45Rhi plasmacytoid DCs (pDCs). An increase in the numbers of pDCs was observed in the lungs of RSV-infected as well as RSV-infected and ova-exposed mice. To mention, pDCs are reported to downregulate Th1 responses. Consistent to this, augmentation of Th2 cytokine responses was observed in the RSV-infected and ova-exposed mice. Thus, polarization of lung DCs towards pDC due to RSV infection may modulate lung cytokine responses leading to susceptibility to asthma in later life. Reversal or inhibition of this skewed polarization may be considered as a clinical strategy in the post-RSV infection asthma. In this connection, we have reported that GMCSF and IL4 treatment expands lung dendritic cell population with enhanced Th1 responses.
Effects of Lactose-b-sitosterol And b-sitosterol On Ovalbumin–induced Asthmatic Mice (39.3)
Authors: Kim In Sik , Yuk Ji Eun , Yun Chi-Young , Lee Ji-Sook , Kim Dong-Hee , Kim Joo-Whan , Yang Eun Ju
Published: January 2023

Asthma is a chronic lung inflammation and patients suffering from it annually increases. Both β-sitosterol (BS) and β-sitosterol glucoside exist in a variety of plants. However, the precise role of BS and β-sitosterol glucoside in asthma has not been understood well. The aim of this study was to investigate the inhibitory effects of BS and lactose-BS (L-BS) on the pathophysiological process in asthma. For this purpose, BALB/c mice were immunized through i.p. injection with OVA and aluminum hydroxide on days 1 and 14. From days 21 to 27, the mice were exposed to 5% ovalbumin solution aerosolized for 1 hr per day. Total cells and eosinophils in BAL fluid markedly decreased after L-BS or BS treatment (p < 0.05) and ROS production also lessened as compared to asthma control. Both L-BS and BS strongly mitigated mucus hypersecretion by goblet hyperplasia and inflammation by eosinophil infiltration. These effects of L-BS were superior to those of BS. Both L-BS and BS inhibited the mRNA and protein expression of IL-4 and IL-5 in the lung tissue and BALF, respectively. Ovalbumin-specific IgE in BALF was uniquely inhibited by L-BS (p < 0.05). This finding provides the possibility of L-BS and BS as therapeutic drugs in asthma and it may contribute the need to improve current therapeutic drugs.

Acknowledgment This work was supported by RIC(R) grants from Traditional and BioMedical Research Center, Daejeon University (RRC04730, 2005) by ITEP.
Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma (39.7)
Authors: Hardy Charles Linton , Nguyen Hong-An , Yao Jun , De Kretser David M , Rolland Jennifer M , Anderson Gary P , Phillips David J , O’Hehir Robyn E
Published: January 2023

Background: Activin A is a member of the TGF-β superfamily implicated in asthmatic inflammation and tissue remodelling. In vitro, activin A bioactivity is neutralized by the soluble binding protein follistatin.

Objective: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization in acute and chronic allergic airway inflammation.

Methods: Ovalbumin-sensitized mice were subjected to acute or chronic allergen challenge protocols. Kinetics and distribution of activin A and follistatin were measured in lung tissue and BAL in relation to airway eosinophilia, Th2 cytokines, mucus production and collagen deposition.

Results: In both models peak BAL activin A and follistatin concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in draining lymph nodes; follistatin and activin A immunoreactivity were lost in airway epithelium in parallel with goblet cell metaplasia. In the chronic model inflammation preceded remodelling associated with elevated BAL activin A and TGF-β. Exogenous follistatin inhibited acute allergic airway inflammation.

Conclusion: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Follistatin has potential as a therapeutic to limit airway remodelling.
Differential accumulation of IgA ASC in sublingual vs. submaxillary salivary glands is directly correlated with the level of CCL28 expression. (40.4)
Authors: Law Yuetching , Hill Erica D , Distelhorst Kathryn , Wilson Eric
Published: January 2023

Optimal function of the immune system is highly dependent on efficient homing and accumulation of leukocytes. The homing of leukocytes has been shown to be dependent on interactions between leukocyte expressed chemokine receptors and adhesion molecules, and tissue expressed chemokines and adhesion molecule ligands. The homing and accumulation of IgA antibody secreting B cells (ASC) to mucosal tissues is thought to be highly dependent upon leukocyte expressed chemokine receptors CCR9 and CCR10 interacting with the chemokines CCL25 and CCL28 respectively. CCL28 has been shown to be involved in the homing of IgA ASC to the gastrointestinal tract and the lactating mammary gland, but has not been demonstrated to play a role, in vivo, in the accumulation of IgA ASC to other mucosal tissues. CCL28 has been shown to be expressed in mouse salivary glands. Here we show that CCL28 is expressed at higher levels in mouse sublingual salivary glands than in submaxillary salivary glands. This higher level of expression in the sublingual gland correlates directly with greater numbers of IgA ASC in the sublingual gland compared with the submaxillary gland. These results demonstrate differential accumulation of IgA ASC into mouse salivary glands and suggest that this difference in lymphocyte accumulation is at least in part the result of differential expression of CCL28.
MyD88-dependent regulation of the polymeric immunoglobulin receptor by colonic microbes (40.5)
Authors: Stefka Andrew T , Bruno Maria EC , Kaetzel Charlotte S
Published: January 2023

Secretory IgA serves as the first line of antigen-specific immunity in the intestine, protecting against pathogens while preventing inflammation in response to commensal microbes. Intestinal epithelial cells (IEC) regulate IgA secretion by controlling expression of the polymeric immunoglobulin receptor (pIgR). Many pro- and anti-inflammatory factors, including pIgR, are transcriptional targets of Toll-like receptor (TLR) signaling in response to molecular patterns from resident microbes. MyD88 is an important TLR signaling adaptor that has been implicated in regulation of intestinal inflammation. To assess the role of MyD88 in IEC gene expression, we compared mRNA levels of pIgR, TNF, and two regulatory factors, A20 and MKP-1, in the small intestines and colons of wild-type and MyD88−/− mice. Gene expression was significantly higher in the colon than in the small intestine of all mice, correlating with the higher microbial load. In the colon, expression of all genes was significantly higher in wild-type than in MyD88−/−mice. We conclude that commensal microorganisms set the tone of intestinal gene expression through MyD88-dependent TLR signaling.

Supported by Crohn’s & Colitis Foundation of America.
Beta7-integrin independent enhancement of mucosal and systemic anti-HIV antibody responses following combined mucosal and systemic gene delivery (41.1)
Authors: Vajdy Michael , Goodsell Amanda , Zhou Fengmin , Singh Manmohan , Malyala Padma , Kazzaz Jina , Greer Catherine , Megede Jan zur , Barnett Susan , O’Hagan Derek , Polo John
Published: January 2023

Vaccination strategies that can block or limit heterosexual HIV transmissions to local and systemic tissues are much sought for. Herein, in a mouse model, we aimed to determine whether the enhancement of antibody responses through mucosal and systemic immunizations, previously observed with protein-based vaccines, applies to immunizations with DNA- or RNA-based vectors. Intra-nasal (i.n.) followed by intra-muscular (i.m.) immunizations (i.n./i.m.) with PLG-DNA microparticles encoding HIV-gag (PLG-DNA-gag) significantly enhanced serum antibody responses, compared i.m., i.n. or i.m. followed by i.n. (i.m./i.n.), immunizations. Moreover, while i.n./i.m., i.n. or i.m./i.n. immunizations with PLG-DNA-gag resulted in genital tract antibody responses, i.m. immunizations alone failed to do so. Importantly, β7-deficient mice developed local and systemic antibody responses following i.n./i.m., or any other routes of immunization similar to wild type mice. To compare the DNA with an RNA delivery system, immunizations were performed with VEE/SIN-gag replicon particles, composed of Venezuelan Equine Encephalitis virus (VEE) replicon RNA and Sindbis surface structure (SIN). i.n./i.m., as compared to any other immunizations, with VEE/SIN-gag resulted in enhanced genital tract, but not serum antibody responses. These data show for the first time that mucosal followed by systemic immunizations with gene delivery systems enhance B cell responses independent of the mucosal homing receptors α4β7 or αEβ7.
Expression and subcellular targeting of a model subunit vaccine in transgenic soybean (41.13)
Authors: Oakes Judy Lynn , Garg Renu , Bost Kenneth L. , Piller Kenneth J.
Published: January 2023

Enterotoxigenic Escherichia coli (ETEC) strains are the leading cause of enteric diseases affecting humans and livestock. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases by delivering naturally encapsulated antigens to effecter sites lining the gastrointestinal tract and eliciting mucosal as well as systemic immunity. Plants, however, have not reached their full potential as a renewable source of oral vaccines, due in part to insufficient levels of recombinant protein accumulation. In an effort to increase the relative amount of subunit vaccine candidate in a practical host system, we targeted the agricultural antigen, FanC, to soybean chloroplasts. We monitored FanC mRNA expression and protein production, demonstrated proper chloroplast accumulation of the protein, and compared the amounts of this chloroplast targeted-protein to accumulation of cytosol-targeted FanC. Surprisingly, cytosol-targeted FanC accumulation was 5-fold greater than chloroplast-targeted FanC accumulation, despite similar FanC mRNA transcription levels in cytosol and chloroplast targeted events. Intraperitoneal vaccination of mice with the chloroplast-derived antigen induced an anti-FanC IgG immune response similar to that generated by vaccination with cytosol-derived and recombinant FanC.
Co-Expression of Yersinia pestis F1- and V-Ags by the Same Salmonella Vaccine Protects Against Nasal Y. pestis Challenge (41.14)
Authors: Trunkle Theresa , Yang Xinghong , Bosio Catherine M. , Pascual David W.
Published: January 2023

To test whether the protective antigens (Ags) to Yersinia pestis can be simultaneously delivered, the Y. pestis cafI operon encoding the F1 Ag, and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vector. F1-Ag expression was controlled under its endogenous promoter from the cafI operon and amply expressed; a chimeric promoter consisting of ptetS and pphoP in the pV55 plasmid showed enhanced V-Ag expression. Immunofluorescence data showed that the single construct, Salmonella-(F1-V)Ags, could co-express both Ags. When orally tested, the Salmonella-(F1-V)Ags vaccine elicited elevated Ab titers to both F1- and V-Ags. Supportive CD4+ T cell responses showed that IFN-γ was elevated in response to F1-Ag restimulation and to a lesser degree, IL-4 and IL-13. V-Ag restimulation showed much less IFN-γ and IL-4 production. IL-10 responses were equivalent to both Ags. IgG1 and IgG2a subclass responses to both Ags were equivalent. Upon nasal challenge, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1-V)Ags protected against 100 LD50 Y. pestis, but mice immunized with Salmonella constructs bearing a single Ag showed impaired efficacies. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect immune protection against Y. pestis.

This work is supported by AI-56286 and NIH 1U54 AI-06537.
Mucosal Immunity to Avian Influenza Following Ocular Immunization with Replication-Deficient Ad5-H5 in Chicken (41.15)
Authors: van GINKEL Frederik W. , Gulley Stephen L. , Toro Haroldo
Published: January 2023

Mucosal immunity plays an important role in the defense against viral pathogens. Limited information is available on the role of mucosal immunity in the chicken Harderian gland (HG) following ocular challenge. Although research has been performed on tears after ocular challenge or immunization of chickens, the mucosal immune response in the HG has not been well defined. To better understand mucosal immunity in HGs, we exposed chickens to a replication competent – free, recombinant adenovirus (Ad5) vaccine expressing the H5 gene of avian influenza (AI) virus. Expression of H5 in the HG after ocular administration of Ad5-H5 was demonstrated by immunohistochemistry. IgA and IgG H5- and Ad5-specific ELISPOT assays were developed to directly measure antibody-secreting B cells in HGs. The IgA and IgG spot-forming cells peaked on day 9 for Ad5 and day 11 for the H5 transgene. Ocular immunization induced Ad5- and H5-specific antibodies in the systemic compartment as measured by ELISA and HI assays, respectively. Increased levels of antibodies were also detected in tears. Western analyses demonstrated the presence of both monomeric and polymeric IgA in tears. RT-PCR analysis of HG derived mRNA confirmed the expression of the polymeric immunoglobulin receptor, confirming the importance of the HG as a mucosal effector site. Mucosal immunity was effectively induced by ocular application of the Ad5-expressed AI vaccine.
Intranasal immunization with Cry1Ac activates differentially lymphocytes in NALT as in NP. (41.16)
Authors: MORENO-FIERROS LETICIA , RODRIGUEZ-MONROY MARCO A.
Published: January 2023

Cry1Ac from B. thuringiensis is a potent mucosal immunogen and adjuvant. Via i.n. route Cry1Ac induces significant antibody responses and is able to improve vaccination against nasal invading pathogens, but their effects on nasal lymphocytes have not been determined. NALT and NP are considered as inductive and effector sites, respectively. We investigated the effect of i.n. immunization with Cry1Ac in the expression of CD69 and CD25 in lymphocytes of NALT and NP. Besides we analyzed effect in the proportion of T cells producing IL2, IL4, IL5, IL10, IFNγ and TNFα. Immunization with Cry1Ac increase considerably the expression of activation markers in both nasal tissues. But as expected for a mucosal effector site, NP contained major proportions of B220, T CD4 and T CD8 cells expressing CD25 and CD69 in comparison to NALT, Likewise, Cry1Ac increased the proportion of T cells spontaneously producing IL2, IFNγ, and IL4, and the effect was higher in NP than in NALT. Present study provides evidence that intranasal immunization with Cry1Ac activates differentially lymphocytes in NALT as in NP. These data support that Cry1Ac is a potent immunogen and a valuable study tool to the induction of nasal immune responses. The new information presented, is important, as it is necessary to have a thorough understanding of the unique molecular and cellular properties of the nasal immune system for the development of successful nasal vaccines

CONACyT 43102 and UNAM DGAPA PAPIIT IN213903 grants