bluuebunny/crossref_metadata_2025 huggingface dataset:
A rapid method for the isolation of Eosinophils without Ficoll or RBC Lysis (38.1)
Authors: Fairhurst Maureen A , Nauer Melany , Thomas Terry E
Published: January 2023

Eosinophils typically comprise 1–5% of blood leucocytes in non-allergic, healthy humans. Allergic and asthmatic reactions result in activation of eosinophils with resultant release of cytoplasmic granules, cytokines and lytic enzymes. Eosinophils are also mediators of responses against parasitic infections. Many protocols for their isolation stress the avoidance of ammonium chloride lysis as this interferes with eosinophil antigen processing, cytokine responses and alters cell morphology.

We describe a rapid and simple method for the enrichment of eosinophils from normal blood that does not require Ficoll or lysis steps and yields good purity and recovery. Blood was collected with heparin and RBC were removed by HetaSep sedimention. The eosinophils were then enriched using immunomagnetic, column-free negative selection (EasySep®). Briefly, cells were labeled with a cocktail of antibodies targeting unwanted cells. These were then coupled to magnetic nanoparticles and the sample was placed in a magnet. The labeled cells were thus removed leaving unlabeled eosinophils. The entire separation procedure can be automated with a pipetting robot (RoboSep®).

Purity of eosinophils assessed by cytospin and Wright’s stain was 98–99%. By flow cytometry (FACS) eosinophils were defined as CD45+CD66b+ and CD16 negative with low side scatter. Final purity assessed by FACS was 94 ± 3%. Recovery averaged 69 ± 21 (n=11).
Basophils function as initiators rather than effectors in IgE-mediated chronic allergic inflammation, independently of T cells and mast cells (38.6)
Authors: Karasuyama Hajime , Mukai Kaori , Obata Kazushige , Ishiwata Kenji , Watanabe Naohiro , Kawano Yohei , Minegishi Yoshiyuki
Published: January 2023

Basophils have often been considered as minor and possibly redundant “circulating mast cells”. We recently demonstrated that basophils play a pivotal role in the development of IgE-mediated chronic allergic inflammation (IgE-CAI), independently of T cells and mast cells. A single subcutaneous injection of multivalent antigens elicited not only immediate- and late-phase ear swelling but also delayed-onset ear swelling with massive eosinophil infiltration in mice sensitized with antigen-specific IgE. Here, we show that basophils function as initiators of the recruitment of eosinophils and neutrophils rather than effectors of inflammation in IgE-CAI. Even though basophils accounted for only 1–2% of the infiltrating cells in the skin legions, treatment of the IgE-sensitized mice with a newly-developed basophil-depleting mAb during the progression of IgE-CAI resulted in suppression of delayed-onset ear swelling with drastic reduction in numbers of not only basophils but also eosinophils and neutrophils infiltrating in the skin legions. Upon activation with IgE and corresponding antigens in vitro, basophils secreted soluble factors that stimulated skin adherent cells to produce soluble factors capable of attracting eosinophils and neutrophils. These findings indicate a novel mechanism of development of chronic allergic inflammation that is initiated and amplified by basophils.
Substance P stimulates human mast cells and together they activate T cells: Inhibition by luteolin (38.8)
Authors: Kempuraj Duraisamy , Iliopoulou Bettina P , Clemons Anthony , Boucher William , Tagen Michael , Vasiadi Magdalini , House Michael , Wolfberg Adam , Theoharides Theoharis C
Published: January 2023

Mast cells are important effector cells in IgE- and Th2-mediated immune responses. Mast cells are located close to substance P (SP) containing nerves. SP can activate connective tissue-type mast cells, but not mucosal mast cells. Here we examined the effect of SP on (a) mediator release from human umbilical cord blood-derived cultured mast cells (hCBMCs) grown in the presence of stem cell factor (SCF), IL-6 plus IL-4; (b) IL-2 from activated T cells alone or together with mast cells. SP dose-dependently released histamine, tryptase and IL-8 (at 10 μM, release was 72.8 ± 7.42%, 5390 ± 954 ng/106 cells and 1140 ± 103 pg/106cells, respectively) compared to controls (6.3 ± 2.4 %, 397 ± 235 ng/106 cells and 583 ± 40 pg/106 cells, respectively). Addition of mast cells to T cells, activated by anti-CD3/anti-CD28, for 24 h stimulated IL-2 production from 38.4 ± 9.2 pg/ml to 1450 ± 63 pg/ml (p<0.05). Addition of SP (10 μM) to mast cells further stimulated activated T cells to release 1747 ± 279 pg/ml (p<0.05) of IL-2. Pretreatment for 15 min with the flavonoid luteolin (10 μM) blocked (>90%) both SP-induced hCBMCs activation and T cell IL-2 release. Mast cells can superactivate T cells and could constitute a new therapeutic target for diseases such as multiple sclerosis. Luteolin could be used to inhibit this interaction.

This work was supported by Theta Biomedical Consulting and Development Co., Inc. (Brookline, MA).
Changes in syk expression in human basophils (HB) induced by a non-IgE-dependent receptor, LIR7 (38.9)
Authors: MacGlashan Donald , Arm Jonathan , Sloane David
Published: January 2023

Expression of syk in HB is highly variable among subjects and correlates with the ability of the cell to respond to FceRI aggregation. Because evidence suggests a post-translational influence on syk expression, we sought ways that syk could be down-regulated by the activation of non-IgE-dependent receptors. HB express LIR7 and aggregation of LIR7 induces mediator secretion kinetically similar to IgE-mediated release. These studies test whether aggregation of LIR7 reduces expression of syk and whether LIR7 operates through syk to induce histamine release (HR). HB were stimulated with mouse anti-LIR7 and anti-mouse Ab (the combination abbreviated to: aLIR7) and assessed for syk phosphorylation and for changes in syk expression. HR was examined ± the presence of a third generation syk inhibitor. aLIR7 induced syk phosphorylation and a modest loss of syk in 18 hour cultures (14±5% loss, n=11, p=0.013) compared to anti-IgE (53±10% loss). There was a correlation between the amount of syk loss induced by anti-IgE and the loss induced by aLIR7 (r=0.63). There was a correlation, r=0.97, between HR induced by anti-IgE and that induced by aLIR7 (means= 43%&25%). The IC50 for inhibition of aLIR7-induced HR was 0.2 μM (IC50 for anti-IgE was 0.038 μM). Each of these data sets is consistent with a dependence of LIR7 signaling on syk and shows that a non-IgE-dependent stimulus can alter syk expression levels.

Supported NIH-AI20253
SMOKING INHIBITS EXPRESSION OF PROINFLAMMATORY CYTOKINES AND ACTIVATION OF IRAK-1, p38 AND NF-κB IN ALVEOLAR MACROPHAGES STIMULATED WITH TLR2 AND TLR4 AGONISTS (40.6)
Authors: Chen Haiyan , Cowan Mark J , Hasday Jeffrey D , Vogel Stefanie N , Medvedev Andrei E
Published: January 2023

Bronchiolitis is common in smokers and we hypothesized that this may be due to impaired sensing of bacterial components via TLR2 and TLR4 by smokers’ alveolar macrophages (AM). AM and PBMC obtained from smokers and non-smoking volunteers were stimulated with TLR2 and TLR4 agonists, Pam3Cys and LPS, and expression of cytokines and activation of intracellular intermediates were examined. Smokers’ AM exhibited suppressed gene expression and secretion of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IFN-γ and chemokines IL-8 and RANTES upon stimulation with LPS and Pam3Cys, whereas expression of anti-inflammatory cytokines IL-10 and IL-1RA was not affected. Comparable expression levels of these cytokines and chemokines were detected in PBMC obtained from smokers and non-smokers, indicating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible phosphorylation of IRAK-1 and p38, and IκB-α degradation were markedly inhibited in smokers’ AM, whereas expression levels of TLR2, TLR4, CD14, MD-2 mRNA, and TLR4 protein, were not significantly changed. These results indicate that smoking induces a state of tolerance in AM manifested by suppression of TLR2/4-induced signaling that may underlie bronchiolitis associated with smoking.

This work was supported by grants from Philip Morris USA Inc. and Philip Morris International, OTRD/University of Maryland, Baltimore, and NIH grants AI059524 (AEM) and AI44936 (SNV).
Epithelial cells trigger frontline immunoglobulin class switching through an SLPI-regulated pathway (41.7)
Authors: Xu Weifeng , He Bing , Chiu April , Chadburn Amy , Shan Meimei , Buldys Malwina , Ding Aihao , Knowles Daniel M , Santini Paul A , Cerutti Andrea
Published: January 2023

Epithelial cells (ECs) recruit B cells expressing class-switched IgG and IgA into mucosal effector sites and transport IgG and IgA antibodies across mucous membranes. Whether ECs initiate IgG and IgA class switching remains unknown. We found that ECs lining tonsillar crypts formed pockets populated by B cells expressing activation-induced cytidine deaminase (AID), a DNA-editing enzyme associated with ongoing class switch DNA recombination (CSR). ECs released AID- and CSR-inducing factors, such as B cell-activating factor of the TNF family (BAFF), after sensing microbial products through Toll-like receptors (TLRs). EC-induced IgG and IgA CSR was further amplified by thymic stromal lymphopoietin (TSLP), an epithelial IL-7-like cytokine that enhanced BAFF production by dendritic cells. Conversely, EC-induced IgG and IgA CSR was restrained by secretory leukocyte protease inhibitor (SLPI), an epithelial homeostatic protein that inhibited BAFF signaling in B cells. Our findings indicate that ECs function as mucosal guardians that orchestrate frontline IgG and IgA class switching through a TLR-inducible SLPI-regulated signaling program.

This work was supported by NIH grants AI057653 (to A.C.) and by NIH T32 training grant AI07621 (to W. X.).
Activation of NKT cells by a-Galactosylceramide treatment licenses lamina propria dendritic cells to abrogate the oral tolerance (42.10)
Authors: kang Chang-Yuil , Chang Jae-Hoon , Lee Jung-Mi , Yoon Hyun-Jun , Lee Kyoo-A , Chung Yeonseok
Published: January 2023

Dendritic cells (DC) play critical roles in Ag presentation and induction of immunity or tolerance. In previous our study, treatment of agonistic anti-CD40 mAb at that time of oral OVA did not abolish the oral tolerance induction whereas injection of α-galactosylceramide (αGC) blocked the oral tolerance. We demonstrate that treatment of αGC but not anti-CD40 mAb resulted in the full maturation of DC in lamina propria of small intestine (SI-LP) at early time window. This phenomenon caused by activated NKT cells which reside in SI-LP. DC in SI-LP (sLP-DC) from αGC-treated mice exhibited a stronger stimulator against allogeneic T cells than those cells from anti-CD40 mAb-treated mice. Ag presentation capability was also increased in sLP-DC by αGC. Importantly, maturated sLP-DC by αGC induced to differentiate of naïve CD4+ T cells into Th1- and Th2 cells toward intestinal OVA. In contrast, sLP-DC by anti-CD40 mAb treatment failed to generate Th differentiation but were involved in the generation of more numbers of Foxp3+ CD4+ T cells than those from αGC-treated group. On the other hand, the fact that recognition of intestinal Ag of naïve CD4+ T cells occurs in MLN, indicating MLN are privileged in triggering the interactions of sLP-DC and CD4+ T cells. Taken together, systemic treatment of αGC resulted in maturation of sLP-DC by NKT cells in the SI-LP and licensed DC educate CD4+ T cells in MLN, which can abrogate oral tolerance.
Polymorphisms in Paan-AG promoter influences NF-κB binding and transcription activity in HEK293 cells (42.12)
Authors: Langat Daudi K , Morales Pedro J , Omwandho Charles O , Fazleabas Asgerally T , Hunt Joan S
Published: January 2023

HLA-G is a protein highly expressed at the human maternal-fetal interface during pregnancy. It is thought to be critical for the survival of the semi-allogenic fetus. The baboon (Papio anubis) expresses an HLA-G-like protein termed Paan-AG in the placenta, and may serve as a model for HLA-G studies. Paan-AG shares many characteristics with HLA-G, including alternative splicing of the mRNA and restricted tissue expression of the protein. Our hypothesis is that the two genes share similar regulatory mechanisms. The objective of the current study was to assess binding of the transcription factor NF-κB to Paan-AG κB elements and determine the effects of binding on Paan-AG promoter activity. We assessed two Paan-AG alleles each containing two κB elements, κB1 and κB2. NF-κB bound both κB1 and κB2 elements in the AG1 allele. In contrast, only κB1 of the AG-2 allele bound to NF-κB; κB2 did not bind. Mutagenesis studies showed that the difference in binding was due to two nucleotide differences in the 3′ end of κB1. The functional activity of the two alleles also differed; AG2 consistently showed higher luciferase activity compared to AG1. Mutating the last two nucleotides in the 3′ end of κB1 resulted in an increase of luciferase activity to levels comparable to that of AG2. Overall, these results suggest that variations in the proximal promoter may influence transcription rates of Paan-AG as reported recently for HLA-G, and provide further evidence of the potential usefulness of the baboon as a model for in vivo HLA-G studies.

Supported by NIH grant HD39878 (JSH).
Immunomodulation of activated immune cells in experimental autoimmune encephalomyelitis (EAE) (42.7)
Authors: Song Fei , Gienapp Ingrid , Shawler Todd , Whitacre Caroline C.
Published: January 2023

The relapse rate is profoundly decreased in female multiple sclerosis (MS) patients during pregnancy. We previously reported that experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is markedly suppressed when disease is induced during late pregnancy. Disease induction during the post partum period is enhanced. In this study, we assess the effects of pregnancy on activated T cells by transferring neuroantigen activated transgenic T cells into pregnant syngeneic wild type recipients (B10.PL). Myelin basic protein (MBP) TCR transgenic (Tg) donor splenocytes are activated in vitro with MBP NAc1-11 for 96 hours. These CD4+CD25+Foxp3- donor cells elicited a strong proliferative response and an increase in Th1 cytokines. The activated T cells were transferred i.v. into pregnant recipients and non-pregnant controls. Control mice showed acute and relapsing EAE, while pregnant recipients showed significantly reduced cumulative disease scores. Furthermore, pregnancy inhibited T cell proliferation and increased the number of B cells post transfer. The role of regulatory/suppressive T cells and inhibitory molecules on antigen presenting cells (B cells and DCs) are under study for their involvement in pregnancy suppression of EAE

(Supported by NIH grant AI 43376).
The role of liver NKT cells in the regulation of peripheral immune responses (42.9)
Authors: Li Wei , Duthie Malcom , Wang Weigang , Carper Katie , Kahn Stuart , Latchman Yvette , Reyes Jorge , Perkins James
Published: January 2023

We have previously shown that the liver plays an important role in oral tolerance induction. The liver can transfer tolerance to OVA from OVA fed mice to naïve mice. In this study we employed an oral tolerance model by OVA feeding to further explore the mechanisms underlying the role of the liver in oral tolerance induction. OVA feeding, either high dose or low dose, expanded NKT cells in the liver which expressed higher levels of CD95L and PD-L1, a moderate level of PD-1 on the surface, peaked at the 2nd dose feeding in the high dose group and at the 3rd dose feeding in the low dose group. The Foxp3+CD4+CD25+ T regulatory cells (Treg) showed a slight increase in the livers and spleens in the low-fed group, but increased only in the livers in the high-fed group. OVA feeding inhibited NKT cell linked T cell proliferation in both low and high dose fed livers and spleens. Elispot assay revealed an increase in IL-4 and IFN-γ production in the low-fed mice, but increased IL-10 in the high-fed mice. Moreover, the DTH responses to OVA stimulation were increased markedly in the NKT−/− mice with high dose feeding, but showed no significant change in the PD-L1−/− mice compared to that in WT mice. Our results indicate that the liver NKT cells are important in OVA-induced oral tolerance, particularly in high dose feeding. The function of NKT cell appears to depend on CD95L and CD4+CD25+Treg, but not on the PD-1/PD-L1 pathway. IL-10 appears dominant in high dose feeding, while IL-4 and IFN-γ show importance in low dose feeding.
Dendritic cell derived exosomes express a Streptococcus pneumoniae capsular polysaccharide type 14 cross-reactive antigen that induces protective immunoglobulin responses against pneumococcal infection in mice (43.10)
Authors: Colino Jesus , Snapper Clifford
Published: January 2023

Exosomes activate T cells in vivo, but whether exosomes are able to induce humoral immune responses is still unknown. We found that dendritic cells, but not other immune cells, constitutively release an exosome-associated glycoconjugate that is cross-reactive with the capsular polysaccharide of Streptococcus pneumoniae type 14 (Cps14-CRA). Cps14-CRA was localized to the cholesterol-enriched microdomains or rafts of the exosomes and was mapped to the β 1→6 branched N-acetyl-lactosamine derivatives of the Cps14-CRA. Injection of CFA primed naïve mice with purified DC exosomes induced Ig anti-Cps14 responses composed predominantly of IgM, IgG3 and IgG1. These responses were associated with protection against a lethal challenge with live S. pneumoniae type 14, but not with type 3 bacteria, and was correlated with the titer of elicited IgM and IgG3 anti-Cps14. These data show for the first time, that exosomes can induce a humoral immune response to an associated unprocessed, autologous antigen. Although anti-Cps14 Ig responses are specifically demonstrated, these could reflect a broader mechanism that modulates both natural immunity and autoimmunity to other glycotopes.
Transcriptional response of human dendritic cells to Borrelia garinii –defective CD38 and CCR7 expression detected (43.18)
Authors: Hartiala Pauliina , Hytönen Jukka , Pelkonen Jenni , Kimppa Katja , West Anne , Suhonen Juha , Lahesmaa Riitta , Viljanen Matti K
Published: January 2023

Lyme borreliosis is a tick-borne disease that can affect several organs and cause a variety of symptoms. In some patients, the infection may become chronic even after antibiotic therapy and cause persisting damage. Dendritic cells (DC) are involved in the initiation of both innate and adaptive immune responses. To study interactions between Borrelia garinii (Bg), one of the causative agents of Lyme borreliosis, and human dendritic cells, we used a cDNA microarray to compare the Bg induced DC transcriptional response to responses induced by lipopolysaccharide (LPS). The Bg induced response consisted of a smaller number of genes than the LPS induced response.

The microarray showed that the ectoenzyme CD38, which has an important role in DC chemotaxis and migration to lymph nodes, was strongly up-regulated by LPS but practically not at all by Bg. This finding was confirmed with quantitative RT-PCR and with flow cytometry at protein level. In addition, RT-PCR showed that the expression of the dominant mediator of DC migration, CCR7, was 11-fold greater in LPS stimulated than in Bg stimulated cells. These findings suggest that Bg may affect crucial DC functions by blocking the up-regulation of important molecules in DC migration, thus affecting further immune responses in Lyme borreliosis infection.

This study was supported by the Academy of Finland (Microbes and Man project)
Mycobacteria Induce Protective Effector Functions in a Subset of Nonprotective Phosphoantigen-reactive γ9δ2 T cells (43.21)
Authors: Spencer Charles T , Abate Getahun , Blazevic Azra , Hoft Daniel F
Published: January 2023

Human γ9δ2 T cells expand and produce protective cytokine and cytolytic responses during mycobacterial infection. γ9δ2 T cells are also stimulated by nonpeptidic phosphoantigens (i.e-IPP, HMB-PP), expressed by intracellular mycobacteria and infected cells. Therefore, purified phosphoantigens could be useful components of new vaccines or immunotherapeutics by stimulating protective γ9δ2 T cells. However, it is unclear whether γ9δ2 T cells induced by phosphoantigens can protect against mycobacterial replication. We show that while BCG-expanded γ9δ2 T cells potently inhibit intracellular mycobacterial growth, IPP/HMB-PP-expanded γ9δ2 T cells fail to inhibit intracellular mycobacteria, although both lyse Daudi targets. TLR co-stimulation during IPP expansion also failed to induce anti-mycobacterial γ9δ2 T cells. TCR spectratyping and CDR3 sequencing demonstrated that BCG-expanded γ9δ2 T cells expressed significantly less TCR sequence diversity than IPP-expanded γ9δ2 T cells. BCG-expanded γ9δ2 T cells respond similarly to BCG- and IPP-stimulation, while IPP-expanded γ9δ2 T cells responded poorly to BCG. BCG appears to stimulate an antigen-specific focusing event in γ9δ2 T cells while IPP acts like a mitogen with broad γ9δ2 T cell reactivity. The antigens and/or co-stimulatory signals required to induce protective γ9δ2 T cells remain to be identified.

Support: NIH R01-AI-48391, VTEU NO1-AI-25464
Early response of alveolar macrophages to Respiratory Syncytial Virus infection (43.23)
Authors: You Dahui , Ripple Michael , Simms Tiffany , Cormier Stephania
Published: January 2023

Respiratory syncytial virus (RSV) infection is the most common cause of lower respiratory tract infection in infants and elderly adults. Previous studies in humans and in animal models suggest that early infection with RSV may predispose to the development of asthma. However, the molecular mechanisms by which RSV induces changes in the immuno-environment of the airway are still largely unknown. The purpose of the present research was to identify the molecular effectors involved in the host response to RSV infection in alveolar macrophages (AMs). Seven day old mice were infected with RSV (4 x 104 TCID50/g body weight). Total RNA was isolated from airway AMs 5 hours post-infection and processed to perform expression analysis using the Affymetrix GeneChip (Mouse Genome 430 2.0s). A total of 424 genes were observed to be differently expressed. Of these, 187 genes were found to be up-regulated and 237 genes to be down-regulated. Many of these genes take part in cell signaling pathways including the IL-6 and p38 MAPK pathways. Interestingly, 10 genes were found to be involved in apoptosis. RSV infection resulted in complex and immediate cellular responses in AMs. Preliminary data suggests that RSV inhibits apoptosis of AMs. IL-6, amphiregulin and matrix metalloproteinase-13 may play roles in the subsequent development of asthma.

This publication was made possible by NIH grant number P20 RR020159 from the LSU/Tulane COBRE-CEIDR Program of the National Center for Research Resources. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.
Preexisting frequency of antigen-specific naive T cell dictates immunodominance of antigen-specific CD8+ T cells (43.25)
Authors: Kuroda Marcelo , Hasegawa Atsuhiko , Liu Huining , Vinet Heather C. , Subbramanian Ramu A.
Published: January 2023

Immunodominance is a common feature of Ag-specific CTL responses to infection or vaccines. Understanding the basis of immunodominance is crucial to understanding cellular immunity and viral evasion mechanisms and will provide a rational approach for improving vaccine design. This study was performed comparing CTLs specific for the SIV Gag p11C (dominant) and SIV Pol p68A (subdominant) epitopes that are consistently generated in Mamu-A*01+ rhesus monkeys exposed to SIV proteins. Additionally, vaccinated monkeys were used to prevent any issues of antigenic variation or dynamic changes in CTL responses by continuous Ag exposure. Analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-α and -β chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of α and β chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Our data suggest that Ag-specific naïve T cell precursor frequency may be predetermined and that this dictates immunodominance of CD8+ T cell responses. These findings will aid in understanding immunodominance and designing new approaches to modulate CTL responses.
B cell expression of TLR2 is critical for optimal induction of humoral immunity to intact Streptococcus pneumoniae (43.27)
Authors: Vasilevsky Sam , Sen Goutam , Chattopadhyay Gouri , Snapper Clifford M.
Published: January 2023

TLR2−/− mice immunized with Streptococcus pneumoniae (Pn) have defective type 1 (IgG3, IgG2b, and IgG2a) humoral immunity, but a similar type 2 (IgG1) response relative to WT mice. Of note, this defect in adaptive immunity was associated with a largely normal innate immune response to Pn. We thus hypothesized that TLR2 plays a critical role directly at the level of the adaptive immune system. We demonstrate that CD4+ T cells from Pn-primed TLR2−/− mice elicit a normal cytokine response after in vitro re-stimulation with Pn protein. In contrast, MyD88−/− mice, exhibiting both defective innate and type 1, but elevated type 2, humoral immunity to Pn, elicit an increased type 2 cytokine response. Bone marrow dendritic cells (BMDC) from TLR2−/− mice have only a modest defect in cytokine secretion, including IL-12, and undergo normal maturation in response to Pn. TLR2−/− BMDC pulsed with Pn and transferred into WT mice elicit a normal Ig response, relative to WT BMDC. In contrast, Pn fails to costimulate in vitro proliferation of purified TLR2−/− B cells activated via multivalent B cell receptor crosslinking, while costimulating WT B cells. Adoptive transfer studies demonstrate that TLR2−/−, but not WT, B cells are defective in eliciting Ig responses to Pn in either a TLR2−/− or WT background. These data are the first to indicate a critical role for B cell expression of TLR2 during a humoral immune response to an intact bacterium.

N.I.H. R01 AI49192
CD4 T cell memory of prior influenza infections regulate new CD4 and CD8 T cell responses (43.3)
Authors: Jelley-Gibbs Dawn M , McKinstry Kai , Strutt Tara , Dibble John , Swain Susan
Published: January 2023

Acute infections are often characterized by short-lived antigen presentation, but influenza infection results in long-lived antigen depots capable of stimulating CD4 and CD8 T cells long after live virus clearance. FACS purification of antigen presenting cells from mice at various times after flu infection, followed by co-culture with naive flu-specific T cells, has demonstrated that dendritic cells are the perpetrators of long-lived flu antigen presentation. However, there may be a reservoir of intact virus that persists at low levels in other cell populations. Regardless of the mechanism of antigen retention, access to long-lived flu antigen presentation is important for the generation of flu-specific memory T cells. Manipulations of our model system that result in enhanced circulation of effector T cells in the periphery of flu infected mice lead to impressive increases in memory T cell generation. Specifically, introduction of flu-experienced memory CD4 T cells during new CD4 and CD8 T cell priming, as well as induction of physical redistribution of newly primed effectors, results in greater effector circulation in peripheral sites and increased numbers of memory T cells. We propose that recirculation of effector T cells during flu infection allows for greater access to long-lived antigen depots, and consequently results in higher numbers of memory T cells.

Funding provided by NIH grant AI46530.
Helminthic infection enhances susceptibility to tuberculosis in a murine coinfection model (43.31)
Authors: Potian Julius Andrew , Bhatt Kamlesh , Liu Zhugong , Gause William , Salgame Padmini
Published: January 2023

Regions of the world with high incidence of Tuberculosis (TB) are also endemic for helminthic diseases. Helminthic infection elicits a profound Th2 immune response, which may alter theTh1 response necessary for the protection to TB. The impactof Th2 responses on the protective immune response elicited to TB is poorly understood. We co-infected BALB/c mice with the nematode Nippostrongylus brasiliensis (Nb) followed by virulent Erdman strain of Mtb via aerosolization. At 4 and 7 weeks post-Mtb infection, co-infected mice displayed significantly higher bacterial burden compared to mice infected with Mtb alone. Furthermore, the enhanced disease progression was accompanied by a shift in the Mtb-induced Th1 response to a Th2 response in the co-infected mice as demonstrated by an increase in Th2 cytokines IL-4 and IL-10. Taken together, theseresults demonstrate that helminth infection alters the ability of the host to mount an effective immune response to TB and modulates the course of tuberculosis disease. Clinically, elucidating the mechanisms which underlie alteration of immunity to TB will facilitate in the better design of TB vaccines and therapies.
Immune complex-induced enhancement on presentation of bacterial antigens by dendritic cells is mediated by activating Fcγ receptor III (43.36)
Authors: Herrada Andres Alonso , Contreras Francisco , Tobar Jaime , Pacheco Rodrigo , Kalergis Alexis
Published: January 2023

Dendritic cells (DCs) are capable of initiating adaptive immune responses against infectious agents by presenting pathogen-derived antigens on MHC molecules to naïve T cells. We have previously shown that Salmonella enterica serovar Typhimurium (S. Typhimurium), is able to survive inside DCs and interfere with their function by avoiding activation of bacteria-specific T cells. In contrast, when S. Typhimurium is targeted to Fcγ receptors on the DC surface, bacteria are degraded and their antigens presented to T cells. However, the specific Fcγ receptor responsible of restoring presentation of antigens remains unknown. Here we show that IgG-coated S. Typhimurium was targeted to lysosomes, degraded and its antigens presented on MHC molecules only when the low affinity activating FcγRIII was expressed on DCs. FcγRIII-mediated enhancement of Ag presentationled to a robust activation of T cells specific for bacteria-expressed antigens. Laser confocal and electron microscopy analyses revealed that IgG-coated S. Typhimurium was rerouted to the lysosomal pathway through a FcγRIII-dependent mechanism. Our data support the notion that the capacity of enhancing priming of bacteria-specific T cells by DCs can be attributed as a novel function for the activating FcγRIII.

This work was supported by grants FONDECYT 1030557 and 1050979, FONDEF D04I1075 and Millennium Nucleus on Immunology and Immunotherapy.
Lung resident memory CD4 T cells provide optimal protective immunity to influenza challenge (43.4)
Authors: Teijaro John , Ndjembi Modesta P , Lorenzo Lani , Chandran smita , Farber Donna
Published: January 2023

Diverse subsets of CD4 memory T cells in lymphoid and non-lymphoid tissue are known to persist after infection; however, the ability of these subsets to mediate protective immunity against viral challenge is not known. We previously showed that a population of influenza hemagglutinin (HA)-specific memory CD4 T cells exhibits diverse migration properties with spleen and lung-derived memory CD4 T cells exhibiting tissue-specific homing. Here, we examined the ability of purified lung and spleen-resident HA-specific memory CD4 T cells to mediate protection upon influenza challenge. We found that lung-derived memory CD4 T cells facilitated more rapid clearance of virus from infected lung compared to equal numbers of spleen-derived memory T cells. Moreover, viral clearance in mice receiving lung memory occurred with reduced weight loss pathology, whereas naive mice or mice receiving splenic HA-specific memory T cells displayed precipitous weight loss and ruffled appearance. We found intrinsic functional differences in proliferation and effector capacity of lung compared to spleen-derived memory cells, including reduced overall expansion and IL-2 production from lung versus spleen subsets. Our results demonstrate an enhanced ability of lung-derived memory CD4 T cells to mediate protective responses with minimal pathology compared to lymphoid memory T cells, with important implications for vaccine design.

Supported by NIH-AI-50632
Catalytic Antibodies to Staphylococcus aureus (43.41)
Authors: Brown Eric Luis , Smith Keri , Paul Sudhir , Duncan Katherine
Published: January 2023

Of growing concern are the steadily increasing numbers of community-acquired methicillin resistant Staphylococcus aureus infections combined with the need for novel antibiotic independent therapeutic strategies. We propose to test the hypothesis that catabolic antibodies (CABs) may play a role in protecting against S. aureus infections. CABs are immunoglobulins endowed with a capacity to catalyze the hydrolysis of specific antigenic protein/polypeptide substrates. A single molecule of a catabolic antibody can degrade thousands of antigen molecules, therefore, a CAB response against bacterial pathogens would provide a very effective means to control infection. To date, a role for catabolic antibodies in defense against bacterial infections has not been examined. To investigate the role of CABs we focused on the S. aureus extracellular fibrinogen binding protein (Efb) protein, which contributes to virulence in wound infection. We found that polyclonal preparations of normal human IgG and IgM showed significant cleavage of Efb. Of interest, IgG was more potent than IgM at hydrolyzing Efb. Further investigation of the cleavage of Efb by IgG and IgM was conducted throught the analysis of N-terminal sequencing. To test the functionality of the CAB response to Efb, we incubated Efb with normal IgG or IgM, then used the products of this reaction in a complement activation assay. Both IgG and IgM cleavage resulted in the prevention of Efb-induced blocking of the complement cascade. Our results suggest that CAB may play an important role in the immune response against S. aureus infection.
The precarious balance of immune response to Mycobacterium tuberculosis … are T regulatory cells predictive of infection outcome in a non-human primate model of tuberculosis? (43.5)
Authors: Green Angela M. , Bongers Kale S. , Flynn JoAnne L.
Published: January 2023

A majority of persons infected with M.tb contain but do not eliminate the bacteria, while a minority, develop active tuberculosis. Recent studies in humans suggest an increased frequency of CD4+CD25+ Treg cells in peripheral blood correlates with active TB. To determine whether an increased frequency of CD4+FoxP3+ Treg cells in the periphery is predictive of active disease, we used the non-human primate model of TB, the only animal model that mimics both human latent infection and active disease. Cynomolgus macaques (24) were infected with low dose M.tb and followed until disease state (active or latent) was declared (6–8 months). All animals demonstrated a decrease in the frequency of Tregs following infection until ~ 2 months p.i. without a change in frequency of total CD4+ T cells. Concurrently, the frequency of Tregs and total CD4 T cells in the airways increased up to ~ 2 months p.i. Prior to and 8 weeks p.i., monkeys that eventually presented as latently infected had a significantly higher frequency of Tregs within the CD4 compartment (p< 0.05 and p< 0.005 respectively) than monkeys that eventually had active disease. Between 12–20 weeks, there was a trend toward a higher frequency of Tregs in the monkeys with active disease. Our results suggest frequency of Treg in the periphery may predict or serve as a marker for infection outcome, depending on the stage of infection. NIH grant: RO1 AI50732-01
Expression of PD1 Differs on T Cells from a Pathogenic Versus Nonpathogenic Primate Models of AIDS (43.50)
Authors: Rogers Kenneth , Onlamoon N , Hudson K , Bryan P , Mayne A E , Mori K , Pattanapanyasat K , Villinger F , Ansari A A
Published: January 2023

In most chronic infections antigen specific memory T cells experience a progressive immune exhaustion. Recent reports of HIV infection demonstrate increased expression of PD1 on exhausted T cells correlating with viral loads, and in vitro restoration of function by blockade of PD1 interactions with its ligands. We characterized PD1 in a rhesus macaque model of SIV infection where AIDS develops versus an asymptomatic natural host, sooty mangabeys. Infected animals of both species showed an increase in frequency of total PD1+ CD4+ cells, but not CD8+, which correlated with viral loads in macaques, but not in mangabeys. Furthermore this increase was noted in naïve mangabey CD4+ cells, but central memory macaque CD4+ cells. In macaques, antigen specific CD8+ T cells had increased PD1 expression (MFI) that was reduced with antiviral therapy. While the blockade of PD1-PDL1 interactions in vitro enhanced overall SIV antigen specific responses, individual responses to specific peptides were found enhanced, unchanged or even diminished. These data support a potential for role PD1 in the phenomena of “immune exhaustion” and a rationale for its study using monkey models of AIDS.
B7-H1 Expression on Gastric Epithelial Cells Induces Development of T Regulatory Cells during Helicobacter pylori Infection (43.52)
Authors: Beswick Ellen J , Pinchuk Irina V , Das Soumita , Reyes Victor E
Published: January 2023

During H. pylori infection, T cells are recruited to the gastric mucosa, but the host response is unable to clear the infection. We have previously shown that during infection H. pylori induces B7-H1 on the gastric epithelium, and interacting T cells are thus anegized. Recently, T regulatory cells (Treg) with a CD4+ CD25high FoxP3+ phenotype were found at an increased frequency in the H. pylori-infected gastric mucosa. Since B7-H1 promotes T cell anergy during H. pylori infection and Treg cells are present in the infected gastric mucosa, we hypothesized that B7-H1 expression by gastric epithelial cells during H. pylori infection promotes development of T regulatory cells. In order to investigate this, we examined by flow cytometry the induction of the Treg phenotype when naïve T cells were incubated with H. pylori-exposed gastric epithelial cells. The number of CD4+CD25+FoxP3+ cells increased together with the production of IL-10 and TGF-β in the cultures. The frequency of Treg cells along with IL-10 and TGF-β production was markedly decreased when B7-H1 was blocked with monoclonal antibodies. The function of these Treg cells was investigated by sorting them for use in co-cultures with activated T cells, which effectively decreased proliferation of the activated T cells. B7-H1 may thereby play a crucial role in the development of CD4+CD25+FoxP3+ Treg cells and the inadequate T cell response leading to chronic infection.
Helminth secretory product Ribonuclease T2 is a Th2-inducing agent (43.8)
Authors: Jankovic Dragana , Steinfelder Svenja , Andersen John F. , Sher Alan
Published: January 2023

Helminth-induced Th2 polarization is associated with down-regulated dendritic cell (DC) function but the underlying molecular mechanism remains elusive due to the poor characterization of helminth molecules with Th2-inducing activity. During Schistosoma mansoni infection egg, but not worm, Ag are a target of a strong Th2 response. Moreover, when injected in naïve mice schistosome eggs or water-soluble egg extract (SEA) have an intrinsic ability to promote development of Th2 cells. Using as a read-out an in vitro model of CD4+ T cell priming with CD11c+ splenic DC we demonstrated that SEA fractions containing molecule(s) with an approximate MW of 30 kD selectively promote Th2 polarization and down-regulate DC functions. Moreover, similar positive fractions were isolated from preparations containing egg excretory/secretory molecules. The N-terminal sequence of the latter identified the S. mansoni enzyme Ribonuclease T2 as the active component and this was further confirmed with the recombinant protein. S. mansoni ribonuclease is the first chemically defined protein with Th2-promoting activity known to act on dendritic cells.
Association between TLR4 SNPs (Asp299Gly/Thr399Ile) and non-IgE mediated food allergy (NFA) with or without recurrent otitis media (OM) and chronic rhinosinusitis (CRS). (44.25)
Authors: Jyonouchi Harumi , Geng Lee , Cushing-Ruby Agnes , Cheongeun Oh , Hester Jeff D
Published: January 2023

Co-segragating missense mutations of Asp299Gly and Thr399Ile in TLR4 are found in 10% of general population. Heterozygousity of these mutations is implicated with hyporesponsiveness to a TLR4 agonist (LPS). This study addressed the association between heterozygousity of these TLR4 alleles and NFA with or without recurrent OM/CRS. The study included 203 children: inflammatory bowel disease (IBD; N=11 median 14.5 y), OM/CRS/no NFA (N=21, median 7.3 y), NFA alone (N=64, median 3.8 y), OM/CRS+NFA (N=30, median 5.5 y) and controls (N=78, median 6.9 y). Production of proinflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-12) by peripheral blood mononuclear cells and TLR2 Arg753Gln allele were also assessed. In this study, 20/203 (9.85%) and 7/203 (3.4%) subjects were heterozygous for TLR4 Asp299Gly/Thr399Ile and TLR2 Arg753Gln alleles, respectively. Heterozygousity of these TLR4 alleles were associated with OM/CRS+NFA (57.1% vs 9.7%, p<0.00001), IgE mediated FA (p<0.001), and atopy (p<0.02) but not with OM/CRS/no NFA or IBD. Such an association was not found in TLR2 Arg753Gln allele. LPS responses did not differ irrespective with these TLR2/4 mutated alleles. Among 21 children with OM/CRS/no NFA, 16 revealed other immunodeficiencies. Thus individuals heterozygous for TLR4 Asp299Gly/Thr399Ile alleles may be predisposed to OM/CRS/NFA and possibly IgE mediated FA.

Funded by Jonty Foundation.
Innate responses at the DC level determine the outcome of Leishmania infections (44.28)
Authors: Vargas-Inchaustegui Diego Antonio , Xin Lijun , Eaves-Pyles Tonyia , Soong Lynn
Published: January 2023

Although Leishmania braziliensis (Lb) and L. amazonensis (La) are endemic in the same geographic regions in South America, their infections can lead to very different clinical manifestations. Given the important role of dendritic cells (DCs) in leishmanial infections and activation of parasite-specific CD4 T cells, we hypothesize that Lb infection efficiently triggers innate responses in DCs and macrophages and that this innate immunity, in conjunction with the subsequent Th1 responses, leads to rapid killing of parasites in the local tissue. To test this hypothesis, we infected murine bone marrow-derived DCs, as well as murine and human macrophages, with Lb and La and measured infection rate, cell activation status, cytokine secretion, and activation of the JAK/STAT signaling pathway. Our key findings are:

although both species of parasites were comparable in infecting mouse DCs and human and mouse macrophages, La parasites tended to have prolonged survival within the cell;Lb-infected DCs displayed an activated phenotype (CD11chighCD45RB−CD83+CD40+CD80+), while La-infected cells showed a “semi-activation” status, with much lower numbers of activated DCs;Lb-infected DCs produced 3-fold higher levels of IL-12p40 and were more potent in priming naïve CD4+ T cells when compared to La-infected counterparts;although IL-10 production was detectable in Lb-infected DCs, its level was considerably lower than that of IL-12p40, suggesting the potential of Lb-infected DCs in activating Th1-type responses;microarray analysis revealed up-regulation of several genes (e.g., IFN-g and IFN regulatory factors) in Lb-infected DCs.

Collectively, study indicates that innate responses at the DC level determine the outcome of Leishmania infections.
Ribosomal protein L22 inhibits induction of type I IFN and faciliates the replication of VSV (44.33)
Authors: yang eunjung , Choi In-Hong
Published: January 2023

L22 is a 14.8-kDa protein constituent of the large ribosomal subunit, which is not conserved in prokaryotes and does not share RNA binding motifs with any other known protein such as PKR and RIG-I/Mda5. L22 associates with human telomerase, the small viral RNA HVP-1 in Herpesvirus papio–infected baboon cells, the 39X region within the 39-untranslated region (UTR) of the human Hepatitis C virus (HCV) genomic RNA, poly (ADPribose) polymerase in Drosophila melanogaster, and the Herpes simplex–infected cell proteins (ICP) 4 and ICP 22 in human cells. In uninfected human B cells, L22 is located in the nucleoli and cytoplasm, whereas following EBV infection, L22 is found in the nucleoplasm. In transformed B cells, EBER 1 is quantitatively bound by L22 (>95%), whereas only 30%–50% of the L22 protein associates with this RNA and inhibits regulation of cellular activities by the Epstein-Barr virus small RNA EBER-1. However, the function of L22 both in the ribosome and in these several RNP complexes remains unknown.

Here we investigated whether L22 has an effect on antiviral response; the replication of VSV and activation of eIF2. First, the replication of VSV was evaluated after transfection of L22 in A549 cells. When the cells were infected with VSV (MOI =1), a significant enhancement of replication of virus was detected. The phosphorylation of the α subunit of eIF2 complex was inhibited by transfection of L22, which is usually followed by VSV infection through PKR activation. Our data also showed L22 inhibited phosphorylation of IRF3 and STAT1. Therefore, we suggest that ribosomal protein L22 negatively regulates type I IFN response in VSV infection model, which facilitates the replication of VSV.

This study was supported by Brain Korea 21 Project for Medical Science, Yonsei University.
AGE AND GENDER-RELATED SPONTANEOUS BACTERIAL KERATITIS IN CD36KO MICE (44.40)
Authors: Barcia Rita N , Whiston Emily , Lee Karen , Heimer Susan , Masli Sharmila , Marshak-Rothstein Ann , Gregory Meredith , Ksander Bruce R
Published: January 2023

Purpose: CD36 is a scavenger receptor with many ligands such as fatty acids, phospholipids, and TSP-1. While its main function is to clear apoptotic cells, CD36 is also required for full activation of TLR 2 triggered by Staphylococcus aureus. Although S. aureus is found in the normal flora of the ocular surface, mice do not spontaneously develop bacterial keratitis. Since both TLR 2 and CD36 are expressed in the cornea, we hypothesize that CD36 is required to prevent S. aureus keratitis.

Methods: Corneas of CD36KO and wild-type C57BL/6 mice were screened via slit lamp. Corneas containing lesions were sectioned, stained, and examined microscopically. Bacterial load in corneal tissue was determined and Staphylococcus was identified by gram stain and ChromAgar. Serum was collected and analyzed for ANA (anti-nuclear antibodies).

Results: CD36KO mice displayed spontaneous corneal lesions that increased in frequency and severity with age. High levels of Staphylococcus were found in corneas with severe lesions, and not controls. Severe corneal lesions showed a massive CD45+ infiltrate in the anterior part of the cornea. The frequency of corneal lesions in the colony was 74% (female) and only 26% (males). ANAs were found in the serum of older female mice with corneal lesions, but not in the controls.

Conclusion: CD36KO mice develop spontaneous bacterial keratitis. The infection may be secondary to autoimmunity since the highest frequency of keratitis occurred in older female mice with ANA. This is the first report of a spontaneous bacterial keratitis in mice.