bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple domains of the MBD2 protein are required for transcriptional repression in B cells. (P4382)
Authors: Dege Carissa , Ramirez Julita , Hagman James
Published: January 2023

We demonstrated previously that the mb-1 (Igα; Cd79a) promoter is demethylated progressively during its activation in early B cell development. Demethylation of promoter CpGs is initiated by binding of the transcription factor, Early B cell Factor 1 (EBF1). EBF1 increases local accessibility and demethylation of mb-1 promoter DNA, which allows for binding by Pax5 prior to transcription. This process is inhibited by Mi-2/NuRD chromatin remodeling complexes. This was evidenced by knockdown of the ATPase subunit of Mi-2/NuRD, Mi-2β (CHD4). Recent work in the laboratory has focused on other components of the Mi-2/NuRD complex to assess their importance for association of Mi-2/NuRD with methylated DNA. A subset of Mi-2/NuRD complexes contains Methyl Binding Domain protein 2 (MBD2). MBD2 binds methylated, but not hydroxymethylated or unmethylated cytosines. We have demonstrated that knockdown of MBD2 using shRNA potentiated mb-1 transcriptional activation only in the presence of EBF1 and Pax5. MBD2 is important for recruiting Mi-2/NuRD complexes to gene targets and for maintenance of heterochromatin. MBD2 comprises arginine-glycine (RG)-rich, Methyl CpG binding (MBD) and C-terminal (CTD) domains. Through mutational analysis, we have demonstrated that multiple domains of MBD2 are important for transcriptional attenuation of the mb-1 gene in B cells. We are now investigating how MBD2 binding is inhibited to de-repress the B cell program in early B cell progenitors.
CTLA4-mediated inhibitory effect on osteoclastogenesis by Tregs in oral cancer (P2047)
Authors: Tomihara Kei , Arai Naoya , Noguchi Makoto
Published: January 2023

Purpose: Despite regulatory T cells (Tregs) have been demonstrated to inhibit inflammatory bone destruction through suppressing osteoclast formation in the bone microenvironment of inflammatory diseases, very little work has been done to characterize Tregs in the microenvironment of cancer bone invasion. In the present study, we investigated the mechanism of interaction between Tregs and osteoclasts in a murine model of oral cancer to elucidate the role of Tregs in the bone invasion of oral cancer. Materials and Methods: SCCVII mouse oral squamous cell carcinoma cells were administered into the right masseter region of a C3H/HeN mouse. After 14 days of tumor inoculation, mice were sacrificed and Tregs were purified from the tumors. Tregs were then cocultured with bone marrow CD11b positive cells in the absence or presence of RANKL and M-CSF for 4 days. Secreted tartrate-resistant acid phosphatase (TRACP) 5b was determined as a marker of osteoclast number. Results: Attenuation of osteoclast-derived TRACP5b was observed in the coculture of tumor derived Tregs and bone marrow CD11b positive cells, and this attenuation of TRACP5b was ameliorated by neutralization of cytotoxic T-lymphocyte-associated antigens-4 (CTLA4). Conclusion: These results indicate that Tregs may play a protective role in bone invasion of oral cancer via suppression of osteoclastogenesis, and CTLA4 may be a key regulator for the suppression of osteoclasts by Tregs in oral cancer.
Myd88 may contribute in several ways to progression of murine mammary carcinoma (P2077)
Authors: Kurt Robert , Carey Maureen , Eidelman Alec , Liberti Maria , Ricca Jacob , Tucker Kyle
Published: January 2023

Previously we reported that Myd88 contributed to growth, metastasis, and expression of CCL2 and CCL5 by mammary carcinoma implying that constitutive signaling through Myd88 contributes to tumor progression. To begin to decipher downstream effects of Myd88 signaling in the tumor cells we used RNA interference and a Myd88 inhibitory peptide and then screened for expression of genes involved in signaling and the cell cycle. Some of the alterations that were found included TLR signaling components such as Tirap, Tollip, Irak1 and Irak2 suggesting a potential feedback mechanism for Myd88 in TLR signaling in the tumor cells. Many of the genes that were modulated upon Myd88 inhibition may contribute to multiple downstream effects such as inflammation, migration and growth. For instance, genes utilized for the NFKb and MAPK pathways including NF-KB1, IKB-beta, MEK, JNK and p38 exhibited reduced expression upon Myd88 inhibition. Genes encoding proteins that could regulate the cell cycle such as p53, cyclins B, D, and F were also modulated, and a role for Myd88 in cell cycle progression was confirmed by cell cycle analysis which showed that Myd88 was important for progression to the G2 phase of the cell cycle. Collectively, these data imply that Myd88 may contribute to tumor progression by regulating several pathways (apoptosis, proliferation, migration and/or inflammation), and underscores the importance of delineating the role of Myd88 in murine mammary carcinoma.
T cells from DNAM-1 deficient mice display immunological synapse defects that impair anti-tumour immunity (P2009)
Authors: Oliaro Jane , Rambsbottom Kelly , Russell Sarah , Smyth Mark
Published: January 2023

DNAX accessory molecule 1 (DNAM-1/CD226) is an adhesion molecule expressed on all CD8+ cytotoxic T cells (CTLs) that acts as an accessory molecule to promote CTL functions such as activation, differentiation, migration and effector function. DNAM-1 interacts with LFA-1, a critical molecule for immunological synapse formation between T cells and antigen presenting cells, and for cytotoxic killing of target/tumour cells. Indeed, mice lacking DNAM-1 display abnormal T cell responses and anti-tumour activity; however, the mechanism involved is not well understood. Here, we investigate the immunological synapse between OT1 DNAM-1 deficient T cells and antigen presenting cells, and show that DNAM-1 deficiency results in reduced proliferation and expansion of T cells in response to antigen presentation, which correlates with a decrease in cytokine production. Furthermore, we demonstrate that activated OT1 DNAM-1 deficient T cells show reduced stable conjugations with tumour cells and decreased recruitment of LFA-1 and lipid rafts to the immunological synapse, which correlates with reduced killing in vitro. This polarity defect may explain why DNAM-1 deficient mice cannot clear tumours in vivo and highlights the importance of the immunological synapse in T cell mediated anti-tumour immunity.
Generation of cellular responses following FcR targeting in the mucosa in a Francisella tularensis model. (P4288)
Authors: Bitsaktsis Constantine , Gosselin Edmund
Published: January 2023

Numerous studies have demonstrated that targeting antigens to FcγR can increase their immunogenicity and generate protective immune responses in various infection models. Previously, we have shown that targeting fixed Francisella tularensis bacteria to FcγRI and III in mice, via intranasal immunization with immune complexes, enhances specific immune responses that are protective against this pathogen. In this study we have utilized our F. tularensis model to dissect the "in vivo" cellular immune responses generated following FcγR targeting. Our results demonstrate for the first time that targeting fixed Francisella tularensis to FcγR: 1. increases the recruitment and activation of dendritic cells in the lungs of immunized mice, 2. increases the frequency and number of effector memory CD4+ T cells during F. tularensis infection in an IL-12-dependent manner, and 3. increases the frequency and number of IFN-γ producing effector memory T cells upon "in vitro" re-stimulation. These data, collectively, confirm the effectiveness of FcγR targeting as a vaccine strategy against infections with mucosal pathogens, and for the first time demonstrate the "in vivo" immunological mode of action involved in the observed protection.
Pathogenesis of emerging severe fever with thrombocytopenia syndrome virus in a non-human primate model (P3021)
Authors: Jin Cong , Liang Mifang , Han Ying , Jiang Hong , Li Chuan , Zhu Hua , Wei Qiang , Qin Chuan , Li Dexin
Published: January 2023

Severe Fever with Thrombocytopenia Syndrome (SFTS) emerged as an infectious disease in 2010 in rural China, and its causative agent SFTS virus (SFTSV) is a new member of the family Bunyaviridae, genus phlebovirus, most closely related to the heartland virus recently discovered in Missouri, the US. To develop effective vaccines and therapeutics against this emerging pathogen, animal disease models are urgently needed. Here, we report the first findings of measurable clinical disease in non-human primates infected with SFTSV. In the primate model, the abrupt fever, thrombocytopenia, and leukocytopenia were duplicated. Substantially enhanced aspartate aminotransferase, creatinine, and lactate dehydrogenase in serum was observed, indicating pathological lesions in organs. Viremia peaked around 3 to 5 days after the inoculation of SFTSV. Viral RNA was detected in the spleen, lymph node, liver, kidney, and intestine. Virus-specific IgM and IgG antibody responses were induced, and cytokines IFN-γ and eotaxin were significantly enhanced in the acute phase. Scattered necrosis and degeneration of hepatocytes in liver, as well as glomerular hypercellularity, mesangial thickening, and congestion in Bowman’s space in kidney were observed after 4 weeks of SFTSV infection. Thus, we established a non-human primate model which resembles major clinical features of the human SFTS disease, and further studied virus-specific immune responses and histopathological changes in this primate model.
Antigen-specific immunoglobulin responses correlate with plasma viral load in SIV/SHIV infected controller macaques (P3049)
Authors: Pahar Bapi , Kenway-Lynch Carys , Pan Diganta , Das Arpita , Marx Preston , Wang Xiaolei , Srivastav Sudesh , Veazey Ronald
Published: January 2023

There is increasing evidence suggesting an unexpected potential for non-neutralizing antibodies to prevent HIV infection. Consequently, identification of functional B cell epitopes for HIV is also important for developing preventative and therapeutic strategies. We therefore explored the antigen-specific immune responses following SIV or SHIV infection in rhesus macaques to determine functional B cell epitopes and identify factors that may influence control of plasma viremia. Functional B cell epitopes are a minimum of 10 amino acids in length for SIV and occur in similar regions despite differences in the route of mucosal SIV inoculation. Peptide-specific IgG responses generate primarily against gp41, V1-V3 regions of gp120, and the NC and p6 regions of Gag. Controller macaques showed much greater IgG responses to Nef, Tat, and p15 and p27 regions of Gag protein compared to absent or minimal responses in non-controller macaques. Antigen-specific IgM and IgA responses share similar epitopes to that of IgG, however the responses are much more limited. Moreover, antigen-specific IgA responses did not correlate with plasma viremia. Our study has shown that Env-specific IgG responses are not reliable prognostic indicators, unlike Gag, Nef, and Tat-specific IgG responses for SIV/SHIV controllers. Therefore, generation of early functional Gag, Nef and Tat-specific IgG responses may be important therapeutic strategies for the control or prevention of HIV/AIDS.
Type I IFN suppresses Type II IFN triggered antimicrobial responses in humans (P1277)
Authors: Teles Rosane , Graeber Thomas , Modlin Robert
Published: January 2023

The mechanism by which Type I IFNs contribute to immune suppression in bacterial infections in humans was investigated by studying leprosy as a model. The Type I IFN, IFN-β, as well as an inducible regulatory pathway involving sequential induction of IL-10 were differentially expressed in progressive lepromatous (L-lep) vs. the self-limited tuberculoid (T-lep) lesions. A common IFN-β gene signature was detected in the peripheral blood of active tuberculosis patients and the skin lesions of L-lep patients, with approximately half of the common IFN-β induced genes also induced by IL-10. Contrary to the expression of Type I IFNs and inducible genes, the Type II IFN, IFN-γ, and downstream genes that comprise antimicrobial pathways were differentially expressed in T-lep vs. L-lep lesions. The IFN-γ-induced macrophage antimicrobial response was inhibited by IFN-β and IL-10, by a mechanism involving blocking the generation of bioactive 1,25-dihyroxyvitamin D as well as inhibiting induction of antimicrobial peptides cathelicidin and DEFB4. Together, these data identify the ability of IFN-β to induce IL-10 as a mechanism by which Type I IFNs inhibit Type II IFN induced host defense pathways and thereby contribute to the pathogenesis of bacterial infections in humans.
T cell-suppression during acute Toxoplasma gondii infection does not involve APC and is a consequence of Treg-mediated IL-2 deprivation and death of proliferating T cells (P1279)
Authors: Salinas Nohemí , Olguín Jonadab , Castellanos Carlos , Saavedra Rafael
Published: January 2023

Foxp3+ Treg (Treg) mediate the suppression of T-cell proliferation observed during the acute Toxoplasma gondii infection in mice. This suppression in T-cell proliferation is related to an IL-2 mechanism. We studied the suppression mechanisms of Treg on T cells in a model of toxoplasmosis using transgenic Foxp3EGFP mice. We analyzed if APC were involved during Treg-suppression of T-cell. Removal of APC from spleen cells from infected mice did not modify suppression and further elimination of Treg led to a complete restored proliferation, demonstrating that APC are not involved in Treg-mediated suppression. Low IL-2 levels were detected in supernatants of APC-free T cells from infected mice. IL-2 levels and T-cell proliferation were restored when Treg and T cells were separated by transwells, indicating that Treg require contact to induce suppression. Although Treg did not inhibit il-2 mRNA levels in T cells, they were able to reduce proliferation of CTLL-2 cells in the IL-2 bioassay, strongly suggesting that Treg act by IL-2 deprivation. Kinetic analysis of T cell proliferation showed that T cells from infected mice die after a few rounds of division in vitro, but addition of rIL-2 or removal of Treg abolished this effect. We conclude that suppression of T-cell proliferation during acute T. gondii infection is mediated by Treg in the absence of APC by IL-2 deprivation, requiring cell contact between T cells and Treg, thus leading to death of proliferating T cells.
Role of histone acetylation, cAMP signaling, and mitogen-activated protein kinase pathways in butyrate-induced host defense peptide gene expression in chicken HD11 macrophage cells. (P1283)
Authors: Sunkara Lakshmi , Jiang Weiyu , Zhang Guolong
Published: January 2023

Host defense peptides (HDPs), critical components of innate immunity of animals, act as direct antimicrobials and immunomodulators. We found that, butyrate, a short-chain fatty acid and histone deacetylase inhibitor, enhances HDP gene expression and reduce Salmonella enteritidis colonization in chickens. However, the molecular mechanisms of butyrate-mediated HDP gene induction remain elusive. Here, we dissected the role of histone acetylation, cAMP signaling, and mitogen-activated protein kinases (MAPKs) in HDP expression in butyrate treated HD11cells. Our real-time PCR results revealed that, in addition to butyrate, several other histone deacetylase inhibitors also enhanced the expression of AvBD9, a chicken beta-defensin gene. In contrast, histone acetyltransferase inhibitors suppressed butyrate-mediated AvBD9 gene induction. Moreover, inhibition of p38 MAP kinase or c-Jun N-terminal kinase, but not extracellular signal regulated kinase, diminished AvBD9 gene expression with butyrate. Interestingly, agonists of the cAMP signaling pathway increased AvBD9 expression either alone or synergistically with butyrate. Collectively, our results showed that histone acetylation, and MAPKs and cAMP pathways play an important role in butyrate-regulated AvBD9 gene expression. A detailed understanding of the transcriptional regulatory mechanisms of HDP expression may facilitate the development of antibiotic-free strategies to disease control and prevention in both animals and humans.
tRNA-derived RNA fragments: A new family of regulatory molecules controlling respiratory syncytial virus replication. (P1401)
Authors: Bao Xiaoyong , Wang Qingrong , Lee Inhan , Ren Junping , Lee Yong Sun
Published: January 2023

The discovery of small non-coding RNAs (sncRNAs) with regulatory functions is a recent breakthrough in biology. Among sncRNAs, microRNA (miRNA), derived from host or virus, has emerged as elements with high importance in control of viral replication and host responses. However, the expression pattern and functional aspects of other types of sncRNAs, following viral infection, are unexplored. In order to define expression patterns of sncRNAs, as well as to discover novel regulatory sncRNAs in response to viral infection, we applied deep sequencing to cells infected with human respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in babies. RSV infection leads to abundant production of tRNA-derived RNA fragments (tRFs) that are ~30 nucleotides (nts) and correspond to the 5’-half of mature tRNAs. At least one tRF, which is derived from tRNA-Glu-CTC, represses target mRNA in the cytoplasm and promotes RSV replication. This demonstrates that this tRF is not a random by-product of tRNA degradation but a functional molecule. The biogenesis of this tRF is also specific, as it is mediated by the endonuclease angiogenin, not by other nucleases. In summary, our study presents novel information on the induction of a functional tRF by viral infection.
Endothelial-expressed leukocyte-specific protein 1 regulates intravascular and extravascular directionality of chemotaxing neutrophils (P5094)
Authors: Qadri Syed , Xu Najia , Hossain Mokarram , Liu Lixin
Published: January 2023

The endothelial-expressed Ca2+- and F-actin-binding leukocyte-specific protein 1 (LSP1) was shown to play a pivotal role in the regulation of endothelial transmigratory cup formation, microvascular permeability increases, and transendothelial migration of leukocytes. Due to the complexity of leukocyte-endothelial interactions, the cell-specific role of LSP1 is incompletely understood. To elucidate the orchestration of neutrophil dynamics by endothelial LSP1 in vivo, intravital microscopy and time-lapsed video analysis were performed in the microvasculature of cremaster muscle in LSP1-deficient chimeric mice. Absence of LSP1 (Lsp1−/−) in non-hematopoietic tissue resulted in significantly impaired chemotactic directionality (chemotaxis index) but not the neutrophil migration velocity during CXCL2-elicited intraluminal crawling and extravascular migration as compared to the responses in wild type (Lsp1+/+) endothelial cells. The velocity of migration of Lsp1−/− neutrophils in extravascular tissue was significantly lower in both Lsp1+/+ and Lsp1−/− chimeric mice as compared to that of Lsp1+/+ neutrophils in these mice. Analysis of integrin expression of transmigrated neutrophils from CXCL2-induced peritonitis in chimeric mice revealed blunted expression of α6 and β1 on neutrophils only in mice with Lsp1−/− endothelial cells. In conclusion, non-hematopoietic endothelial LSP1 is a novel regulator of neutrophil directionality during neutrophil recrutment.
Signaling via neurokinin-1 receptor stimulates mast cell pro-inflammatory and Th2 biasing functions (P6008)
Authors: Sumpter Tina , Ho Chin , Tkacheva Olga , Shufesky William , Morelli Adrian , Larregina Adriana
Published: January 2023

Pro-inflammatory mast cells (MC) are crucial for initiation and perpetuation of anaphylaxis and atopic diseases. TNFα secretion by activated MC is critical for MC-mediated inflammation. Efficient MC activation depends on exogenous stimuli triggering endogenous mediators, such as pro-inflammatory neuropeptides. Surprisingly, studies linking neuromediators to MC are lacking. Here we analyzed the role of the neuropeptides, substance-P (SP) and hemokinin-1 (HK-1), in the regulation of IgE-mediated MC inflammation. In vitro, we show that MC express the shared receptor for SP and HK-1, the neurokinin-1 receptor (NK1R). FcϵRI ligation of bone marrow derived MC (BMMC) from WT mice increased HK-1 and NK1R mRNA and protein levels but had no effect on SP. NK1R-/- BMMC had significantly decreased degranulation and TNFα release suggesting that autocrine HK-1 promoted MC inflammatory functions. Mechanistic analysis demonstrated that NK1R-/- BMMC had impaired Akt-dependent NFκB activation. In vivo, NK1R-/- mice showed significantly decreased IgE-mediated cutaneous and systemic anaphylaxis and reduced development of chronic asthma, marked by reduced pulmonary eosinophilia, reduced Th2 skewing and low levels of TNFα in bronchial fluid. We conclude that MC utilize a positive feedback loop whereby autocrine HK-1 induces NK1R signaling that, in turn, potentiates TNFα secretion in the course of IgE-mediated anaphylactic and atopic disorders.
Functional expression of the novel C5a receptor C5L2 in human mast cells (P6014)
Authors: Pundir Priyanka , Kulka Marianna
Published: January 2023

C5a regulates immune responses by chemoattracting and activating immune cells, i.e. mast cells (MC). Its postulated that C5a regulates MC function by activating a G protein-coupled receptor (GPCR), however it can bind to C5aR, classical GPCR, or C5L2, not G protein coupled, and thought to activate distinct signaling. The presence and role of C5L2 in MC remain unknown. We hypothesized that human MC express functional C5L2. C5aR and C5L2 expression was analyzed by qPCR and flow cytometry. Degranulation, cytokine/chemokine production, adhesion, and migration were measured using standard assays. Lentivirus short hairpin (sh)RNA was utilized to create a stable C5L2-deficient (LAD2-C5L2ko) and a control MC cell line (LAD2-cntr). LAD2 expressed cell-surface C5L2 but not C5aR. C5a stimulated LAD2 degranulation (25%), TNF (22±1.3pg/ml), GM-CSF (15±0.4pg/ml), MCP1 (53±3.6pg/ml) and IP-10 (32±4.3pg/ml) production, adherence to fibronectin (466±92%), and chemotaxis (148.93±0.94%). Pre-treatment with pertussis toxin, wortmanin, U-73122, and Ro-31-8220 inhibited adhesion. Exposure to C5a reduced cell-surface C5L2 expression by ~45%. LAD2-C5L2ko showed decreased expression of GM-CSF and MIP-1β compared to LAD2-cntr. LAD2-C5L2ko showed reduced adhesion and chemotaxis compared to LAD2-cntr. Therefore, we demonstrated that C5a activates LAD2 through C5L2 which is sensitive to G protein signaling inhibitors and provide the first evidence of the crucial role of C5L2 in regulating MC functions.
The role of germinal centers in T cell receptor revision (P1421)
Authors: Higdon Lauren , Fink Pamela
Published: January 2023

Vβ5 transgenic CD4 T cells are tolerized to the endogenous superantigen Mtv-8 by either deletion or TCR revision. Revision occurs when a peripheral CD4 T cell downregulates Vβ5 and undergoes RAG-mediated recombination and surface expression of an endogenous TCR. Given that revision occurs in germinal centers, and that follicular helper T cells, or Tfh, in Vβ5 Tg mice increase in an Mtv-8-dependent manner, we are studying whether revising cells have a Tfh phenotype and whether germinal center interactions are required for revision. Revising cells have a transcription factor and surface phenotype resembling that of Tfh, with low Blimp-1 and elevated Bcl-6, CXCR5, PD-1, IL-21 receptor, and OX-40 expression. The frequency of post-revision T cells increases in mice heterozygous for Blimp-1, a transcriptional repressor of Tfh formation, but revision intermediates are unaffected. The adaptor molecule SAP is required for prolonged B and T cell interactions in the germinal center, and SAP null mice have reduced numbers of post-revision T cells, but no reduction in revision intermediates. These results suggest that revising T cells share some traits with Tfh and that Tfh formation and prolonged B-T cell interactions are required for the completion, but not initiation, of TCR revision. Ongoing studies will analyze revision in the absence of Bcl-6, a transcription factor required for Tfh formation, and TNFR1, a receptor required for the formation of germinal center structures.
SHP-2 is dispensable for effector CD8+ T cell and iNKT cell development (P1429)
Authors: Miah S. M. Shahjahan , Salter Alexander , Reilly Emma , Yang Wentian , Brossay Laurent
Published: January 2023

Src homology region 2 domain-containing phosphatase-2 (SHP-2) is ubiquitously expressed and is required for gastrulation, hematopoiesis, and embryonic development. Constitutive deletion of SHP-2 in mice is embryonically lethal. During development and maturation, certain subsets of T cells express a variety of inhibitory receptors known to associate with SHP-2. These subsets include iNKT cells, Ly49+CD8+ T cells with a memory phenotype, and exhausted T cells during chronic infection. We hypothesized that conditional SHP-2 deletion would have major effects on T cell development by altering the thresholds for positive and negative selection. Surprisingly, and in contrast to a previous report, we found that SHP-2-deficient CD8+ T cells and iNKT cells develop normally in the thymus and exhibit proper maturation in the periphery. Therefore, SHP-2 is dispensable for development and differentiation of both effector CD8+ T cells and iNKT cells.
Bacteriophage adhered to mucus provide a novel mucosal immune system (P3166)
Authors: Barr Jeremy , Auro Rita , Furlan Mike , Whiteson Katrine , Talago Natasha , Paul Lauren , Erb Marcella , Pogliano Joe , Stotland Aleksandr , Wolkowicz Roland , Cutting Andrew , Doran Kelly , Salamon Peter , Youle Merry , Rohwer Forest
Published: January 2023

Mucosal surfaces serve as a primary entry point for multiple pathogens and are therefore principal sites of immune defense. Here we demonstrate through in vitro and in silico studies that increased phage adherence to the host mucosal layer, provides a novel immune defense mechanism. We show that compared to the surrounding environment, phage-to-bacteria ratios were increased on all mucosal surfaces sampled ranging from cnidarians to humans. This increased phage abundance protects the underlying epithelium from bacterial infection. Enrichment of phage on mucus occurs via interactions between host mucin glycoproteins and phage immunoglobulin-like protein domains exposed on phage capsids. Metagenomic analysis found these immunoglobulin-like proteins present in many environments, particularly those adjacent to mucosal surfaces. Preliminary glycan microarrays and 2D gel electrophoresis show that phage adherence can rapidly adapt to hosts mucus glycan profiles, and in response, the host may regulate its mucus glycosylation to select for a beneficial phage community. This adaptation between phage and host provide a mechanism for the manipulation and selection of the mucosal microbiota. Based on these observations, we present the Bacteriophage Adherence to Mucus (BAM) model describing a phage-derived mucosal immunity with potential applicability to all mucosal surfaces, thus opening a novel field of immunological study.
Amphiregulin is essential for UVB mediated immunosuppression. (P6231)
Authors: Meulenbroeks Chantal , van Weelden Huib , Rutten Victor , Willemse Ton , Sijts Alice , Zaiss Dietmar
Published: January 2023

UVB exposure of the skin is associated with amelioration of inflammatory diseases, which has been shown to be mediated by activated Tregs. Since we have recently shown that Tregs, for optimal functioning, are dependent on signals induced by the EGF-like growth factor AREG, we tested whether UVB-mediated immunosuppression is dependent on AREG. We irradiated C57BL/6 wt and AREG-/- mice with UVB light and sensitized them with DNFB. Mice then were challenged and ear swelling was measured. Our results show that UVB light irradiation suppressed ear swelling in wt but not in AREG-/- mice. To determine at what point AREG expression would be essential, we transferred lymphocytes isolated from lymph nodes of irradiated and sensitized wt mice into naïve wt or AREG-/- mice, that then were sensitized with DNFB and challenged. In contrast to wt recipients, AREG-/- mice lacked suppression upon challenge. Similar results were found when lymphocytes, derived from UVB-irradiated CD4cre x EGF-Rflox/flox or wt mice, were transferred into wt mice. Mice that had received lymphocytes from irradiated and sensitized wt mice were fully able to suppress challenge induced ear swelling, while those that had received lymphocytes derived from UVB-irradiated CD4cre x EGF-Rflox/flox showed a significantly decreased ability to suppress swelling. Our data shows that AREG at the site of inflammation is essential for UVB irradiation-induced and Treg mediated immunosuppression.
The investigation of the role of Acanthamoeba as an allergen in allergic airway inflammation. (P6218)
Authors: Park Mi Kyung , Cho Min Kyoung , Kang Shin Ae , Park Hye Kyung , Yu Hak Sun
Published: January 2023

Asthma patient rapidly have increase recently, but some of these patients do not react to known allergens in skin prick tests, which suggest the presence of unknown environmental allergens. Although Acanthamoeba are abundant in the environment (it also have been often found in human airway cavities) and possess strong proteases enough to elicit allergic airway inflammation, no study has been conducted to determine whether Acanthamoeba can elicit allergic reactions. We repeatedly inoculated Acanthamoeba trophozoites or excretory-secretory (ES) proteins intranasally into mice, and evaluated symptoms and airway immune responses. We found that Acanthamoeba trophozoites or ES proteins reproducibly elicited immune responses resembling allergic airway inflammation. After boiling, which removed protease activity, treatment no longer elevate Th2 related chemokine gene expression in mouse lung epithelial cells, allergic airway inflammation symptoms and Th2 immune responses were remarkably reduced by de-activating proteases. These responses might be not fully elicited through protease activated receptor 2. Furthermore, asthma patients were found to have higher Acanthamoeba specific IgE titers than normal controls. This study suggests that it might be possible to cause allergic airway inflammation by Acanthamoeba (or their products).
Characterization of a weakly expressed KIR2DL1 allele supports a positive role for the KIR distal promoter in proximal KIR promoter activation. (P1357)
Authors: Anderson Stephen , Li Hongchuan , Huehn Andrew , Cooley Sarah , Miller Jeffrey
Published: January 2023

The variegated expression of the human KIR family of class I MHC receptors provides an interesting model system for the study of stochastic activation of gene transcription. Previous studies have linked distal KIR promoter transcription to the initiation of KIR expression from the proximal promoter. In order to identify novel genetic alterations associated with decreased KIR expression, a group of 182 donors was characterized for KIR gene content, KIR transcripts, and FACS analysis of KIR surface expression. An individual was discovered that possessed a single copy of the KIR2DL1 gene but had a low level of gene expression by either FACS or Q-PCR. Complete sequencing of the KIR2DL1 gene confirmed the presence of an intact coding region. Analysis of the three known promoter regions revealed a cluster of 3 single nucleotide polymorphisms (SNPs) in the distal promoter region approximately 1 kb upstream from the start of KIR2DL1 translation. These SNPs are also found in the distal promoter region of the non-transcribed KIR2DL5*002 allele as well as the KIR3DP1 pseudogene. One of these SNPs creates a binding site for the inhibitory ZEB1 transcription factor that overlaps a c-Myc binding site previously implicated in the activation of KIR genes by distal transcription. We therefore propose that the generation of this novel ZEB1-binding site antagonizes the Myc-associated activation of the KIR2DL1 gene by the distal promoter.
Multiplex IP-FCM reveals network-scale protein-protein interaction differences between pro-immunogenic and pro-tolerogenic TCR signaling (P1130)
Authors: Smith Stephen , Neier Steven , Davis Tessa , McCluskey James , Burrows Scott , Sicotte Hughes , Gil-Pages Diana , Pittelkow Mark , Neuhauser Claudia , Schrum Adam
Published: January 2023

T cell receptor signaling involves interactions of various kinases, phosphatases, ubiquitin ligases and adaptor proteins. However, the network-scale protein-protein interactions (PPIs) that mediate signaling, as well as the key differences between pro-immunogenic (agonist) and pro-tolerogenic (antagonist) signals, remain unclear. We have developed a novel method to examine large networks of signaling PPIs, multiplex immunoprecipitation measured by flow cytometry (mIP-FCM). Using multiple classes of Luminex® microspheres, we created a 96-well-formatted matrix array capable of measuring 484 PPIs in a single overnight assay. The array is focused on the most proximal signaling proteins associated with the TCR/CD3 complex. As a model system, we have used Jurkat cells that express the public LC13 TCR, stimulated by antigen presenting cells loaded with either LC13 agonist (FLRGRAYGL) or antagonist (FLRGRFYGL) peptide. Time-course studies have revealed both quantitative and kinetic differences between the two types of signal. We present our latest results mapping the PPI network that mediates TCR signaling. Our results have significantly impacted our understanding of the difference between pro-immunogenic and pro-tolerogenic TCR signals.
Single cell mass cytometry of human peripheral blood reveals an endogenous immune response to surgical trauma (P1144)
Authors: Fragiadakis Gabriela , Gaudilliere Brice , Angst Martin , Nolan Garry
Published: January 2023

An integral component of understanding immune function is measuring how the system responds to perturbation. We hypothesized that studying the immune response to the physiological stress of surgical trauma in vivo would uncover endogenous immune signaling networks critical for the function of the human immune system. Mass cytometry enables the measurement of 45 parameters on single immune cells with antibodies to both surface antigens and intracellular signaling proteins. Upon analyzing peripheral blood from patients at several time points pre- and post-surgery, we discovered that surgical trauma produces distinct population size changes, phenotypic changes, and intracellular signaling responses. Single cell analysis of immune signaling networks identified serial waves of six signaling proteins (STAT1, STAT3, STAT5, P38, S6 and CREB) that coordinated the functional response of the innate and adaptive immune systems. In addition, a subset of myeloid cells expands from 1% to 8% of hematopoietic cells in the 24 hours following surgery in all patients. These cells signal through STAT3, STAT1, CREB, and p38, and are phenotypically similar to previously described myeloid-derived suppressor cells. This systems-level single cell analysis generates a personalized surgical immune response profile of unprecedented resolution. In future work we plan to determine the relationship between these profiles and differences in clinical outcomes.
Transcriptomic and cistromic comparison of the effects of glucocorticoid receptor activation in B-1 and B-2 cells. (P1118)
Authors: Diehl Cody , Spann Nathan , Kaikkonen Minna , Glass Christopher , Witztum Joseph
Published: January 2023

Glucocorticoids are widely used clinically to potently modulate the activity of many immune cells. They bind to the glucocorticoid receptor, which functions as a ligand inducible transcription factor capable of either transactivation or trans-repression of target genes. Surprisingly, little is known regarding the molecular mechanisms by which glucocorticoids modulate B cells in general and B-1 cells in particular. We have previously reported that the glucocorticoid receptor is prominently expressed in both mouse B-1 and B-2 cells. Ex-vivo treatment of primary B-1 cells with dexamethasone inhibits proliferation and antibody secretion as well as strongly induces apoptosis. Utilizing RNA- sequencing the effect of glucocorticoid receptor activation on genome-wide mRNA transcript levels was analyzed and compared in quiescent and activated primary B-1 and B-2 cells. Furthermore, the genome-wide binding of glucocorticoid receptor was analyzed in B-1 and B-2 cells using chromatin immunoprecipitation and deep sequencing. While in general the dexamethasone dependent changes in glucocorticoid receptor binding and mRNA levels were similar between B-1 and B-2 cells, reflecting B cell specific effects, there were notable differences as well, possibly reflecting lineage specific differences between B-1 and B-2 cells. Current work is exploring the reasons for these differential responses in B-1 and B-2 cells to glucocorticoid receptor ligation, and the consequences to B cell function.
The adaptor protein HSH2 regulates the terminal differentiation of B cells resulting in qualitative and quantitative differences in the humoral response (P1120)
Authors: Justement Louis , King R.
Published: January 2023

Endogenous expression of the adaptor protein HSH2 is regulated in a dynamic manner during B cell maturation and differentiation. Developing B cells lack detectable HSH2, whereas T1 and T2 B cells in the periphery exhibit increasing levels of expression. Mature follicular B cells exhibit decreased expression of HSH2 compared to T2 cells and expression is further downregulated in germinal center B cells. In contrast, marginal zone B cells and B1a/b B cells exhibit high-level HSH2 expression. Regulation of HSH2 expression plays a critical role in determining the outcome of the humoral immune response as demonstrated using HSH2 transgenic (Tg) and HSH2 hypomorph (HSH2-Lo) mice. Constitutive expression of HSH2 in B cells results in decreased serum Ig titers for all subclasses with the exception of IgA. HSH2-Tg mice immunized with T-dependent or T-independent antigens exhibit a moderate decrease in the production of antigen-specific IgM, whereas class switched isotypes are significantly decreased compared to control mice. In contrast, HSH2-Lo mice exhibit accelerated and enhanced class switching in response to challenge with antigen. Analysis of HSH2 Tg versus HSH2-Lo B cell activation in vitro demonstrated that altered HSH2 expression does not affect class switching, but it does lead to altered expression of the transcriptional regulatory proteins Bach2 and Blimp-1. Thus, HSH2 appears to play an important role in regulating terminal differentiation of B cells.
Skin pathology in Wiskott-Aldrich syndrome mice is caused by hyperactive CD8+ T cells and increased cross-presentation by DCs (P1048)
Authors: Baptista Marisa , Dahlberg Carin , Liedén Agne , Kuo I-Chun , Sunahara Karen , Wallin Robert , Forsell Mattias , Snapper Scott , Eidsmo Liv , Scheynius Annika , Karlsson Mikael , Susanne Nylén , Westerberg Lisa
Published: January 2023

It remains puzzling how immunodeficiency with hypo-responsive cells can be associated with increased immune cell activation to seemingly harmless substances such as allergens. Here, we have investigated skin pathology in the severe primary immunodeficiency Wiskott-Aldrich syndrome (WAS). Wildtype and WAS protein-deficient (WASp KO) mice were challenged with the allergen Der p 2 to induce eczema or infected with Leishmania major to induce robust skin inflammation. Der p 2 challenge of wildtype mice induced eczema with epidermal hyperplasia and egress of Langerhans cells from the skin. WASp KO mice had increased accumulation of mature DCs in the skin and, in contrast to wildtype cells, Der p 2 induced expansion of WASp KO CD8+ T cells that produced IFNγ. Similar to challenge with Der p 2 and unlike wildtype mice, WASp KO mice infected with L. major showed a skewed immune response with increased expansion of IFNγ-producing CD8+ T cells in the draining lymph node. Finally, we show that WASp KO DCs have increased capacity to cross-present exogenous antigen and activate CD8+ T cells. Our results imply that WASp deficiency in skin inflammation causes a breach in tolerance and skews the immune system to DC mediated CD8+ T cell responses at incorrect sites.
Pulmonary vaccination against influenza virus induces a protective CD8 T cell response, which is regulated by APCs within the lungs (P6154)
Authors: Hemann Emily , Kang Sang-Moo , Legge Kevin
Published: January 2023

Recently there has been a renewed interest in the development of novel pulmonary influenza A virus (IAV) vaccines. CD8 T cells play an important role in clearance of IAV, and our prior studies demonstrate a key role for dendritic cells (DCs) in activation of IAV-specific CD8 T cells in the lymph nodes and maintenance in the lungs during IAV infections. Herein, we determined the role of pulmonary vaccine-induced CD8 T cells in protection from IAV challenge and the requirements for generation and maintenance of such CD8 T cell immunity. Our data demonstrates vaccination with IAV virus-like particles (VLPs) containing IAV HA and M1 leads to a significant increase in HA533-specific CD8 T cells. These VLP-specific CD8 T cells mediate protection of mice challenged with lethal doses of either homo- or heterosubtypic IAVs containing the HA533 epitope. Our preliminary results suggest interactions between DCs and effector T cells within the lungs are required following vaccination for maximal pulmonary VLP-specific CD8 T cell responses. Currently, we are investigating the contribution of inflammation and the APC subsets required in generation, boosting, and maintenance of vaccine-specific CD8 T cells during pulmonary vaccination. Collectively, our results highlight an important role for CD8 T cell mediated protection after i.n. vaccination and the seminal role pulmonary APCs play in activating and maintaining local vaccine-specific CD8 T cell responses against IAV in the lungs.
Age-associated cross-reactive antibody-dependent cellular cytotoxicity to 2009 H1N1 influenza prior to the pandemic (P6160)
Authors: Jegaskanda Sinthujan , Laurie Karen , Amarasena Thakshila , Winnall Wendy , Kramski Marit , De Rose Robert , Barr Ian , Brooks Andrew , Reading Patrick , Kent Stephen
Published: January 2023

During the 2009 H1N1 influenza pandemic (H1N1pdm) older individuals were partially protected from disease. Prior seasonal H1N1 infections likely played a role by the induction of neutralizing antibodies and T cell immunity towards 2009-H1N1pdm. Non-neutralizing antibodies with other effector functions such as antibody-dependent cellular cytotoxicity (ADCC) may have assisted in the immunity observed. We tested sera from a cohort of 177 individuals aged 1-72 years old for ADCC antibodies to H1N1pdm HA either immediately prior to, or following, 2009 H1N1 pandemic by our ADCC NK cell activation assay. Almost all individuals in the >45 age group (28/30 subjects) had ADCC antibodies towards the H1N1pdm prior to the 2009 H1N1 pandemic, whereas approximately half of the individuals in the 1-14 (11/32) and 15-45 (9/16) had ADCC antibodies prior to the 2009 pandemic. The H1N1pdm-specific ADCC antibodies were able to mediate the killing of H1N1pdm-infected respiratory epithelial cells. Additionally, ADCC to H1N1pdm HA correlated with ADCC to the antigenically similar 1918 HA protein in subjects post-2009 influenza pandemic and subjects >45 years had higher ADCC towards a range of seasonal H1N1 HA proteins compared to younger subjects. We conclude that ADCC antibodies may have contributed to the protection exhibited in older individuals during the 2009-H1N1 pandemic. This has significant implications for improved vaccination strategies against future influenza pandemics.
Cross-strain human TCR signatures and their importance in universal immunity against influenza (P6164)
Authors: Loh Liyen , Valkenberg Sophie , Wang George , Thomas Paul , Kedzierska Katherine
Published: January 2023

In contrast to the current antibody-mediated and strain-specific influenza vaccines, T-cells directed at conserved internal proteins elicit broad immunity against seasonal and pandemic influenza viruses. Here, we dissected the optimal human influenza-specific CD8+ T-cell populations across different HLAs, both at functional and clonal levels, to provide insights into universal immunity against influenza. We found that although sporadic variations can be found across many peptides, mutations ‘escaping’ T-cell recognition get fixed only for selected CD8+ T-cell populations. Using a human single-cell multiplex RT-PCR for TCRαβ, we found that specific TCRs can recognize multiple variants derived from different influenza strains. For example, a dominant public TCR clone found at high frequency within M158+CD8+ T-cell responses in all HLA-A*0201+ individuals can recognize the naturally-occurring variants of the peptide. This suggests that the globally common HLA-A2+ population have universal T-cell immunity to any M158 variant, which can, at least in part, explain high conservation level for M158. In addition, our current analyses across other HLAs including HLA-A*01, greatly extend these findings. Our study provides insights into the diversity of the CD8 T cell response against influenza and may help unravel beneficial targets needed for a successful universal vaccine against both season and pandemic influenza strains.
Altered T-bet dominance in interferon-γ decoupled CD4+ T cells with attenuated cytokine storm and preserved memory in influenza (P6144)
Authors: Dutta Avijit , Miaw Shi-Chuen , Yu Jhang-Sian , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Chuang Sheng-Hao , Yen Ming-I , Huang Ching-Tai
Published: January 2023

Cytokine storm has been postulated as one of the major causes of mortality in patients with severe respiratory viral infections such as influenza. With the help of an influenza antigen- specific mouse experimental system, we report that CD4+ T cells contribute effector cytokines leading to lung inflammation in acute influenza. Although virus can no longer be detected from tissues 14 days after infection, virus-derived antigen continues to drive a CD4+ T cell response after viral clearance. Antigen specific CD4+ T cells proliferate and evolve into memory CD4+ T cells efficiently, but the production of effector cytokines is seriously hampered during this phase. This decoupling of proliferation and effector cytokine production doesn’t appear in conjunction with increased suppression by regulatory T cells or decreased induction of transcription factors. Rather, GATA-3 and ROR-γt levels are elevated when compared to cells that have effector cytokine production. T-bet dominance over GATA-3 and ROR-γt decreases with the disarmament of effector cytokine production. Importantly, upon re-infection, these decoupled cells produce elevated levels of IFN-γ and were effective in virus eradication. These results provide a mechanism through altered T-bet expression to dampen the cytokine storm without impeding the generation of memory T cells in influenza virus infection.